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We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large ‘rosehip’-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1–SST–CALB2–PVALB–) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

In 1986, electron microscopy was used to reconstruct by hand the entire nervous system of a roundworm, the nematode Caenorhabditis elegans 1 . Since this landmark study, high-throughput electron-microscopic techniques have enabled reconstructions of much larger mammalian brain circuits at synaptic resolution 2 , 3 . Nevertheless, it remains unknown how the structure of a synapse relates to its physiological transmission strength—a key limitation for inferring brain function from neuronal wiring diagrams. Here we combine slice electrophysiology of synaptically connected pyramidal neurons in the mouse somatosensory cortex with correlated light microscopy and high-resolution electron microscopy of all putative synaptic contacts between the recorded neurons. We find a linear relationship between synapse size and strength, providing the missing link in assigning physiological weights to synapses reconstructed from electron microscopy. Quantal analysis also reveals that synapses contain at least 2.7 neurotransmitter-release sites on average. This challenges existing release models and provides further evidence that neocortical synapses operate with multivesicular release 4 – 6 , suggesting that they are more complex computational devices than thought, and therefore expanding the computational power of the canonical cortical microcircuitry. Electrophysiology combined with correlated light and electron microscopy confirms the long-standing assumption that the size of a synapse is proportional to its strength, and reveals that neocortical synapses may have greater computational capacity than thought.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (LM) and electron microscopy (EM). Here we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100-nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.Kuan, Phelps, et al. used synchrotron X-ray imaging and deep learning to map dense neuronal wiring in fly and mouse tissue, enabling examination of individual cells and connectivity in circuits governing motor control and perceptual decision-making.

The posterior parietal cortex exhibits choice-selective activity during perceptual decision-making tasks 1 – 10 . However, it is not known how this selective activity arises from the underlying synaptic connectivity. Here we combined virtual-reality behaviour, two-photon calcium imaging, high-throughput electron microscopy and circuit modelling to analyse how synaptic connectivity between neurons in the posterior parietal cortex relates to their selective activity. We found that excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity. In turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif. This motif was present even between neurons with activity peaks in different task epochs. We developed neural-circuit models of the computations performed by these motifs, and found that opponent inhibition between neural populations with opposite selectivity amplifies selective inputs, thereby improving the encoding of trial-type information. The models also predict that opponent inhibition between neurons with activity peaks in different task epochs contributes to creating choice-specific sequential activity. These results provide evidence for how synaptic connectivity in cortical circuits supports a learned decision-making task. Excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity and, in turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif that supports decision-making.

Live fluorescent imaging of murine hippocampal slices shows that NMDAR-dependent glutamate signalling leads to postsynaptic BDNF release, with associated signalling of its receptor, TrkB, on the same dendritic spine, suggesting autocrine BDNF signalling. Mechanisms of neuronal plasticity Secreted messenger molecules such as brain-derived neurotrophic factor (BDNF) are known to participate in various forms of neuronal plasticity, such as long-term synaptic potentiation (LTP) and associated changes in dendritic spine morphology, but the exact sites of BDNF release and action remain poorly defined. Two papers from Ryohei Yasuda's lab, published in this issue of Nature , tackle this question. Stephen Harward et al . use live fluorescent imaging of murine hippocampal slices to show that NMDAR-dependent glutamate signalling leads to postsynaptic BDNF release, with associated signalling of its receptor, TrkB, on the same dendritic spine, suggesting autocrine BDNF signalling. In the second study Nathan Hedrick et al . find that the three small GTPases Rac1, RhoA and Cdc42 are differentially involved in structural long-term potentiation of rodent dendritic spines, simultaneously ensuring signal specificity while also priming the system for plasticity. Taken together these results suggest molecular mechanisms for both signal specificity at single spines and synaptic cross-talk, a unique biochemical computation involved in neuronal plasticity and learning. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity 1 , 2 , 3 , 4 , 5 , 6 , including structural long-term potentiation (sLTP) 7 , 8 , which is a correlate of an animal’s learning 7 , 9 , 10 , 11 , 12 . However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy 13 , 14 , 15 , 16 , we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction 9 , 14 , 15 , 16 , we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR ( N -methyl- d -aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin 17 , 18 , 19 . We demonstrate that this postsynaptic BDNF–TrkB signalling pathway is necessary for both structural and functional LTP 20 . Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR–CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

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.

How do neurons encode long-term memories? Bilateral imaging of neuronal activity in the mouse hippocampus reveals that, from one day to the next, ~40% of neurons change their responsiveness to cues, but thereafter only 1% of cells change per day. Despite these changes, neuronal responses are resilient to a lack of exposure to a previously completed task or to hippocampus lesions. Unlike individual neurons, the responses of which change after a few days, groups of neurons with inter- and intrahemispheric synchronous activity show stable responses for several weeks. The likelihood that a neuron maintains its responsiveness across days is proportional to the number of neurons with which its activity is synchronous. Information stored in individual neurons is relatively labile, but it can be reliably stored in networks of synchronously active neurons.

The authors uncovered a pathway from the lateral amygdala to the auditory cortex (ACx) of mice that is essential for auditory fear memory retrieval. Simultaneous imaging of pre- and postsynaptic structures in ACx in vivo revealed an increased rate of synapse formation in this pathway after auditory fear conditioning. Neural circuits underlying auditory fear conditioning have been extensively studied. Here we identified a previously unexplored pathway from the lateral amygdala (LA) to the auditory cortex (ACx) and found that selective silencing of this pathway using chemo- and optogenetic approaches impaired fear memory retrieval. Dual-color in vivo two-photon imaging of mouse ACx showed pathway-specific increases in the formation of LA axon boutons, dendritic spines of ACx layer 5 pyramidal cells, and putative LA–ACx synaptic pairs after auditory fear conditioning. Furthermore, joint imaging of pre- and postsynaptic structures showed that essentially all new synaptic contacts were made by adding new partners to existing synaptic elements. Together, these findings identify an amygdalocortical projection that is important to fear memory expression and is selectively modified by associative fear learning, and unravel a distinct architectural rule for synapse formation in the adult brain.

Microglia have emerged as critical regulators of synapse development and circuit formation in the healthy brain. To date, examination of microglia in such processes has largely been focused on excitatory synapses. Their roles, however, in the modulation of GABAergic interneuron synapses—particularly those targeting the axon initial segment (AIS)—during development remain enigmatic. Here, we identify a synaptogenic/growth-promoting role for microglia in regulating pyramidal neuron (PyN) AIS synapse formation by chandelier cells (ChCs), a unique interneuron subtype whose axonal terminals, called cartridges, selectively target the AIS. We show that a subset of microglia contacts PyN AISs and ChC cartridges and that such tripartite interactions, which rely on the unique AIS cytoskeleton and microglial GABAB1 receptors, are associated with increased ChC cartridge length and bouton number and AIS synaptogenesis. Conversely, microglia depletion or disease-induced aberrant microglia activation impairs the proper development and maintenance of ChC cartridges and boutons, as well as AIS synaptogenesis. These findings unveil key roles for homeostatic, AIS-associated microglia in regulating proper ChC axonal morphogenesis and synaptic connectivity in the neocortex.

In the cerebral cortex, local circuits consist of tens of thousands of neurons, each of which makes thousands of synaptic connections. Perhaps the biggest impediment to understanding these networks is that we have no wiring diagrams of their interconnections. Even if we had a partial or complete wiring diagram, however, understanding the network would also require information about each neuron's function. Here we show that the relationship between structure and function can be studied in the cortex with a combination of in vivo physiology and network anatomy. We used two-photon calcium imaging to characterize a functional property—the preferred stimulus orientation—of a group of neurons in the mouse primary visual cortex. Large-scale electron microscopy of serial thin sections was then used to trace a portion of these neurons’ local network. Consistent with a prediction from recent physiological experiments, inhibitory interneurons received convergent anatomical input from nearby excitatory neurons with a broad range of preferred orientations, although weak biases could not be rejected. Untangling neural nets in the visual system Connectivity forms the basis of functional computations performed by neural circuits, but it is notoriously difficult to follow the complex structural wiring between neurons to the function of individual cells. Now, using a combination of functional imaging and three-dimensional serial electron-microscopic reconstruction at an unprecedented scale, two groups present detailed representations of the connectivity of single cells in the mouse visual system. Davi Bock et al . in Clay Reid's lab investigate connectivity in the primary visual cortex, and find that inhibitory neurons receive input from excitatory cells with widely varying functions, consistent with predictions from recent physiological studies of the mouse cortex. Kevin Briggman, Moritz Helmstaedter and Winfried Denk show that direction-selective ganglion cells receive more synapses from a starburst amacrine cell dendrite if their preferred directions are opposites, suggesting that the directional sensitivity of retinal ganglion cells arises from the asymmetry in their wiring with amacrine cells. To date, various aspects of connectivity have been inferred from electron microscopy (EM) of synaptic contacts, light microscopy of axonal and dendritic arbors, and correlations in activity. However, until now it has not been possible to relate the complex structural wiring between neurons to the function of individual cells. Using a combination of functional imaging and three-dimensional serial EM reconstruction at unprecedented scale, two papers now describe the connectivity of single cells in the mouse visual system. This study investigates the connectivity of inhibitory interneurons in primary visual cortex.