The effects of locomotion on sensory-evoked haemodynamic responses in the cortex of awake mice

Investigating neurovascular coupling in awake rodents is becoming ever more popular due, in part, to our increasing knowledge of the profound impacts that anaesthesia can have upon brain physiology. Although awake imaging brings with it many advantages, we still do not fully understand how voluntary locomotion during imaging affects sensory-evoked haemodynamic responses. In this study we investigated how evoked haemodynamic responses can be affected by the amount and timing of locomotion. Using an awake imaging set up, we used 2D-Optical Imaging Spectroscopy (2D-OIS) to measure changes in cerebral haemodynamics within the sensory cortex of the brain during either 2s whisker stimulation or spontaneous (no whisker stimulation) experiments, whilst animals could walk on a spherical treadmill. We show that locomotion alters haemodynamic responses. The amount and timing of locomotion relative to whisker stimulation is important, and can significantly impact sensory-evoked haemodynamic responses. If locomotion occurred before or during whisker stimulation, the amplitude of the stimulus-evoked haemodynamic response was significantly altered. Therefore, monitoring of locomotion during awake imaging is necessary to ensure that conclusions based on comparisons of evoked haemodynamic responses (e.g., between control and disease groups) are not confounded by the effects of locomotion.

Learning enhances behaviorally relevant representations in apical dendrites

Learning alters cortical representations and improves perception. Apical tuft dendrites in Layer 1, which are unique in their connectivity and biophysical properties, may be a key site of learning-induced plasticity. We used both two-photon and SCAPE microscopy to longitudinally track tuft-wide calcium spikes in apical dendrites of Layer 5 pyramidal neurons as mice learned a tactile behavior. Mice were trained to discriminate two orthogonal directions of whisker stimulation. Reinforcement learning, but not repeated stimulus exposure, enhanced tuft selectivity for both directions equally, even though only one was associated with reward. Selective tufts emerged from initially unresponsive or low-selectivity populations. Animal movement and choice did not account for changes in stimulus selectivity. Enhanced selectivity persisted even after rewards were removed and animals ceased performing the task. We conclude that learning produces long-lasting realignment of apical dendrite tuft responses to behaviorally relevant dimensions of a task.

Cortical Spreading Depression Can Be Triggered by Sensory Stimulation in Primed Wild Type Mouse Brain: a Mechanistic Insight to Migraine Aura Generation

Background: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight how migraine starts in an otherwise healthy brain, remains largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. Methods: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low dose of Na+/K+-ATPase blocker ouabain that does not induce repetitive CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake by astrocytes, with shRNA. Stimulation-induced CSDs and extracellular K+ changes were monitored in vivo electrophysiologically or with a K+-sensitive fluoroprobe (IPG-4). Results: After priming with ouabain, photic stimulation increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p<0.001). Whisker stimulation was less effective (14.9 vs. 2.4%, p=0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested but triggered stimulus-induced CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during stimulation after subthreshold ouabain or shRNA treatment unlike controls. In line with higher CSD susceptibility, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the incidence of stimulus-induced CSDs, we applied an A1-receptor (DPCPX) or GABA-A (bicuculine) antagonist over the occipital cortex, because adenosine formed during stimulation or inhibitory interneuron activity can reduce CSD susceptibility. DPCPX induced CSDs or CSD-like small-DC shifts during photic stimulation, whereas bicuculine was not effective. Conclusions: Our findings indicate that normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has the potential to trigger a CSD when co-existing conditions can bring extracellular K+ and glutamate concentrations over threshold via reduced uptake of K+ and glutamate (e.g. inefficient fueling of α2-Na+/K+-ATPase due to reduced glycogen breakdown) or facilitated glutamate release (e.g. reduced presynaptic adenosinergic inhibition).

State-Dependent Synchrony and Functional Connectivity in the Primary and Secondary Whisker Somatosensory Cortices

Synchronized activity plays an important role in sensory coding and memory and is a hallmark of functional network connectivity. However, the effect of sensory activation on synchronization and cortical functional connectivity is largely unknown. In this study, we investigated the effect of whisker activation on synchronization and functional connectivity of the primary (wS1) and secondary (wS2) whisker somatosensory cortices at the single-cell level. The results showed that during the spontaneous pre-stimulus state, neurons tended to be functionally connected with nearby neurons which shared similar tuning characteristics. Whisker activation using either ramp-and-hold stimulation or artificial whisking against sandpaper has significantly reduced the average overall pairwise synchronization and functional connectivity within the wS1 barrel and wS2 cortices. Whisker stimulation disconnected approximately a third of neuronal pairs that were functionally connected during the unstimulated state. Nearby neurons with congruent tuning properties were more likely to remain functionally connected during whisker activation. The findings of this study indicated that cortical somatosensory networks are organized in non-random small world networks composed of neurons sharing relatively similar tuning properties. Sensory whisker activation intensifies these properties and further subdivides the cortical network into smaller more functionally uniform subnetworks, which possibly serve to increase the computational capacity of the network.

Neuronal Avalanches Across the Rat Somatosensory Barrel Cortex and the Effect of Single Whisker Stimulation

Since its first experimental signatures, the so called “critical brain hypothesis” has been extensively studied. Yet, its actual foundations remain elusive. According to a widely accepted teleological reasoning, the brain would be poised to a critical state to optimize the mapping of the noisy and ever changing real-world inputs, thus suggesting that primary sensory cortical areas should be critical. We investigated whether a single barrel column of the somatosensory cortex of the anesthetized rat displays a critical behavior. Neuronal avalanches were recorded across all cortical layers in terms of both multi-unit activities and population local field potentials, and their behavior during spontaneous activity compared to the one evoked by a controlled single whisker deflection. By applying a maximum likelihood statistical method based on timeseries undersampling to fit the avalanches distributions, we show that neuronal avalanches are power law distributed for both multi-unit activities and local field potentials during spontaneous activity, with exponents that are spread along a scaling line. Instead, after the tactile stimulus, activity switches to a transient across-layers synchronization mode that appears to dominate the cortical representation of the single sensory input.

Role of endothelium-pericyte signaling in capillary blood flow response to neuronal activity

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs’ action on peri-capillary pericytes.

Olfactory modulation of barrel cortex activity during active whisking and passive whisker stimulation

Rodents depend on olfaction and touch to meet many of their fundamental needs. The joint significance of these sensory systems is underscored by an intricate coupling between sniffing and whisking. However, the impact of simultaneous olfactory and tactile inputs on sensory representations in the cortex remains elusive. To study these interactions, we recorded large populations of barrel cortex neurons using 2-photon calcium imaging in head-fixed mice during olfactory and tactile stimulation. We find that odors alter barrel cortex activity in at least two ways, first by enhancing whisking, and second by central cross-talk that persists after whisking is abolished by facial nerve sectioning. Odors can either enhance or suppress barrel cortex neuronal responses, and while odor identity can be decoded from population activity, it does not interfere with the tactile representation. Thus, barrel cortex represents olfactory information which, in the absence of learned associations, is coded independently of tactile information.

Abstract 66: No Evidence of Functional Peri-Infarct Circuit Remapping in Mouse Somatosensory Cortex After Ischemic Stroke

Following ischemic stroke, many patients exhibit partial spontaneous recovery, suggesting that the brain has endogenous mechanisms to recover lost functions. Evidence supports a role for peri-infarct cortex in recovery as this area undergoes structural, physiologic, and transcriptomic changes following stroke. It has been hypothesized that these changes promote circuit rewiring, leading spared neurons in the peri-infarct cortex to “remap” and subsume the function previously performed by neurons in the ischemic core. However, direct evidence for remapping at the single neuron level is lacking. To test this, we targeted photothrombotic (PT) strokes to an individual barrel (C1) in the barrel field of mouse primary somatosensory cortex (S1BF). We then performed longitudinal in vivo two-photon (2P) calcium imaging in Thy1 -GCaMP6s transgenic mice and recorded whisker-evoked responses of individual layer 2/3 neurons in the adjacent D3 barrel. Before stroke, ~30% of active neurons in the D3 barrel respond to stimulation of the D3 whisker and ~8% of neurons respond to the C1 whisker. Based on the remapping hypothesis, we predicted that the percentage of C1 whisker-responsive neurons in the spared D3 barrel would increase after stroke; however, we found that only ~2% of neurons in the D3 barrel responded to C1 whisker stimulation one month after stroke. We also tested the effect of forced-use therapy on recovery by plucking all whiskers, except the C1 whisker corresponding to the infarcted barrel, following stroke. Still, we found that forced-use therapy did not lead to an increased percentage of C1 whisker-responsive neurons, but it did enhance the responses to C1 whisker stimulation in surviving C1-responsive neurons in the peri-infarct cortex. These results suggest that at the circuit level recovery may occur through potentiation of spared homotopic neurons rather than remapping of neurons to perform new functions.

Somatosensory processing deficits and altered cortico-hippocampal connectivity in Shank3b-/- mice

Abnormal tactile response is considered an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. However, the neural underpinnings of these somatosensory abnormalities are still poorly understood. Here we investigated, in Shank3b-/- adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents' interaction with the surrounding environment. To this aim, we assessed whisker-dependent behaviors in Shank3b-/- adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b-/- mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Notably, sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA in situ hybridization, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutant mice. Together, these findings suggest that impaired crosstalk between hippocampus and S1 might underlie Shank3b-/- hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable form of dysfunctional somatosensory processing in ASD.