Local Perturbations of Cortical Excitability Propagate Differentially Through Large-Scale Functional Networks.

Electrophysiological recordings have established that GABAergic interneurons regulate excitability, plasticity, and computational function within local neural circuits. Importantly, GABAergic inhibition is focally disrupted around sites of brain injury. However, it remains unclear whether focal imbalances in inhibition/excitation lead to widespread changes in brain activity. Here, we test the hypothesis that focal perturbations in excitability disrupt large-scale brain network dynamics. We used viral chemogenetics in mice to reversibly manipulate parvalbumin interneuron (PV-IN) activity levels in whisker barrel somatosensory cortex. We then assessed how this imbalance affects cortical network activity in awake mice using wide-field optical neuroimaging of pyramidal neuron GCaMP dynamics as well as local field potential recordings. We report 1) that local changes in excitability can cause remote, network-wide effects, 2) that these effects propagate differentially through intra- and interhemispheric connections, and 3) that chemogenetic constructs can induce plasticity in cortical excitability and functional connectivity. These findings may help to explain how focal activity changes following injury lead to widespread network dysfunction.

Electrically coupled inhibitory interneurons constrain long-range connectivity of cortical networks.

Spontaneous infra-slow brain activity (ISA) exhibits a high degree of temporal synchrony, or correlation, between distant brain regions. The spatial organization of ISA synchrony is not explained by anatomical connections alone, suggesting that active neural processes coordinate spontaneous activity. Inhibitory interneurons (IINs) form electrically coupled connections via the gap junction protein connexin 36 (Cx36) and networks of interconnected IINs are known to influence neural synchrony over short distances. However, the role of electrically coupled IIN networks in regulating spontaneous correlation over the entire brain is unknown. In this study, we performed OIS imaging on Cx36-/- mice to examine the role of this gap junction in ISA correlation across the entire cortex. We show that Cx36 deletion increased long-distance intra-hemispheric anti-correlation and inter-hemispheric correlation in spontaneous ISA. This suggests that electrically coupled IIN networks modulate ISA synchrony over long cortical distances.

Visual experience sculpts whole-cortex spontaneous infraslow activity patterns through an Arc-dependent mechanism.

Decades of work in experimental animals has established the importance of visual experience during critical periods for the development of normal sensory-evoked responses in the visual cortex. However, much less is known concerning the impact of early visual experience on the systems-level organization of spontaneous activity. Human resting-state fMRI has revealed that infraslow fluctuations in spontaneous activity are organized into stereotyped spatiotemporal patterns across the entire brain. Furthermore, the organization of spontaneous infraslow activity (ISA) is plastic in that it can be modulated by learning and experience, suggesting heightened sensitivity to change during critical periods. Here we used wide-field optical intrinsic signal imaging in mice to examine whole-cortex spontaneous ISA patterns. Using monocular or binocular visual deprivation, we examined the effects of critical period visual experience on the development of ISA correlation and latency patterns within and across cortical resting-state networks. Visual modification with monocular lid suturing reduced correlation between left and right cortices (homotopic correlation) within the visual network, but had little effect on internetwork correlation. In contrast, visual deprivation with binocular lid suturing resulted in increased visual homotopic correlation and increased anti-correlation between the visual network and several extravisual networks, suggesting cross-modal plasticity. These network-level changes were markedly attenuated in mice with genetic deletion of Arc, a gene known to be critical for activity-dependent synaptic plasticity. Taken together, our results suggest that critical period visual experience induces global changes in spontaneous ISA relationships, both within the visual network and across networks, through an Arc-dependent mechanism.

Effective Connectivity Measured Using Optogenetically Evoked Hemodynamic Signals Exhibits Topography Distinct from Resting State Functional Connectivity in the Mouse.

Brain connectomics has expanded from histological assessment of axonal projection connectivity (APC) to encompass resting state functional connectivity (RS-FC). RS-FC analyses are efficient for whole-brain mapping, but attempts to explain aspects of RS-FC (e.g., interhemispheric RS-FC) based on APC have been only partially successful. Neuroimaging with hemoglobin alone lacks specificity for determining how activity in a population of cells contributes to RS-FC. Wide-field mapping of optogenetically defined connectivity could provide insights into the brain's structure-function relationship. We combined optogenetics with optical intrinsic signal imaging to create an efficient, optogenetic effective connectivity (Opto-EC) mapping assay. We examined EC patterns of excitatory neurons in awake, Thy1-ChR2 transgenic mice. These Thy1-based EC (Thy1-EC) patterns were evaluated against RS-FC over the cortex. Compared to RS-FC, Thy1-EC exhibited increased spatial specificity, reduced interhemispheric connectivity in regions with strong RS-FC, and appreciable connection strength asymmetry. Comparing the topography of Thy1-EC and RS-FC patterns to maps of APC revealed that Thy1-EC more closely resembled APC than did RS-FC. The more general method of Opto-EC mapping with hemoglobin can be determined for 100 sites in single animals in under an hour, and is amenable to other neuroimaging modalities. Opto-EC mapping represents a powerful strategy for examining evolving connectivity-related circuit plasticity.

Optical imaging of disrupted functional connectivity following ischemic stroke in mice.

Recent human neuroimaging studies indicate that spontaneous fluctuations in neural activity, as measured by functional connectivity magnetic resonance imaging (fcMRI), are significantly affected following stroke. Disrupted functional connectivity is associated with behavioral deficits and has been linked to long-term recovery potential. FcMRI studies of stroke in rats have generally produced similar findings, although subacute cortical reorganization following focal ischemia appears to be more rapid than in humans. Similar studies in mice have not been published, most likely because fMRI in the small mouse brain is technically challenging. Extending functional connectivity methods to mouse models of stroke could provide a valuable tool for understanding the link between molecular mechanisms of stroke repair and human fcMRI findings at the system level. We applied functional connectivity optical intrinsic signal imaging (fcOIS) to mice before and 72 h after transient middle cerebral artery occlusion (tMCAO) to examine how graded ischemic injury affects the relationship between functional connectivity and infarct volume, stimulus-induced response, and behavior. Regional changes in functional connectivity within the MCA territory were largely proportional to infarct volume. However, subcortical damage affected functional connectivity in the somatosensory cortex as much as larger infarcts of cortex and subcortex. The extent of injury correlated with cortical activations following electrical stimulation of the affected forelimb and with functional connectivity in the somatosensory cortex. Regional homotopic functional connectivity in motor cortex correlated with behavioral deficits measured using an adhesive patch removal test. Spontaneous hemodynamic activity within the infarct exhibited altered temporal and spectral features in comparison to intact tissue; failing to account for these regional differences significantly affected apparent post-stroke functional connectivity measures. Thus, several results were strongly dependent on how the resting-state data were processed. Specifically, global signal regression alone resulted in apparently distorted functional connectivity measures in the intact hemisphere. These distortions were corrected by regressing out multiple sources of variance, as performed in human fcMRI. We conclude that fcOIS provides a sensitive imaging modality in the murine stroke model; however, it is necessary to properly account for altered hemodynamics in injured brain to obtain accurate measures of functional connectivity.

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice.

The accumulation of aggregated amyloid-ß (Aß) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation-glial fibrillary acid protein (Gfap) and vimentin (Vim)-in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap(-/-)Vim(-/-) mice had twice the plaque load of APP/PS1 Gfap(+/+)Vim(+/+) mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aß levels were unchanged, suggesting that the deletions had no effect on APP processing or Aß generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap(-/-)Vim(-/-) astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap(+/+)Vim(+/+) mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.

In a hub-and-spoke network, spoke-administered thrombolysis reduces mechanical thrombectomy procedure time and number of passes.

BACKGROUND: The utility of intravenous thrombolysis (IVT) prior to mechanical thrombectomy (MT) in large vessel occlusion stroke (LVO) is controversial. Some data suggest IVT increases MT technical difficulty. Within our hub-and-spoke telestroke network, we examined how spoke-administered IVT affected hub MT procedure time and pass number. METHODS: Patients presenting to 25 spoke hospitals who were transferred to the hub and underwent MT from 2018 to 2020 were identified from a prospectively maintained database. MT procedure time, fluoroscopy time, and pass number were obtained from operative reports. RESULTS: Of 107 patients, 48 received IVT at spokes. Baseline characteristics and NIHSS were similar. The last known well (LKW)-to-puncture time was shorter among IVT patients (4.3 ± 1.9 h vs. 10.5 ± 6.5 h, p < 0.0001). In patients that received IVT, mean MT procedure time was decreased by 18.8 min (50.5 ± 29.4 vs. 69.3 ± 46.7 min, p = 0.02) and mean fluoroscopy time was decreased by 11.3 min (21.7 ± 15.8 vs. 33.0 ± 30.9 min, p = 0.03). Furthermore, IVT-treated patients required fewer MT passes (median 1 pass [IQR 1.0, 1.80] vs. 2 passes [1.0, 2.3], p = 0.0002) and were more likely to achieve reperfusion in ≤2 passes (81.3% vs. 59.3%, p = 0.01). An increased proportion of IVT-treated patients achieved TICI 2b-3 reperfusion after MT (93.9% vs. 83.8%, p = 0.045). There were no associations between MT procedural characteristics and LKW-to-puncture time. CONCLUSION: Within our network, hub MT following spoke-administered IVT was faster, required fewer passes, and achieved improved reperfusion. This suggests spoke-administered IVT does not impair MT, but instead may enhance it.

Spoke-administered thrombolysis improves large vessel occlusion early recanalization: the real-world experience of a large academic hub-and-spoke telestroke network.

Introduction: Intravenous thrombolysis (IVT) prior to mechanical thrombectomy (MT) for large vessel occlusion (LVO) stroke is increasingly controversial. Recent trials support MT without IVT for patients presenting directly to MT-capable "hub" centers. However, bypassing IVT has not been evaluated for patients presenting to IVT-capable "spoke" hospitals that require hub transfer for MT. A perceived lack of efficacy of IVT to result in LVO early recanalization (ER) is often cited to support bypassing IVT, but ER data for IVT in patients that require interhospital transfer is limited. Here we examined LVO ER rates after spoke-administered IVT in our hub-and-spoke stroke network. Methods: Patients presenting to 25 spokes before hub transfer for MT consideration from 2018-2020 were retrospectively identified from a prospectively maintained database. Inclusion criteria were pre-transfer CTA-defined LVO, ASPECTS ≥6, and post-transfer repeat vessel imaging. Results: Of 167 patients, median age was 69 and 51% were female. 76 received spoke IVT (+spokeIVT) and 91 did not (-spokeIVT). Alteplase was the only IVT used in this study. Comorbidities and NIHSS were similar between groups. ER frequency was increased 7.2-fold in +spokeIVT patients [12/76 (15.8%) vs. 2/91 (2.2%), P<0.001]. Spoke-administered IVT was independently associated with ER (aOR=11.5, 95% CI=2.2,99.6, p<0.05) after adjusting for timing of last known well, interhospital transfer, and repeat vessel imaging. Interval NIHSS was improved in patients with ER (median -2 (IQR -6.3, -0.8) vs. 0 (-2.5, 1), p<0.05). Conclusion: Within our network, +spokeIVT patients had a 7.2-fold increased ER relative likelihood. This real-world analysis supports IVT use in eligible patients with LVO at spoke hospitals before hub transfer for MT.

Dysarthria-Facial Paresis and Rostral Pontine Ischemic Stroke.

We describe an acute, postoperative dysarthria-facial paresis. While the rare stroke syndrome has been described previously, we present an under-described clinical nuance to its presentation with a particularly clear imaging correlation. A 78-year-old, right-handed man with a past medical history of aortic stenosis presented after a transcatheter aortic valve replacement. Immediately postoperatively, no neurological deficits were noted. That evening, he described his speech as "drunken." He was later noted to have a right lower facial droop in addition to the speech change. His speech exhibited labial, lingual, and (to a lesser degree) guttural dysarthria. At the patient's request due to claustrophobia, he received 2 mg of oral lorazepam prior to cranial imaging. Afterwards, he was sleepy but arousable, yet was unable to put pen to paper when asked to write. Right lower facial paresis persisted, but he now demonstrated a right pronator drift, which resolved after 14 h without other evolution to his clinical examination. Brainstem lesions above the level of the pontine facial nucleus may present with central facial paresis contralateral to the lesion. An associated dysarthria may have both labial and lingual features in the absence of tongue or pharyngeal weakness. Our review of reported cases of dysarthria in isolation, dysarthria in combination with facial paresis, and facial paresis finds that all presentations may result from cortical, subcortical, or brainstem involvement. Stroke mechanisms are most commonly thromboembolic or small-vessel-ischemic in either the anterior or posterior circulations.

Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke.

Understanding circuit-level manipulations that affect the brain's capacity for plasticity will inform the design of targeted interventions that enhance recovery after stroke. Following stroke, increased contralesional activity (e.g. use of the unaffected limb) can negatively influence recovery, but it is unknown which specific neural connections exert this influence, and to what extent increased contralesional activity affects systems- and molecular-level biomarkers of recovery. Here, we combine optogenetic photostimulation with optical intrinsic signal imaging to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice either recovered spontaneously or received chronic optogenetic excitation of right S1FP over the course of 4 weeks. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of stimulus-evoked activity in the unaffected limb. This maneuver also prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited transcriptional changes in several genes relevant for recovery. Our results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of cortical activity and several neuroplasticity-related genes after stroke, and highlight the importance of site selection for targeted therapeutic interventions after focal ischemia.

Characterizing reasons for stroke thrombectomy ineligibility among potential candidates transferred in a hub-and-spoke network.

Background: Access to endovascular thrombectomy (EVT) is relatively limited. Hub-and-spoke networks seek to transfer appropriate large vessel occlusion (LVO) candidates to EVT-capable hubs. However, some patients are ineligible upon hub arrival, and factors that drive transfer inefficiencies are not well described. We sought to quantify EVT transfer efficiency and identify reasons for EVT ineligibility. Methods: Consecutive EVT candidates presenting to 25 spokes from 2018-2020 with pre-transfer CTA-defined LVO and ASPECTS ≥6 were identified from a prospectively maintained database. Outcomes of interest included hub EVT, reasons for EVT ineligibility, and 90-day modified Rankin Scale (mRS) ≤2. Results: Among 258 patients, the median age was 70 years (IQR 60-81); 50% were female. 56% were ineligible for EVT after hub arrival. Cited reasons were large established infarct (49%), mild symptoms (33%), recanalization (6%), distal occlusion (5%), sub-occlusive lesion (3%), and goals of care (3%). Late window patients [last known well (LKW) >6 hours] were more likely to be ineligible (67% vs 43%, P<0.0001). EVT ineligible patients were older (73 vs 68 years, p=0.04), had lower NIHSS (10 vs 16, p<0.0001), longer LKW-hub arrival time (8.4 vs 4.6 hours, p<0.0001), longer spoke Telestroke consult-hub arrival time (2.8 vs 2.2 hours, p<0.0001), and received less intravenous thrombolysis (32% vs 45%, p=0.04) compared to eligible patients. EVT ineligibility independently reduced the odds of 90-day mRS≤2 (aOR=0.26, 95%CI=0.12,0.56; p=0.001) when controlling for age, NIHSS, and LKW-hub arrival time. Conclusions: Among patients transferred for EVT, there are multiple reasons for ineligibility upon hub arrival, with most excluded for infarct growth and mild symptoms. Understanding factors that drive transfer inefficiencies is important to improve EVT access and outcomes.

Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain.

Alternative translation initiation and stop codon readthrough in a few well-studied cases have been shown to allow the same transcript to generate multiple protein variants. Because the brain shows a particularly abundant use of alternative splicing, we sought to study alternative translation in CNS cells. We show that alternative translation is widespread and regulated across brain transcripts. In neural cultures, we identify alternative initiation on hundreds of transcripts, confirm several N-terminal protein variants, and show the modulation of the phenomenon by KCl stimulation. We also detect readthrough in cultures and show differential levels of normal and readthrough versions of AQP4 in gliotic diseases. Finally, we couple translating ribosome affinity purification to ribosome footprinting (TRAP-RF) for cell-type-specific analysis of neuronal and astrocytic translational readthrough in the mouse brain. We demonstrate that this unappreciated mechanism generates numerous and diverse protein isoforms in a cell-type-specific manner in the brain.

Sensory deprivation after focal ischemia in mice accelerates brain remapping and improves functional recovery through Arc-dependent synaptic plasticity.

Recovery after stroke, a major cause of adult disability, is often unpredictable and incomplete. Behavioral recovery is associated with functional reorganization (remapping) in perilesional regions, suggesting that promoting this process might be an effective strategy to enhance recovery. However, the molecular mechanisms underlying remapping after brain injury and the consequences of its modulation are poorly understood. Focal sensory loss or deprivation has been shown to induce remapping in the corresponding brain areas through activity-regulated cytoskeleton-associated protein (Arc)-mediated synaptic plasticity. We show that targeted sensory deprivation via whisker trimming in mice after induction of ischemic stroke in the somatosensory cortex representing forepaw accelerates remapping into the whisker barrel cortex and improves sensorimotor recovery. These improvements persisted even after focal sensory deprivation ended (whiskers allowed to regrow). Mice deficient in Arc, a gene critical for activity-dependent synaptic plasticity, failed to remap or recover sensorimotor function. These results indicate that post-stroke remapping occurs through Arc-mediated synaptic plasticity and is required for behavioral recovery. Furthermore, our findings suggest that enhancing perilesional cortical plasticity via focal sensory deprivation improves recovery after ischemic stroke in mice.

Designing a large field-of-view two-photon microscope using optical invariant analysis.

Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter [Formula: see text]. Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image [Formula: see text] lateral and [Formula: see text] axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.

Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice.

The interplay between hemodynamic-based markers of cortical activity (e.g. fMRI and optical intrinsic signal imaging), which are an indirect and relatively slow report of neural activity, and underlying synaptic electrical and metabolic activity through neurovascular coupling is a topic of ongoing research and debate. As application of resting state functional connectivity measures is extended further into topics such as brain development, aging and disease, the importance of understanding the fundamental physiological basis for functional connectivity will grow. Here we extend functional connectivity analysis from hemodynamic- to calcium-based imaging. Transgenic mice (n = 7) expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketamine/xylazine) and awake states. Sequential LED illumination (λ = 454, 523, 595, 640nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. Functional connectivity network maps were constructed for infraslow (0.009-0.08Hz), intermediate (0.08-0.4Hz), and high (0.4-4.0Hz) frequency bands. At infraslow and intermediate frequencies, commonly used in BOLD fMRI and fcOIS studies of functional connectivity and implicated in neurovascular coupling mechanisms, GCaMP6 and HbO2 functional connectivity structures were in high agreement, both qualitatively and also quantitatively through a measure of spatial similarity. The spontaneous dynamics of both contrasts had the highest correlation when the GCaMP6 signal was delayed with a ~0.6-1.5s temporal offset. Within the higher-frequency delta band, sensitive to slow wave sleep oscillations in non-REM sleep and anesthesia, we evaluate the speed with which the connectivity analysis stabilized and found that the functional connectivity maps captured putative network structure within time window lengths as short as 30 seconds. Homotopic GCaMP6 functional connectivity maps at 0.4-4.0Hz in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. This structure is potentially explained in part by observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas. During awake imaging, this spatio-temporal quality is altered, and a more complex and detailed functional connectivity structure is observed. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and neurovascular coupling and provide a framework for subsequent studies of neurological disease such as stroke.