The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Red blood cells stabilize flow in brain microvascular networks.

Capillaries are the prime location for oxygen and nutrient exchange in all tissues. Despite their fundamental role, our knowledge of perfusion and flow regulation in cortical capillary beds is still limited. Here, we use in vivo measurements and blood flow simulations in anatomically accurate microvascular network to investigate the impact of red blood cells (RBCs) on microvascular flow. Based on these in vivo and in silico experiments, we show that the impact of RBCs leads to a bias toward equating the values of the outflow velocities at divergent capillary bifurcations, for which we coin the term "well-balanced bifurcations". Our simulation results further reveal that hematocrit heterogeneity is directly caused by the RBC dynamics, i.e. by their unequal partitioning at bifurcations and their effect on vessel resistance. These results provide the first in vivo evidence of the impact of RBC dynamics on the flow field in the cortical microvasculature. By structural and functional analyses of our blood flow simulations we show that capillary diameter changes locally alter flow and RBC distribution. A dilation of 10% along a vessel length of 100 µm increases the flow on average by 21% in the dilated vessel downstream a well-balanced bifurcation. The number of RBCs rises on average by 27%. Importantly, RBC up-regulation proves to be more effective the more balanced the outflow velocities at the upstream bifurcation are. Taken together, we conclude that diameter changes at capillary level bear potential to locally change the flow field and the RBC distribution. Moreover, our results suggest that the balancing of outflow velocities contributes to the robustness of perfusion. Based on our in silico results, we anticipate that the bi-phasic nature of blood and small-scale regulations are essential for a well-adjusted oxygen and energy substrate supply.

Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging.

Quantitative imaging of oxygen distributions in tissue can provide invaluable information about metabolism in normal and diseased states. Two-photon phosphorescence lifetime microscopy (2PLM) has been developed to perform measurements of oxygen in vivo with micron-scale resolution in 3D; however, the method's potential has not yet been fully realized due to the limitations of current phosphorescent probe technology. Here, we report a new sensor, Oxyphor 2P, that enables oxygen microscopy twice as deep (up to 600 µm below the tissue surface) and with ∼60 times higher speed than previously possible. Oxyphor 2P allows longitudinal oxygen measurements without having to inject the probe directly into the imaged region. As proof of principle, we monitored oxygen dynamics for days following micro-stroke induced by occlusion of a single capillary in the mouse brain. Oxyphor 2P opens up new possibilities for studies of tissue metabolic states using 2PLM in a wide range of biomedical research areas.

Vascular density and distribution in neocortex.

An amazingly wide range of complex behavior emerges from the cerebral cortex. Much of the information processing that leads to these behaviors is performed in neocortical circuits that span throughout the six layers of the cortex. Maintaining this circuit activity requires substantial quantities of oxygen and energy substrates, which are delivered by the complex yet well-organized and tightly-regulated vascular system. In this review, we provide a detailed characterization of the most relevant anatomical and functional features of the cortical vasculature. This includes a compilation of the available data on laminar variation of vascular density and the topological aspects of the microvascular system. We also review the spatio-temporal dynamics of cortical blood flow regulation and oxygenation, many aspects of which remain poorly understood. Finally, we discuss some of the important implications of vascular density, distribution, oxygenation and blood flow regulation for (laminar) fMRI.

Intravitreal AAV-Delivery of Genetically Encoded Sensors Enabling Simultaneous Two-Photon Imaging and Electrophysiology of Optic Nerve Axons.

Myelination of axons by oligodendrocytes is a key feature of the remarkably fast operating CNS. Oligodendrocytes not only tune axonal conduction speed but are also suggested to maintain long-term axonal integrity by providing metabolic support to the axons they ensheath. However, how myelinating oligodendrocytes impact axonal energy homeostasis remains poorly understood and difficult to investigate. Here, we provide a method of how to study electrically active myelinated axons expressing genetically encoded sensors by combining electrophysiology and two-photon imaging of acutely isolated optic nerves. We show that intravitreal adeno-associated viral (AAV) vector delivery is an efficient tool to achieve functional sensor expression in optic nerve axons, which is demonstrated by measuring axonal ATP dynamics following AAV-mediated sensor expression. This novel approach allows for fast expression of any optical sensor of interest to be studied in optic nerve axons without the need to go through the laborious process of producing new transgenic mouse lines. Viral-mediated biosensor expression in myelinated axons and the subsequent combination of nerve recordings and sensor imaging outlines a powerful method to investigate oligodendroglial support functions and to further interrogate cellular mechanisms governing axonal energy homeostasis under physiological and pathological conditions.

The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 2: Capillary Networks.

Brain metabolism is highly dependent on continuous oxygen supply. Cortical microvascular networks exhibit heterogeneous blood flow, leading to non-uniform tissue oxygenation and capillary hemoglobin saturation. We recently proposed capillary outflow saturation heterogeneity (COSH) to represent effects of heterogeneity on oxygen supply to tissue regions most vulnerable to hypoxia, and showed that diffusive oxygen exchange among red blood cells within capillaries and among capillaries (diffusive interaction) significantly reduces COSH in simplified geometrical configurations. Here, numerical simulations of oxygen transport in capillary network geometries derived from mouse somatosensory cortex are presented. Diffusive interaction was found to reduce COSH by 41 to 62% compared to simulations where diffusive interaction was excluded. Hemoglobin saturation drop across the microvascular network is strongly correlated with red blood cell transit time, but the coefficient of variation of saturation drop is approximately one third lower. Unexpectedly, the radius of the tissue cylinder supplied by a capillary correlates weakly with the anatomical tissue cylinder radius, but strongly with hemoglobin saturation. Thus, diffusive interaction contributes greatly to the microcirculation's ability to achieve tissue oxygenation, despite heterogeneous capillary transit time and hematocrit distribution. These findings provide insight into the effects of cerebral small vessel disease on tissue oxygenation and brain function.

Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons.

Sensory stimulation evokes intracellular calcium signals in astrocytes; however, the timing of these signals is disputed. Here, we used novel combinations of genetically encoded calcium indicators for concurrent two-photon imaging of cortical astrocytes and neurons in awake mice during whisker deflection. We identified calcium responses in both astrocyte processes and endfeet that rapidly followed neuronal events (∼120 ms after). These fast astrocyte responses were largely independent of IP3R2-mediated signaling and known neuromodulator activity (acetylcholine, serotonin, and norepinephrine), suggesting that they are evoked by local synaptic activity. The existence of such rapid signals implies that astrocytes are fast enough to play a role in synaptic modulation and neurovascular coupling. VIDEO ABSTRACT.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity.

Design and performance of an ultra-flexible two-photon microscope for in vivo research.

We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.

Improving estimates of the cerebral metabolic rate of oxygen from optical imaging data.

The cerebral metabolic rate of oxygen (CMRO2) is an important measure of brain function. Since it is challenging to measure directly, especially dynamically, a number of neuroimaging techniques aim to infer activation-induced changes in CMRO2 from indirect data. Here, we employed a mathematical modelling approach, based on fundamental biophysical principles, to investigate the validity of the widely-used method to calculate CMRO2 from optical measurements of cerebral blood flow and haemoglobin saturation. In model-only simulations and simulations of in vivo data changes in CMRO2 calculated in this way differed substantially from the changes in CMRO2 directly imposed on the model, under both steady state and dynamic conditions. These results suggest that the assumptions underlying the calculation method are not appropriate, and that it is important to take into account, under steady state conditions: 1) the presence of deoxyhaemoglobin in arteriolar vessels; and 2) blood volume changes, especially in veins. Under dynamic conditions, the model predicted that calculated changes in CMRO2 are moderately correlated with the rate of oxygen extraction--not consumption--during the initial phase of stimulation. However, during later phases of stimulation the calculation is dominated by the change in blood flow. Therefore, we propose that a more sophisticated approach is required to estimate CMRO2 changes from these types of data.

Extra permeability is required to model dynamic oxygen measurements: evidence for functional recruitment?

Neural activation triggers a rapid, focal increase in blood flow and thus oxygen delivery. Local oxygen consumption also increases, although not to the same extent as oxygen delivery. This 'uncoupling' enables a number of widely-used functional neuroimaging techniques; however, the physiologic mechanisms that govern oxygen transport under these conditions remain unclear. Here, we explore this dynamic process using a new mathematical model. Motivated by experimental observations and previous modeling, we hypothesized that functional recruitment of capillaries has an important role during neural activation. Using conventional mechanisms alone, the model predictions were inconsistent with in vivo measurements of oxygen partial pressure. However, dynamically increasing net capillary permeability, a simple description of functional recruitment, led to predictions consistent with the data. Increasing permeability in all vessel types had the same effect, but two alternative mechanisms were unable to produce predictions consistent with the data. These results are further evidence that conventional models of oxygen transport are not sufficient to predict dynamic experimental data. The data and modeling suggest that it is necessary to include a mechanism that dynamically increases net vascular permeability. While the model cannot distinguish between the different possibilities, we speculate that functional recruitment could have this effect in vivo.

Arteries dominate volume changes during brief functional hyperemia: evidence from mathematical modelling.

Variations in local neural activity are accompanied by rapid, focal changes in cerebral blood flow and volume. While a range of observations have shown that dilation occurs in cerebral arteries, there is conflicting evidence about the significance of volume changes in post-arteriole vessels. Here, we reconcile the competing observations using a new mathematical model of the hemodynamic response. First, we followed a 'top down' approach, without constraining the model, but using experimental observations at progressively more detailed scales to ensure physiological behaviour. Then, we blocked dilation of post-arteriole vessels, and predicted observations at progressively more aggregated scales (a 'bottom up' approach). Predictions of blood flow, volume, velocity, and vessel diameter changes were consistent with experimental observations. Interestingly, the model predicted small, slow increases in capillary and venous diameter in agreement with recent in vivo data. Blocking dilation in these vessels led to erroneous volume predictions. The results are further evidence that arteries make up the majority of blood volume increases during brief functional activation. However, dilation of capillaries and veins appears to be increasingly significant during extended stimulation. These are important considerations when interpreting results from different neurovascular imaging modalities.