Balázs Rózsa

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging–derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.

Spine neck plasticity regulates compartmentalization of synapses

Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood. We used time-lapse super-resolution stimulated emission depletion (STED) imaging in combination with fluorescence recovery after photobleaching (FRAP) measurements, two-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices. We report a diversity of spine morphologies that argues against common categorization schemes and establish a close link between compartmentalization and spine morphology, wherein spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after long-term potentiation. These morphological changes are predicted to lead to a substantial drop in spine head excitatory postsynaptic potential (EPSP) while preserving overall biochemical compartmentalization.

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation–evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke

Microglia are the main immune cells of the brain and contribute to common brain diseases. However, it is unclear how microglia influence neuronal activity and survival in the injured brain in vivo. Here we develop a precisely controlled model of brain injury induced by cerebral ischaemia combined with fast in vivo two-photon calcium imaging and selective microglial manipulation. We show that selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases.

Distance-Dependent Scaling of Calcium Transients Evoked by Backpropagating Spikes and Synaptic Activity in Dendrites of Hippocampal Interneurons

Although interactions between backpropagating action potentials and synaptic stimulations have been extensively studied in pyramidal neurons, dendritic propagation and the summation of these signals in interneurons are not nearly as well known. In this study, two-photon imaging was used to explore the basic properties of dendritic calcium signaling in CA1 stratum radiatum interneurons. In contrast to hippocampal pyramidal neurons, the backpropagating action potential-evoked calcium transients in dendrites of interneurons underwent a distance-dependent increment. Although, in proximal dendrites, an increment could be attributed to a smaller dendrite diameter, distal dendrites did not show such dependence. Calcium responses in interneurons had a smaller amplitude, slower rise time, and decay than in pyramidal neurons. To explore the factors underlying the difference, we compared the calcium-binding capacity in interneurons and in pyramidal neurons. Our finding that endogenous calcium buffers had a higher level in interneurons may primarily explain the different kinetics and amplitudes of calcium transients. Synaptic stimulation-evoked calcium transients were also larger at distant dendritic locations. The spread of these signals was restricted to 12-13 μm long dendritic compartments. Supporting the reported lack of long-term potentiation in these interneurons, we found only sublinear or linear summations of calcium responses to coincident synaptic inputs and backpropagating spikes.

Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules

Tracing cells that project to one neuron Feature extraction is a prominent characteristic of cortical neurons involved in the early stages of sensory processing. Wertz et al. retrogradely marked an injected neuron and its direct inputs to reveal the network mechanisms that mediate their response. Neurons within each presynaptic network layer of single direction-selective cells showed similar motion direction preferences. In some networks, layer-specific functional modules were identical to the orientation preference of the postsynaptic neuron. Presynaptic neurons, however, displayed a general bias toward the stimulus feature that elicited a response in the postsynaptic neuron. Science, this issue p. 70 Orientation-sensitive visual neurons receive input from presynaptic neurons with similar stimulus preferences. Individual cortical neurons can selectively respond to specific environmental features, such as visual motion or faces. How this relates to the selectivity of the presynaptic network across cortical layers remains unclear. We used single-cell–initiated, monosynaptically restricted retrograde transsynaptic tracing with rabies viruses expressing GCaMP6s to image, in vivo, the visual motion–evoked activity of individual layer 2/3 pyramidal neurons and their presynaptic networks across layers in mouse primary visual cortex. Neurons within each layer exhibited similar motion direction preferences, forming layer-specific functional modules. In one-third of the networks, the layer modules were locked to the direction preference of the postsynaptic neuron, whereas for other networks the direction preference varied by layer. Thus, there exist feature-locked and feature-variant cortical networks.

Differential distribution of NCX1 contributes to spine–dendrite compartmentalization in CA1 pyramidal cells

Compartmentalization of Ca2+ between dendritic spines and shafts is governed by diffusion barriers and a range of Ca2+ extrusion mechanisms. The distinct contribution of different Ca2+ clearance systems to Ca2+ compartmentalization in dendritic spines versus shafts remains elusive. We applied a combination of ultrastructural and functional imaging methods to assess the subcellular distribution and role of NCX1 in rat CA1 pyramidal cells. Quantitative electron microscopic analysis of preembedding immunogold reactions revealed uniform densities of NCX1 along the shafts of apical and basal dendrites, but densities in dendritic shafts were approximately seven times higher than in dendritic spines. In line with these results, two-photon imaging of synaptically activated Ca2+ transients during NCX blockade showed preferential action localized to the dendritic shafts for NCXs in regulating spine–dendrite coupling.

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons

Inhibitory interneurons are considered to be the controlling units of neural networks, despite their sparse number and unique morphological characteristics compared with excitatory pyramidal cells. Although pyramidal cell dendrites have been shown to display local regenerative events—dendritic spikes (dSpikes)—evoked by artificially patterned stimulation of synaptic inputs, no such studies exist for interneurons or for spontaneous events. In addition, imaging techniques have yet to attain the required spatial and temporal resolution for the detection of spontaneously occurring events that trigger dSpikes. Here we describe a high-resolution 3D two-photon laser scanning method (Roller Coaster Scanning) capable of imaging long dendritic segments resolving individual spines and inputs with a temporal resolution of a few milliseconds. By using this technique, we found that local, NMDA receptor-dependent dSpikes can be observed in hippocampal CA1 stratum radiatum interneurons during spontaneous network activities in vitro. These NMDA spikes appear when approximately 10 spatially clustered inputs arrive synchronously and trigger supralinear integration in dynamic interaction zones. In contrast to the one-to-one relationship between computational subunits and dendritic branches described in pyramidal cells, here we show that interneurons have relatively small (∼14 μm) sliding interaction zones. Our data suggest a unique principle as to how interneurons integrate synaptic information by local dSpikes.

Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes.

Organic electrochemical transistors are integrated on depth probes to achieve localized electrical stimulation of neurons. The probes feature a mechanical delamination process which leaves only a 4 μm thick film with embedded transistors inside the brain. This considerably reduces probe invasiveness and correspondingly improves future brain-machine interfaces.

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.