Jeffrey M. LeDue

Spontaneous cortical activity alternates between motifs defined by regional axonal projections

Using millisecond-timescale voltage-sensitive dye imaging in lightly anesthetized or awake adult mice, we show that a palette of sensory-evoked and hemisphere-wide activity motifs are represented in spontaneous activity. These motifs can reflect multiple modes of sensory processing, including vision, audition and touch. We found similar cortical networks with direct cortical activation using channelrhodopsin-2. Regional analysis of activity spread indicated modality-specific sources, such as primary sensory areas, a common posterior-medial cortical sink where sensory activity was extinguished within the parietal association area and a secondary anterior medial sink within the cingulate and secondary motor cortices for visual stimuli. Correlation analysis between functional circuits and intracortical axonal projections indicated a common framework corresponding to long-range monosynaptic connections between cortical regions. Maps of intracortical monosynaptic structural connections predicted hemisphere-wide patterns of spontaneous and sensory-evoked depolarization. We suggest that an intracortical monosynaptic connectome shapes the ebb and flow of spontaneous cortical activity.

In vivo Large-Scale Cortical Mapping Using Channelrhodopsin-2 Stimulation in Transgenic Mice Reveals Asymmetric and Reciprocal Relationships between Cortical Areas

We have mapped intracortical activity in vivo independent of sensory input using arbitrary point channelrhodopsin-2 (ChR2) stimulation and regional voltage sensitive dye imaging in B6.Cg-Tg (Thy1-COP4/EYFP)18Gfng/J transgenic mice. Photostimulation of subsets of deep layer pyramidal neurons within forelimb, barrel, or visual primary sensory cortex led to downstream cortical maps that were dependent on synaptic transmission and were similar to peripheral sensory stimulation. ChR2-evoked maps confirmed homotopic connections between hemispheres and intracortical sensory and motor cortex connections. This ability of optogentically activated subpopulations of neurons to drive appropriate downstream maps suggests that mechanisms exist to allow prototypical cortical maps to self-assemble from the stimulation of neuronal subsets. Using this principle of map self-assembly, we employed ChR2 point stimulation to map connections between cortical areas that are not selectively activated by peripheral sensory stimulation or behavior. Representing the functional cortical regions as network nodes, we identified asymmetrical connection weights in individual nodes and identified the parietal association area as a network hub. Furthermore, we found that the strength of reciprocal intracortical connections between primary and secondary sensory areas are unequal, with connections from primary to secondary sensory areas being stronger than the reciprocal.

Activation of Neuronal NMDA Receptors Triggers Transient ATP-Mediated Microglial Process Outgrowth

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron–microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron–microglial communication mediated by ATP.

Optogenetic Mapping after Stroke Reveals Network-Wide Scaling of Functional Connections and Heterogeneous Recovery of the Peri-Infarct

We used arbitrary point channelrhodopsin-2 (ChR2) stimulation and wide-scale voltage sensitive dye (VSD) imaging in mice to map altered cortical connectivity at 1 and 8 weeks after a targeted cortical stroke. Network analysis based on optogenetic stimulation revealed a symmetrical sham network with distinct sensorimotor and association groupings. This symmetry was disrupted after stroke: at 1 week after stroke, we observed a widespread depression of optogenetically evoked activity that extended to the non-injured hemisphere; by 8 weeks, significant recovery was observed. When we considered the network as a whole, scaling the ChR2-evoked VSD responses from the stroke groups to match the sham group mean resulted in a relative distribution of responses that was indistinguishable from the sham group, suggesting network-wide down-scaling and connectional diaschisis after stroke. Closer inspection revealed that connections that had little connectivity with the peri-infarct, such as contralateral visual areas, tended to escape damage, whereas some connections near the peri-infarct were more severely affected. When connections within the peri-infarct were isolated, we did not observe equal down-scaling of responses after stroke. Peri-infarct sites that had weak connection strength in the sham condition tended to have the greatest relative post-stroke recovery. Our findings suggest that, during recovery, most cortical areas undergo homeostatic upscaling, resulting in a relative distribution of responses that is similar to the pre-stroke (sham) network, albeit still depressed. However, recovery within the peri-infarct zone is heterogeneous and these cortical points do not follow the recovery scaling factor expected for the entire network.

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+‐sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two‐photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single‐cell patch‐loading strategy to image Ca2+‐sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L‐ or T‐type voltage‐gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+. The Ca2+ influx dependency of the spontaneous signals in patch‐loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093–2103

Mesoscale infraslow spontaneous membrane potential fluctuations recapitulate high-frequency activity cortical motifs

Neuroimaging of spontaneous, resting-state infraslow (<0.1 Hz) brain activity has been used to reveal the regional functional organization of the brain and may lead to the identification of novel biomarkers of neurological disease. However, these imaging studies generally rely on indirect measures of neuronal activity and the nature of the neuronal activity correlate remains unclear. Here we show, using wide-field, voltage-sensitive dye imaging, the mesoscale spatiotemporal structure and pharmacological dependence of spontaneous, infraslow cortical activity in anaesthetized and awake mice. Spontaneous infraslow activity is regionally distinct, correlates with electroencephalography and local field potential recordings, and shows bilateral symmetry between cortical hemispheres. Infraslow activity is attenuated and its functional structure abolished after treatment with voltage-gated sodium channel and glutamate receptor antagonists. Correlation analysis reveals patterns of infraslow regional connectivity that are analogous to cortical motifs observed from higher-frequency spontaneous activity and reflect the underlying framework of intracortical axonal projections.

Optogenetic Stimulation of GABA Neurons can Decrease Local Neuronal Activity While Increasing Cortical Blood Flow

We investigated the link between direct activation of inhibitory neurons, local neuronal activity, and hemodynamics. Direct optogenetic cortical stimulation in the sensorimotor cortex of transgenic mice expressing Channelrhodopsin-2 in GABAergic neurons (VGAT-ChR2) greatly attenuated spontaneous cortical spikes, but was sufficient to increase blood flow as measured with laser speckle contrast imaging. To determine whether the observed optogenetically evoked gamma aminobutyric acid (GABA)-neuron hemodynamic responses were dependent on ionotropic glutamatergic or GABAergic synaptic mechanisms, we paired optogenetic stimulation with application of antagonists to the cortex. Incubation of glutamatergic antagonists directly on the cortex (NBQX and MK-801) blocked cortical sensory evoked responses (as measured with electroencephalography and intrinsic optical signal imaging), but did not significantly attenuate optogenetically evoked hemodynamic responses. Significant light-evoked hemodynamic responses were still present after the addition of picrotoxin (GABA-A receptor antagonist) in the presence of the glutamatergic synaptic blockade. This activation of cortical inhibitory interneurons can mediate large changes in blood flow in a manner that is by and large not dependent on ionotropic glutamatergic or GABAergic synaptic transmission. This supports the hypothesis that activation of inhibitory neurons can increase local cerebral blood flow in a manner that is not entirely dependent on levels of net ongoing neuronal activity.

Optogenetic approaches for functional mouse brain mapping

To better understand the connectivity of the brain, it is important to map both structural and functional connections between neurons and cortical regions. In recent years, a set of optogenetic tools have been developed that permit selective manipulation and investigation of neural systems. These tools have enabled the mapping of functional connections between stimulated cortical targets and other brain regions. Advantages of the approach include the ability to arbitrarily stimulate brain regions that express opsins, allowing for brain mapping independent of behavior or sensory processing. The ability of opsins to be rapidly and locally activated allows for investigation of connectivity with spatial resolution on the order of single neurons and temporal resolution on the order of milliseconds. Optogenetic methods for functional mapping have been applied in experiments ranging from in vitro investigation of microcircuits, to in vivo probing of inter-regional cortical connections, to examination of global connections within the whole brain. We review recently developed functional mapping methods that use optogenetic single-point stimulation in the rodent brain and employ cellular electrophysiology, evoked motor movements, voltage sensitive dyes (VSDs), calcium indicators, or functional magnetic resonance imaging (fMRI) to assess activity. In particular we highlight results using red-shifted organic VSDs that permit high temporal resolution imaging in a manner spectrally separated from Channelrhodopsin-2 (ChR2) activation. VSD maps stimulated by ChR2 were dependent on intracortical synaptic activity and were able to reflect circuits used for sensory processing. Although the methods reviewed are powerful, challenges remain with respect to finding approaches that permit selective high temporal resolution assessment of stimulated activity in animals that can be followed longitudinally.

Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aβ plaques by iGluSnFR two-photon imaging

Amyloid-β (Aβ) plaques, a hallmark of Alzheimers disease (AD), are surrounded by regions of neuronal and glial hyperactivity. We use in vivo two-photon and wide-field imaging of the glutamate sensor iGluSnFR to determine whether pathological changes in glutamate dynamics in the immediate vicinity of Aβ deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment. In regions close to Aβ plaques chronic states of high spontaneous glutamate fluctuations are observed and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas. GLT-1 expression is reduced around Aβ plaques and upregulation of GLT-1 expression and activity by ceftriaxone partially restores glutamate dynamics to values in control regions. We conclude that the toxic microenvironment surrounding Aβ plaques results, at least partially, from enhanced glutamate levels and that pharmacologically increasing GLT-1 expression and activity may be a new target for early therapeutic intervention.

Mapping cortical mesoscopic networks of single spiking cortical or sub-cortical neurons

Understanding the basis of brain function requires knowledge of cortical operations over wide-spatial scales, but also within the context of single neurons. In vivo, wide-field GCaMP imaging and sub-cortical/cortical cellular electrophysiology were used in mice to investigate relationships between spontaneous single neuron spiking and mesoscopic cortical activity. We make use of a rich set of cortical activity motifs that are present in spontaneous activity in anesthetized and awake animals. A mesoscale spike-triggered averaging procedure allowed the identification of motifs that are preferentially linked to individual spiking neurons by employing genetically targeted indicators of neuronal activity. Thalamic neurons predicted and reported specific cycles of wide-scale cortical inhibition/excitation. In contrast, spike-triggered maps derived from single cortical neurons yielded spatio-temporal maps expected for regional cortical consensus function. This approach can define network relationships between any point source of neuronal spiking and mesoscale cortical maps. DOI: http://dx.doi.org/10.7554/eLife.19976.001