Scientific Understanding of Consciousness
Consciousness as an Emergent Property of Thalamocortical Activity

Synaptic Motor Traces in the Motor Cortex


Nature  525, 333–338 (17 September 2015)

Labelling and optical erasure of synaptic memory traces in the motor cortex

Akiko Hayashi-Takagi,

PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut 06032, USA

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA

Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA


Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.

Optogenetics is a powerful tool for controlling neuronal action potentials, and has been used to demonstrate the crucial role of cell assemblies in representing memory traces. However, owing to the limitations of spatial resolution of probes currently available, manipulation of individual dendritic spines, the major sites of excitatory synapses, has been unfeasible, hindering the comprehensive understanding of synaptic reorganization during learning. Thus, for spine-specific light control, we took advantage of the structural properties of spines: the tight correlation between spine volume and function. Because the prolonged activation of the small GTPase Rac1 induces spine shrinkage, we used a photoactivatable form of Rac1 (PaRac1) to induce spine shrinkage, which allowed us to control synaptic transmission with light. Moreover, since it has been suggested for a long time that the memory trace is allocated to specific neurons and spines of neurocircuits; here we targeted PaRac1 to the activated synapses (activated synapse targeting PaRac1, AS-PaRac1) to establish a novel method, termed ‘synaptic optogenetics’, to visualize and manipulate the memory trace.

Current models of learning and memory suggest that structural plasticity of spines is the underlying mechanism of information storage in the brain. Nonetheless, clear visualization of spine structure in vivo requires the sparse labelling of neurons, and analysis of structural changes in spines is very laborious. In contrast, the AS-PaRac1 signal appears as fluorescence puncta, which allows the detection of potentiated spines far more easily, even at high transfection condition. Moreover, the role of potentiated spines can be directly assessed with photoactivation during behavioural examinations. In this study, we showed that photoactivation of the bilateral M1 cortex disrupted the acquired motor skill. We estimated the number of learning-evoked neurons affected by photoactivation was approximately 4,700 neurons based on the following calculation: (a) × (b) × (c) × (d) × (e), in which (a) represents the density of neurons in the neocortex, 9.2 × 104/mm3; (b) the photoactivated area, fibre core diameter = 500 μm, 0.4 mm2/bilateral; (c) the thickness of cortical layers (II–V) that were infected with AAV, 0.8 mm; (d) AAV infection efficiency, 80%; (e) the percentage of AS-PaRac1-positive neurons upon learning, 20%. On the other hand, due to the limitations of light transmission, the majority of the shrunk spines resided in layer I (up to 100 μm from the dura). The minimal number of learning-evoked spines illuminated by the optical fibre was roughly 410,000 spines in the bilateral M1 cortex based on the following calculation: (d) × (f) × (g) × (h), in which (f) represents the density of excitatory synapses in the mouse neocortex, 6.4 × 108/mm3; (g) learning-evoked potentiation, approximately 2% of the spines in this area; (h) brain volume that received photoactivation: 0.4 mm2 of photoactivation area × 0.1 mm of depth = 0.04 mm3). In the layer I, corticocortical feedback projections mediating top-down influences are concentrated, which strongly excite a subpopulation of pyramidal neurons. Learning-evoked changes in neuronal ensembles via the synaptic reorganization of the M1 cortex directly predict future task performance. As nonlinear information integration primarily occurs in the tuft of dendrites in behaving animals, and activation of several spines in the tuft is sufficient to initiate NMDA spikes for action potential generation. Thus, the shrinkage of potentiated spines in our study (410,000 spines in the dendritic tufts of 4,700 neurons) would be reasonably expected to disrupt the learning-evoked substantial remodelling in a specific neuronal population. Formation of the dense connections in a small neuronal ensemble may be consistent with the formation of functional neuronal clusters in the motor cortex after learning. Thus, synaptic optogenetics might be a powerful tool to uncover the mechanism of synaptic plasticity and its relationships with subsequent behavioural manifestations.



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