David Kleinfeld

ReaChR: A red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation

Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ∼590–630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.

Suppressed neuronal activity and concurrent arteriolar vasoconstriction may explain negative blood oxygenation level-dependent signal.

Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulus-induced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal.

Automatic sorting of multiple unit neuronal signals in the presence of anisotropic and non-Gaussian variability

Neuronal noise sources and systematic variability in the shape of a spike limit the ability to sort multiple unit waveforms recorded from nervous tissue into their single neuron constituents. Here we present a procedure to efficiently sort spikes in the presence of noise that is anisotropic, i.e., dominated by particular frequencies, and whose amplitude distribution may be non-Gaussian, such as occurs when spike waveforms are a function of interspike interval. Our algorithm uses a hierarchical clustering scheme. First, multiple unit records are sorted into an overly large number of clusters by recursive bisection. Second, these clusters are progressively aggregated into a minimal set of putative single units based on both similarities of spike shape as well as the statistics of spike arrival times, such as imposed by the refractory period. We apply the algorithm to waveforms recorded with chronically implanted micro-wire stereotrodes from neocortex of behaving rat. Natural extension of the algorithm may be used to cluster spike waveforms from records with many input channels, such as those obtained with tetrodes and multiple site optical techniques.

Active sensation: insights from the rodent vibrissa sensorimotor system

Rats sweep their vibrissae through space to locate objects in their immediate environment. In essence, their view of the proximal world is generated through pliable hairs that tap and palpate objects. The texture and shape of those objects must be discerned for the rat to assess the value of the object. Furthermore, the location of those objects must be specified with reference to the position of the rats head for the rat to plan its movements. Recent in vivo and in vitro electrophysiological measurements provide insight into the algorithms and mechanisms that underlie these behavioral-based computations.

Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels

It is well known that the density of neurons varies within the adult brain. In neocortex, this includes variations in neuronal density between different lamina as well as between different regions. Yet the concomitant variation of the microvessels is largely uncharted. Here, we present automated histological, imaging, and analysis tools to simultaneously map the locations of all neuronal and non-neuronal nuclei and the centerlines and diameters of all blood vessels within thick slabs of neocortex from mice. Based on total inventory measurements of different cortical regions (∼107 cells vectorized across brains), these methods revealed: (1) In three dimensions, the mean distance of the center of neuronal somata to the closest microvessel was 15 μm. (2) Volume samples within lamina of a given region show that the density of microvessels does not match the strong laminar variation in neuronal density. This holds for both agranular and granular cortex. (3) Volume samples in successive radii from the midline to the ventral-lateral edge, where each volume summed the number of cells and microvessels from the pia to the white matter, show a significant correlation between neuronal and microvessel densities. These data show that while neuronal and vascular densities do not track each other on the 100 μm scale of cortical lamina, they do track each other on the 1–10 mm scale of the cortical mantle. The absence of a disproportionate density of blood vessels in granular lamina is argued to be consistent with the initial locus of functional brain imaging signals.

Traveling electrical waves in cortex: insights from phase dynamics and speculation on a computational role.

The theory of coupled phase oscillators provides a framework to understand the emergent properties of networks of neuronal oscillators. When the architecture of the network is dominated by short-range connections, the pattern of electrical output is predicted to correspond to traveling plane and rotating waves, in addition to synchronized output. We argue that this theory provides the foundation for understanding the traveling electrical waves that are observed across olfactory, visual, and visuomotor areas of cortex in a variety of species. The waves are typically present during periods outside of stimulation, while synchronous activity typically dominates in the presence of a strong stimulus. We suggest that the continuum of phase shifts during epochs with traveling waves provides a means to scan the incoming sensory stream for novel features. Experiments to test our theoretical approach are presented.

Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes

We have identified large‐scale patterns of electrical activity in cortical circuits that occur in response to stimulation of peripheral receptors. Our focus was on primary (S1) vibrissal cortex of anesthetized rat, and we used optical techniques in conjunction with voltage‐sensitive dyes to measure depolarization of the upper layers of cortex. Displacement of one vibrissa produced a field of activity that extends over very many cortical columns in S1. There are multiple, focal maxima within this field. A global maximum is located near the center of the field of activity, and, as determined electrically and histologically, this site maps to the cortical column appropriate for the deflected vibrissa. The amplitude of this component attains a steady‐state value under continuous stimulation. Additional temporal characteristics are revealed by the response to a single displacement; the signal was triphasic and began with a prompt depolarization that was followed by a transient phase of inhibition and a final phase of long‐lasting depolarization. The somatotopy of the other, satellite maxima in the field of activity were established through the reconstruction of the fields of activity produced by individual stimulation of other vibrissae. Local maxima for one vibrissa were seen to overlie the global maximum found for stimulation of nearest‐ and next‐nearest‐neighbor vibrissae. In contrast to the amplitude of the global maxima, the amplitude associated with the local maxima was not maintained with either continuous or infrequent but repetitive stimulation. Finally, the field of activity induced by alternate deflection of two neighboring vibrissae was suppressed in amplitude in comparison to the summed amplitudes of the signals elicited by deflection of each vibrissa alone. We suggest that these patterns of activity are a manifestation of the dynamic interaction among neighboring cortical columns.

Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke.

We present a method to produce vascular disruptions within rat brain parenchyma that targets single microvessels. We used two-photon microscopy to image vascular architecture, to select a vessel for injury and to measure blood-flow dynamics. We irradiated the vessel with high-fluence, ultrashort laser pulses and achieved three forms of vascular insult. (i) Vessel rupture was induced at the highest optical energies; this provides a model for hemorrhage. (ii) Extravasation of blood components was induced near the lowest energies and was accompanied by maintained flow in the target vessel. (iii) An intravascular clot evolved when an extravasated vessel was further irradiated. Such clots dramatically impaired blood flow in downstream vessels, in which speeds dropped to as low as ∼10% of baseline values. This demonstrates that a single blockage to a microvessel can lead to local cortical ischemia. Lastly, we show that hemodilution leads to a restoration of flow in secondary downstream vessels.

Chronic optical access through a polished and reinforced thinned skull

We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.

Penetrating arterioles are a bottleneck in the perfusion of neocortex

Penetrating arterioles bridge the mesh of communicating arterioles on the surface of cortex with the subsurface microvascular bed that feeds the underlying neural tissue. We tested the conjecture that penetrating arterioles, which are positioned to regulate the delivery of blood, are loci of severe ischemia in the event of occlusion. Focal photothrombosis was used to occlude single penetrating arterioles in rat parietal cortex, and the resultant changes in flow of red blood cells were measured with two-photon laser-scanning microscopy in individual subsurface microvessels that surround the occlusion. We observed that the average flow of red blood cells nearly stalls adjacent to the occlusion and remains within 30% of its baseline value in vessels as far as 10 branch points downstream from the occlusion. Preservation of average flow emerges 350 μm away; this length scale is consistent with the spatial distribution of penetrating arterioles. We conclude that penetrating arterioles are a bottleneck in the supply of blood to neocortex, at least to superficial layers.