Spectral-temporal-spatial customization via modulating multimodal nonlinear pulse propagation.

Multimode fibers (MMFs) are gaining renewed interest for nonlinear effects due to their high-dimensional spatiotemporal nonlinear dynamics and scalability for high power. High-brightness MMF sources with effective control of the nonlinear processes would offer possibilities in many areas from high-power fiber lasers, to bioimaging and chemical sensing, and to intriguing physics phenomena. Here we present a simple yet effective way of controlling nonlinear effects at high peak power levels. This is achieved by leveraging not only the spatial but also the temporal degrees of freedom during multimodal nonlinear pulse propagation in step-index MMFs, using a programmable fiber shaper that introduces time-dependent disorders. We achieve high tunability in MMF output fields, resulting in a broadband high-peak-power source. Its potential as a nonlinear imaging source is further demonstrated through widely tunable two-photon and three-photon microscopy. These demonstrations provide possibilities for technology advances in nonlinear optics, bioimaging, spectroscopy, optical computing, and material processing.

Somatosensory cortical signature of facial nociception and vibrotactile touch-induced analgesia.

Pain relief by vibrotactile touch is a common human experience. Previous neurophysiological investigations of its underlying mechanism in animals focused on spinal circuits, while human studies suggested the involvement of supraspinal pathways. Here, we examine the role of primary somatosensory cortex (S1) in touch-induced mechanical and heat analgesia. We found that, in mice, vibrotactile reafferent signals from self-generated whisking significantly reduce facial nociception, which is abolished by specifically blocking touch transmission from thalamus to the barrel cortex (S1B). Using a signal separation algorithm that can decompose calcium signals into sensory-evoked, whisking, or face-wiping responses, we found that the presence of whisking altered nociceptive signal processing in S1B neurons. Analysis of S1B population dynamics revealed that whisking pushes the transition of the neural state induced by noxious stimuli toward the outcome of non-nocifensive actions. Thus, S1B integrates facial tactile and noxious signals to enable touch-mediated analgesia.

An Unexpected Dependence of Cortical Depth in Shaping Neural Responsiveness and Selectivity in Mouse Visual Cortex.

Two-photon imaging studies in mouse primary visual cortex (V1) consistently report that around half of the neurons respond to oriented grating stimuli. However, in cats and primates, nearly all neurons respond to such stimuli. Here we show that mouse V1 responsiveness and selectivity strongly depends on neuronal depth. Moving from superficial layer 2 down to layer 4, the percentage of visually responsive neurons nearly doubled, ultimately reaching levels similar to what is seen in other species. Over this span, the amplitude of neuronal responses also doubled. Moreover, stimulus selectivity was also modulated, not only with depth but also with response amplitude. Specifically, we found that orientation and direction selectivity were greater in stronger responding neurons, but orientation selectivity decreased with depth whereas direction selectivity increased. Importantly, these depth-dependent trends were found not just between layer 2/3 and layer 4 but at different depths within layer 2/3 itself. Thus, neuronal depth is an important factor to consider when pooling neurons for population analyses. Furthermore, the inability to drive the majority of cells in superficial layer 2/3 of mouse V1 with grating stimuli indicates that there may be fundamental differences in the micro-circuitry and role of V1 between rodents and other mammals.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain.

Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Blood-brain barrier disruption (BBB) and release of toxic blood molecules into the brain contributes to neuronal injury during stroke and other cerebrovascular diseases. While pericytes are builders and custodians of the BBB in the normal brain, their impact on BBB integrity during ischemia remains unclear. We imaged pericyte-labeled transgenic mice with in vivo two-photon microscopy to examine the relationship between pericytes and blood plasma leakage during photothrombotic occlusion of cortical capillaries. Upon cessation of capillary flow, we observed that plasma leakage occurred with three times greater frequency in regions where pericyte somata adjoined the endothelium. Pericyte somata covered only 7% of the total capillary length in cortex, indicating that a disproportionate amount of leakage occurred from a small fraction of the capillary bed. Plasma leakage was preceded by rapid activation of matrix metalloproteinase (MMP) at pericyte somata, which was visualized at high resolution in vivo using a fluorescent probe for matrix metalloproteinase-2/9 activity, fluorescein isothiocyanate (FITC)-gelatin. Coinjection of an MMP-9 inhibitor, but not an MMP-2 inhibitor, reduced pericyte-associated FITC-gelatin fluorescence and plasma leakage. These results suggest that pericytes contribute to rapid and localized proteolytic degradation of the BBB during cerebral ischemia. SIGNIFICANCE STATEMENT: Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

Neural correlates of single-vessel haemodynamic responses in vivo.

Neural activation increases blood flow locally. This vascular signal is used by functional imaging techniques to infer the location and strength of neural activity. However, the precise spatial scale over which neural and vascular signals are correlated is unknown. Furthermore, the relative role of synaptic and spiking activity in driving haemodynamic signals is controversial. Previous studies recorded local field potentials as a measure of synaptic activity together with spiking activity and low-resolution haemodynamic imaging. Here we used two-photon microscopy to measure sensory-evoked responses of individual blood vessels (dilation, blood velocity) while imaging synaptic and spiking activity in the surrounding tissue using fluorescent glutamate and calcium sensors. In cat primary visual cortex, where neurons are clustered by their preference for stimulus orientation, we discovered new maps for excitatory synaptic activity, which were organized similarly to those for spiking activity but were less selective for stimulus orientation and direction. We generated tuning curves for individual vessel responses for the first time and found that parenchymal vessels in cortical layer 2/3 were orientation selective. Neighbouring penetrating arterioles had different orientation preferences. Pial surface arteries in cats, as well as surface arteries and penetrating arterioles in rat visual cortex (where orientation maps do not exist), responded to visual stimuli but had no orientation selectivity. We integrated synaptic or spiking responses around individual parenchymal vessels in cats and established that the vascular and neural responses had the same orientation preference. However, synaptic and spiking responses were more selective than vascular responses--vessels frequently responded robustly to stimuli that evoked little to no neural activity in the surrounding tissue. Thus, local neural and haemodynamic signals were partly decoupled. Together, these results indicate that intrinsic cortical properties, such as propagation of vascular dilation between neighbouring columns, need to be accounted for when decoding haemodynamic signals.

Hidden complexity of synaptic receptive fields in cat V1.

In the primary visual cortex (V1), Simple and Complex receptive fields (RFs) are usually characterized on the basis of the linearity of the cell spiking response to stimuli of opposite contrast. Whether or not this classification reflects a functional dichotomy in the synaptic inputs to Simple and Complex cells is still an open issue. Here we combined intracellular membrane potential recordings in cat V1 with 2D dense noise stimulation to decompose the Simple-like and Complex-like components of the subthreshold RF into a parallel set of functionally distinct subunits. Results show that both Simple and Complex RFs exhibit a remarkable diversity of excitatory and inhibitory Complex-like contributions, which differ in orientation and spatial frequency selectivity from the linear RF, even in layer 4 and layer 6 Simple cells. We further show that the diversity of Complex-like contributions recovered at the subthreshold level is expressed in the cell spiking output. These results demonstrate that the Simple or Complex nature of V1 RFs does not rely on the diversity of Complex-like components received by the cell from its synaptic afferents but on the imbalance between the weights of the Simple-like and Complex-like synaptic contributions.

The shape of dendritic arbors in different functional domains of the cortical orientation map.

The neocortex is organized into macroscopic functional maps. However, at the microscopic scale, the functional preference and degree of feature selectivity between neighboring neurons can vary considerably. In the primary visual cortex, adjacent neurons in iso-orientation domains share the same orientation preference, whereas neighboring neurons near pinwheel centers are tuned to different stimulus orientations. Moreover, several studies have found greater orientation selectivity in iso-orientation domains than in pinwheel centers. These differences suggest that neurons sample local inputs in a spatially homogenous fashion and independently of the location of their soma on the orientation map. Here we determine whether dendritic geometry is affected by neuronal position on the orientation map. We labeled individual layer 2/3 pyramidal neurons with fluorescent dyes in specific domains of the orientation map in cat primary visual cortex and imaged their dendritic trees in vivo by two-photon microscopy. We found that the circularity and uniformity of dendritic trees is independent of somatic position on the orientation map. Moreover, the dendrites of neurons located close to pinwheel centers extend across all orientation domains in an unbiased fashion. Thus, unbiased dendritic trees appear to provide an anatomical substrate for the systematic variations in feature selectivity across the orientation map.

The role of delayed suppression in slow and fast contrast adaptation in V1 simple cells.

The sensitivity and rate of neural coding along the early visual pathways adapt to changes in contrast of the retinal image caused by external motion or self-generated eye movements. To identify the functional mechanisms of fast and slow contrast adaptation at the level of the visual cortex, we randomly varied, over both short and long timescales, the contrast of optimal sinusoidal gratings flashed in the receptive field of simple cells. We found that fast contrast-dependent suppression lagged excitation by ~11 ms and controlled the spike's temporal precision. During slow adaptation to low contrasts, the gain and latency of excitation increased whereas suppression became less visible, resulting in more sensitive but slower and more variable responses. We conclude that delayed suppression controls the response dynamics during both fast and slow contrast adaptation. More generally, we propose that sensory adaptation trades neuronal sensitivity for processing speed by changing the balance between excitation and delayed inhibition.

Animation of natural scene by virtual eye-movements evokes high precision and low noise in V1 neurons.

Synaptic noise is thought to be a limiting factor for computational efficiency in the brain. In visual cortex (V1), ongoing activity is present in vivo, and spiking responses to simple stimuli are highly unreliable across trials. Stimulus statistics used to plot receptive fields, however, are quite different from those experienced during natural visuomotor exploration. We recorded V1 neurons intracellularly in the anaesthetized and paralyzed cat and compared their spiking and synaptic responses to full field natural images animated by simulated eye-movements to those evoked by simpler (grating) or higher dimensionality statistics (dense noise). In most cells, natural scene animation was the only condition where high temporal precision (in the 10-20 ms range) was maintained during sparse and reliable activity. At the subthreshold level, irregular but highly reproducible membrane potential dynamics were observed, even during long (several 100 ms) "spike-less" periods. We showed that both the spatial structure of natural scenes and the temporal dynamics of eye-movements increase the signal-to-noise ratio by a non-linear amplification of the signal combined with a reduction of the subthreshold contextual noise. These data support the view that the sparsening and the time precision of the neural code in V1 may depend primarily on three factors: (1) broadband input spectrum: the bandwidth must be rich enough for recruiting optimally the diversity of spatial and time constants during recurrent processing; (2) tight temporal interplay of excitation and inhibition: conductance measurements demonstrate that natural scene statistics narrow selectively the duration of the spiking opportunity window during which the balance between excitation and inhibition changes transiently and reversibly; (3) signal energy in the lower frequency band: a minimal level of power is needed below 10 Hz to reach consistently the spiking threshold, a situation rarely reached with visual dense noise.

Targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation(1-4), direction(5-7), ocular dominance(8,9) and binocular disparity(9). Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex(10,11). Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined(12). The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines(13) or record activity simultaneously from hundreds of individual neuronal cell bodies(6,14). Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording, and pharmacology.

Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1) hyperpolarization by intracellular current injection, (2) pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3) GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5-10 nA), which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be impractical.

A Re-Examination of Hebbian-Covariance Rules and Spike Timing-Dependent Plasticity in Cat Visual Cortex in vivo.

Spike timing-dependent plasticity (STDP) is considered as an ubiquitous rule for associative plasticity in cortical networks in vitro. However, limited supporting evidence for its functional role has been provided in vivo. In particular, there are very few studies demonstrating the co-occurrence of synaptic efficiency changes and alteration of sensory responses in adult cortex during Hebbian or STDP protocols. We addressed this issue by reviewing and comparing the functional effects of two types of cellular conditioning in cat visual cortex. The first one, referred to as the "covariance" protocol, obeys a generalized Hebbian framework, by imposing, for different stimuli, supervised positive and negative changes in covariance between postsynaptic and presynaptic activity rates. The second protocol, based on intracellular recordings, replicated in vivo variants of the theta-burst paradigm (TBS), proven successful in inducing long-term potentiation in vitro. Since it was shown to impose a precise correlation delay between the electrically activated thalamic input and the TBS-induced postsynaptic spike, this protocol can be seen as a probe of causal ("pre-before-post") STDP. By choosing a thalamic region where the visual field representation was in retinotopic overlap with the intracellularly recorded cortical receptive field as the afferent site for supervised electrical stimulation, this protocol allowed to look for possible correlates between STDP and functional reorganization of the conditioned cortical receptive field. The rate-based "covariance protocol" induced significant and large amplitude changes in receptive field properties, in both kitten and adult V1 cortex. The TBS STDP-like protocol produced in the adult significant changes in the synaptic gain of the electrically activated thalamic pathway, but the statistical significance of the functional correlates was detectable mostly at the population level. Comparison of our observations with the literature leads us to re-examine the experimental status of spike timing-dependent potentiation in adult cortex. We propose the existence of a correlation-based threshold in vivo, limiting the expression of STDP-induced changes outside the critical period, and which accounts for the stability of synaptic weights during sensory cortical processing in the absence of attention or reward-gated supervision.

High-resolution intracellular recordings using a real-time computational model of the electrode.

Intracellular recordings of neuronal membrane potential are a central tool in neurophysiology. In many situations, especially in vivo, the traditional limitation of such recordings is the high electrode resistance and capacitance, which may cause significant measurement errors during current injection. We introduce a computer-aided technique, Active Electrode Compensation (AEC), based on a digital model of the electrode interfaced in real time with the electrophysiological setup. The characteristics of this model are first estimated using white noise current injection. The electrode and membrane contribution are digitally separated, and the recording is then made by online subtraction of the electrode contribution. Tests performed in vitro and in vivo demonstrate that AEC enables high-frequency recordings in demanding conditions, such as injection of conductance noise in dynamic-clamp mode, not feasible with a single high-resistance electrode until now. AEC should be particularly useful to characterize fast neuronal phenomena intracellularly in vivo.