We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1T. We apply the method to optically map synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator we split the laser beam onto several neurons and perform simultaneous optogenetic activation of selected neurons in three dimensions.

Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

Dendritic spines are the primary site of excitatory synaptic input onto neurons, and are biochemically isolated from the parent dendritic shaft by their thin neck. However, due to the lack of direct electrical recordings from spines, the influence that the neck resistance has on synaptic transmission, and the extent to which spines compartmentalize voltage, specifically excitatory postsynaptic potentials, albeit critical, remains controversial. Here, we use quantum-dot-coated nanopipette electrodes (tip diameters ~15–30 nm) to establish the first intracellular recordings from targeted spine heads under two-photon visualization. Using simultaneous somato-spine electrical recordings, we find that back propagating action potentials fully invade spines, that excitatory postsynaptic potentials are large in the spine head (mean 26 mV) but are strongly attenuated at the soma (0.5–1 mV) and that the estimated neck resistance (mean 420 MΩ) is large enough to generate significant voltage compartmentalization. Nanopipettes can thus be used to electrically probe biological nanostructures.

Synaptic refinement via the elimination of inappropriate synapses and strengthening of appropriate ones is crucially important for the establishment of specific, topographic neural circuits. The mechanisms driving these processes are poorly understood, particularly concerning inhibitory projections. Here, we address the refinement of an inhibitory topographic projection in the auditory brainstem in functional and anatomical mapping studies involving patch-clamp recordings in combination with minimal and maximal stimulation, caged glutamate photolysis, and single axon tracing. We demonstrate a crucial dependency of the refinement on Ca V 1.3 calcium channels: Ca V 1.3 Ϫ/Ϫ mice displayed virtually no elimination of projections up to hearing onset. Furthermore, strengthening was strongly impaired, in line with a reduced number of axonal boutons. The mediolateral topography was less precise and the shift from a mixed GABA/ glycinergic to a purely glycinergic transmission before hearing onset did not occur. Together, our findings provide evidence for a Ca V 1.3-dependent mechanism through which both inhibitory circuit formation and determination of the neurotransmitter phenotype are achieved.

Within the Ca v 1 family of voltage-gated calcium channels, Ca v 1.2 and Ca v 1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Ca v 1.3 subunits in the activity-dependent development of the auditory brainstem. We used Ca v 1.3-deficient (Ca v 1.3 Ϫ/Ϫ ) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25-59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Ca v 1.3 Ϫ/Ϫ mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the K v 1.2 subtype. Fura2 imaging provided evidence for functional Ca v 1.3 channels in the LSO of wild-type mice. Our results imply that Ca v 1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Ca v 1.3 Ϫ/Ϫ mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Ca v 1.3 channels in the neurons plus lack of peripheral input.

Key points Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10–12 and P27–49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB–LSO inputs, imprecise MNTB–LSO topography and weaker MNTB–LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild‐types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB–LSO refinement during a critical prehearing period. Abstract Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super‐numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB–LSO circuit refinement after P20. Moreover, MNTB–LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10–12) and young adult (P27–49) otoferlin knockout (Otof KO) mice with respect to MNTB–LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild‐types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB–LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB–LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB–LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild‐types. Collectively, we provide novel insights into functional MNTB–LSO circuit maturation governed by a cochlea‐specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss.

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Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive—a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions.