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Postsynaptic mitochondria are critical for the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We examined a correlative dataset from ferret visual cortex with in vivo two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally and structurally characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with a mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons.

Brain circuits are highly interconnected three-dimensional structures fabricated from components ranging vastly in size; from cell bodies to individual synapses. While neuronal activity can be visualized with advanced light microscopy (LM) techniques, the resolution of electron microscopy (EM) is critical for identifying synaptic connections between neurons. Here, we combine these two techniques, affording the advantage of each and allowing for measurements to be made of the same neural features across imaging platforms. We established an EM-label-free workflow utilizing inherent structural features to correlate in vivo two-photon LM and volumetric scanning EM (SEM) in the ferret visual cortex. By optimizing the volume SEM sample preparation protocol, imaging with the OnPoint detector, and utilizing the focal charge compensation device during serial block-face imaging, we achieved sufficient resolution and signal-to-noise ratio to analyze synaptic ultrastructure for hundreds of synapses within sample volumes. Our novel workflow provides a reliable method for quantitatively characterizing synaptic ultrastructure in functionally imaged neurons, providing new insights into neuronal circuit organization.

Single neocortical neurons are driven by populations of excitatory inputs, which form the basis of neuronal selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity 1 , whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others 2 . Evidence in support of this process includes measurements of synaptic ultrastructure and in vitro and in vivo physiology and imaging studies 3 – 8 . These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neuronal selectivity, whereas weak synaptic inputs are less correlated with the somatic output and modulate activity overall 6 , 7 . Supporting evidence from cortical circuits, however, has been limited to measurements of neighbouring, connected cell pairs, raising the question of whether this prediction holds for a broad range of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy. Using electron microscopy reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses have a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs appears to be reserved for weaker synapses, enhancing the contribution of weak synapses to somatic responses. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths. Live neuron imaging and electron microscopy reconstruction shows that the selectivity of cortical neuron responses to visual stimuli arises from the total number of synapses activated rather than being dominated by a small number of strong synaptic inputs.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03158-8.

Most excitatory synapses in the mammalian brain are contacted by astrocytes, forming the tripartite synapse. This interface is thought to be critical for glutamate turnover and structural or functional dynamics of synapses. While the degree of synaptic contact of astrocytes is known to vary across brain regions and animal species, the implications of this variability remain unknown. Furthermore, precisely how astrocyte coverage of synapses relates to functional properties of individual dendritic spines has yet to be investigated. Here, we characterized perisynaptic astrocyte processes (PAPs) contacting synapses of pyramidal neurons of the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and to sensory-evoked Ca activity. Nearly all synapses were contacted by PAPs, and most were contacted along the axon-spine interface (ASI). Structurally, we found that the degree of PAP coverage scaled with synapse size and complexity. Functionally, we found that PAP coverage scaled with the selectivity of Ca responses of individual synapses to visual stimuli and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca events. Our study shows astrocyte coverage is highly correlated with structural properties of excitatory synapses in the visual cortex and implicates astrocytes as a contributor to reliable sensory activation.

A team of facilitators describe the process and content of portfolios they create for families attending weekly playgroup sessions based on the philosophy and practices of the Parents Interacting with Infants (PIWI) model. The parent–child portfolios are a form of authentic assessment and highlight children’s development within the context of parent–child relationships. Facilitators complete weekly observations of parent–child interactions, which allow them to monitor children’s progress on goals set by parents, evaluate their own practices, and plan future playgroup sessions.

Postsynaptic mitochondria are critical for the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We examined a correlative dataset from ferret visual cortex with in vivo two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally and structurally characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with a mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons.

Postsynaptic mitochondria are critical to the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We examined a correlative dataset from ferret visual cortex with two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy (EM) reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally- and structurally-characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons.

Single neocortical neurons are driven by populations of excitatory inputs, forming the basis of neural selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity, whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others. Evidence in support of this process ranges from measurements of synaptic ultrastructure to slice and in vivo physiology and imaging studies. These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neural selectivity, while weak synaptic inputs are less correlated with the functional properties of somatic output and act to modulate activity overall. Supporting evidence from cortical circuits, however, has been limited to measurements of neighboring, connected cell pairs, raising the question of whether this prediction holds for the full profile of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy (EM). Using EM reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses play a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs, thought to amplify synaptic drive, appears reserved for weaker synapses, further enhancing the contribution of the large number of weak synapses to somatic response. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.