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.

Modulation of brain state, e.g., by anesthesia, alters the correlation structure of spontaneous activity, especially in the delta band. This effect has largely been attributed to the ∼1 Hz slow oscillation that is characteristic of anesthesia and nonrapid eye movement (NREM) sleep. However, the effect of the slow oscillation on correlation structures and the spectral content of spontaneous activity across brain states (including NREM) has not been comprehensively examined. Further, discrepancies between activity dynamics observed with hemoglobin versus calcium (GCaMP6) imaging have not been reconciled. Lastly, whether the slow oscillation replaces functional connectivity (FC) patterns typical of the alert state, or superimposes on them, remains unclear. Here, we use wide-field calcium imaging to study spontaneous cortical activity in awake, anesthetized, and naturally sleeping mice. We find modest brain state-dependent changes in infraslow correlations but larger changes in GCaMP6 delta correlations. Principal component analysis of GCaMP6 sleep/anesthesia data in the delta band revealed that the slow oscillation is largely confined to the first three components. Removal of these components revealed a correlation structure strikingly similar to that observed during wake. These results indicate that, during NREM sleep/anesthesia, the slow oscillation superimposes onto a canonical FC architecture.

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.

The biophysical chemistry of macromolecular complexes confer their functional characteristics. We investigate the mechanisms that make the AB5 holotoxin of Vibrio cholerae (CT) a significantly more pathogenic molecule than the enterotoxin of Escherichia coli (LT) with which it shares 88% similarity and whose structure is homologous with a backbone RMSD of 0.84 Å and imposes its deleterious effects though the same process to constitutively ADP-ribosylate adenylate cyclase. We present computational data that characterizes the impact of amino acid variations in the A2 tail, which helps to explain experimental data that demonstrate CT's higher toxicity. A hydrophobic patch on the B pentamer interface and its interactions with the A subdomain are partially disrupted by the substitution of an aspartic acid (LT) for glycine in CT. CT's holotoxin has less solvent accessible surface area (94 Å(2) vs 54 Å(2)) and higher contact area (280 Å(2) vs 241 Å(2)) with S228, which is a gatekeeper, partially controlling the diffusion of water into the pore. CT excludes water from the top of the central pore whereas LT allows much more water to interact. These biophysical properties of the toxins lead to their differential toxicity and resulting impact to human health.

No abstract

Cross-sectional studies have established a variety of structural, synaptic, and cell physiological changes corresponding to critical periods in cortical development. However, the emergence of functional connectivity (FC) in development has not been fully characterized, and hemodynamic-based measures are vulnerable to any neurovascular coupling changes occurring in parallel. We therefore used optical fluorescence imaging to trace longitudinal calcium FC in the awake, resting-state mouse cortex at 5 developmental timepoints beginning at postnatal day 15 (P15) and ending in early adulthood at P60. Calcium FC displayed coherent functional maps as early as P15, and FC significantly varied in connections between many regions across development, with the developmental trajectory’s shape specific to the functional region. Evaluating 325 seed–seed connections, we found that there was a significant increase in FC between P15 and P22 over the majority of the cortex as well as bilateral connectivity and node degree differences in frontal, motor, and retrosplenial cortices after P22. A rebalancing of inter- and intrahemispheric FC and local-distal FC dominance was also observed during development. This longitudinal developmental calcium FC study therefore provides a resource dataset to the field and identifies periods of dynamic change which cross-sectional studies may target for examination of disease states.

Functional connectivity (FC) is a sensitive metric that provides a readout of whole cortex coordinate neural activity in a mouse model. We examine the impact of experimental SAH modeled through endovascular perforation, and the effectiveness of subsequent treatment on FC, through three key questions: 1) Does the endovascular perforation model of SAH induce deficits in FC; 2) Does exposure to hypoxic conditioning provide protection against these FC deficits and, if so, is this neurovascular protection SIRT1-mediated; and 3) does treatment with the SIRT1 activator resveratrol alone provide protection against these FC deficits? Cranial windows were adhered on skull-intact mice that were then subjected to either sham or SAH surgery and either left untreated or treated with hypoxic post-conditioning (with or without EX527) or resveratrol for 3 days. Mice were imaged 3 days post-SAH/sham surgery, temporally aligned with the onset of major SAH sequela in mice. Here we show that the endovascular perforation model of SAH induces global and network-specific deficits in FC by day 3, corresponding with the time frame of DCI in mice. Hypoxic conditioning provides SIRT1-mediated protection against these network-specific FC deficits post-SAH, as does treatment with resveratrol. Conditioning-based strategies provide multifaceted neurovascular protection in experimental SAH.

Received wisdom in the field of fungal biology holds that the process of editing a genome by transformation and homologous recombination is inherently mutagenic. However, that belief is based on circumstantial evidence. We provide the first direct measurement of the effects of transformation on a fungal genome by sequencing the genomes of 29 transformants and 30 untransformed controls with high coverage. Contrary to the received wisdom, our results show that transformation of DNA segments flanked by long targeting sequences, followed by homologous recombination and selection for a drug marker, is extremely safe. If a transformation deletes a gene, that may create selective pressure for a few compensatory mutations, but even when we deleted a gene, we found fewer than two point mutations per deletion strain, on average. We also tested these strains for changes in gene expression and found only a few genes that were consistently differentially expressed between the wild type and strains modified by genomic insertion of a drug resistance marker. As part of our report, we provide the assembled genome sequence of the commonly used laboratory strain Cryptococcus neoformans var. grubii strain KN99α.