Cerebral cortical functional hyperconnectivity in a mouse model of spinocerebellar ataxia type 8 (SCA8).

Spinocerebellar Ataxia Type 8 (SCA8) is an inherited neurodegenerative disease caused by a bidirectionally expressed CTG●CAG expansion mutation in the ATXN-8 and ATXN8-OS genes. While primarily a motor disorder, psychiatric and cognitive symptoms have been reported. It is difficult to elucidate how the disease alters brain function in areas with little or no degeneration producing both motor and cognitive symptoms. Using transparent polymer skulls and CNS-wide GCaMP6f expression, we studied neocortical networks throughout SCA8 progression using wide-field Ca2+ imaging in a transgenic mouse model of SCA8. We observed that neocortical networks in SCA8+ mice were hyperconnected globally which led to network configurations with increased global efficiency and centrality. At the regional level, significant network changes occurred in nearly all cortical regions, however mainly involved sensory and association cortices. Changes in functional connectivity in anterior motor regions worsened later in the disease. Near perfect decoding of animal genotype was obtained using a generalized linear model based on canonical correlation strengths between activity in cortical regions. The major contributors to decoding were concentrated in the somatosensory, higher visual and retrosplenial cortices and occasionally extended into the motor regions, demonstrating that the areas with the largest network changes are predictive of disease state.

Altered brain state during episodic dystonia in tottering mice decouples primary motor cortex from limb kinematics.

Episodic Ataxia Type 2 (EA2) is a rare neurological disorder caused by a mutation in the CACNA1A gene, encoding the P/Q-type voltage-gated Ca2+ channel important for neurotransmitter release. Patients with this channelopathy exhibit both cerebellar and cerebral pathologies, suggesting the condition affects both regions. The tottering (tg/tg) mouse is the most commonly used EA2 model due to an orthologous mutation in the cacna1a gene. The tg/tg mouse has three prominent behavioral phenotypes: a dramatic episodic dystonia; absence seizures with generalized spike and wave discharges (GSWDs); and mild ataxia. We previously observed a novel brain state, transient low-frequency oscillations (LFOs) in the cerebellum and cerebral cortex under anesthesia. In this study, we examine the relationships among the dystonic attack, GSWDs, and LFOs in the cerebral cortex. Previous studies characterized LFOs in the motor cortex of anesthetized tg/tg mice using flavoprotein autofluorescence imaging testing the hypothesis that LFOs provide a mechanism for the paroxysmal dystonia. We sought to obtain a more direct understanding of motor cortex (M1) activity during the dystonic episodes. Using two-photon Ca2+ imaging to investigate neuronal activity in M1 before, during, and after the dystonic attack, we show that there is not a significant change in the activity of M1 neurons from baseline through the attack. We also conducted simultaneous, multi-electrode recordings to further understand how M1 cellular activity and local field potentials change throughout the progression of the dystonic attack. Neither putative pyramidal nor inhibitory interneuron firing rate changed during the dystonic attack. However, we did observe a near complete loss of GSWDs during the dystonic attack in M1. Finally, using spike triggered averaging to align simultaneously recorded limb kinematics to the peak Ca2+ response, and vice versa, revealed a reduction in the spike triggered average during the dystonic episodes. Both the loss of GSWDs and the reduction in the coupling suggest that, during the dystonic attack, M1 is effectively decoupled from other structures. Overall, these results indicate that the attack is not initiated or controlled in M1, but elsewhere in the motor circuitry. The findings also highlight that LFOs, GSWDs, and dystonic attacks represent three brain states in tg/tg mice.

To be and not to be: wide-field Ca2+ imaging reveals neocortical functional segmentation combines stability and flexibility.

The stability and flexibility of the functional parcellation of the cerebral cortex is fundamental to how familiar and novel information is both represented and stored. We leveraged new advances in Ca2+ sensors and microscopy to understand the dynamics of functional segmentation in the dorsal cerebral cortex. We performed wide-field Ca2+ imaging in head-fixed mice and used spatial independent component analysis (ICA) to identify independent spatial sources of Ca2+ fluorescence. The imaging data were evaluated over multiple timescales and discrete behaviors including resting, walking, and grooming. When evaluated over the entire dataset, a set of template independent components (ICs) were identified that were common across behaviors. Template ICs were present across a range of timescales, from days to 30 seconds, although with lower occurrence probability at shorter timescales, highlighting the stability of the functional segmentation. Importantly, unique ICs emerged at the shorter duration timescales that could act to transiently refine the cortical network. When data were evaluated by behavior, both common and behavior-specific ICs emerged. Each behavior is composed of unique combinations of common and behavior-specific ICs. These observations suggest that cerebral cortical functional segmentation exhibits considerable spatial stability over time and behaviors while retaining the flexibility for task-dependent reorganization.

Wide-field calcium imaging reveals widespread changes in cortical functional connectivity following mild traumatic brain injury in the mouse.

>2.5 million individuals in the United States suffer mild traumatic brain injuries (mTBI) annually. Mild TBI is characterized by a brief period of altered consciousness, without objective findings of anatomic injury on clinical imaging or physical deficit on examination. Nevertheless, a subset of mTBI patients experience persistent subjective symptoms and repeated mTBI can lead to quantifiable neurological deficits, suggesting that each mTBI alters neurophysiology in a deleterious manner not detected using current clinical methods. To better understand these effects, we performed mesoscopic Ca2+ imaging in mice to evaluate how mTBI alters patterns of neuronal interactions across the dorsal cerebral cortex. Spatial Independent Component Analysis (sICA) and Localized semi-Nonnegative Matrix Factorization (LocaNMF) were used to quantify changes in cerebral functional connectivity (FC). Repetitive, mild, controlled cortical impacts induce temporary neuroinflammatory responses, characterized by increased density of microglia exhibiting de-ramified morphology. These temporary neuro-inflammatory changes were not associated with compromised cognitive performance in the Barnes maze or motor function as assessed by rotarod. However, long-term alterations in functional connectivity (FC) were observed. Widespread, bilateral changes in FC occurred immediately following impact and persisted for up to 7 weeks, the duration of the experiment. Network alterations include decreases in global efficiency, clustering coefficient, and nodal strength, thereby disrupting functional interactions and information flow throughout the dorsal cerebral cortex. A subnetwork analysis shows the largest disruptions in FC were concentrated near the impact site. Therefore, mTBI induces a transient neuroinflammation, without alterations in cognitive or motor behavior, and a reorganized cortical network evidenced by the widespread, chronic alterations in cortical FC.

Wide-Field Calcium Imaging of Neuronal Network Dynamics In Vivo.

A central tenet of neuroscience is that sensory, motor, and cognitive behaviors are generated by the communications and interactions among neurons, distributed within and across anatomically and functionally distinct brain regions. Therefore, to decipher how the brain plans, learns, and executes behaviors requires characterizing neuronal activity at multiple spatial and temporal scales. This includes simultaneously recording neuronal dynamics at the mesoscale level to understand the interactions among brain regions during different behavioral and brain states. Wide-field Ca2+ imaging, which uses single photon excitation and improved genetically encoded Ca2+ indicators, allows for simultaneous recordings of large brain areas and is proving to be a powerful tool to study neuronal activity at the mesoscopic scale in behaving animals. This review details the techniques used for wide-field Ca2+ imaging and the various approaches employed for the analyses of the rich neuronal-behavioral data sets obtained. Also discussed is how wide-field Ca2+ imaging is providing novel insights into both normal and altered neural processing in disease. Finally, we examine the limitations of the approach and new developments in wide-field Ca2+ imaging that are bringing new capabilities to this important technique for investigating large-scale neuronal dynamics.

Polymer Skulls With Integrated Transparent Electrode Arrays for Cortex-Wide Opto-Electrophysiological Recordings.

Electrophysiology and optical imaging provide complementary neural sensing capabilities - electrophysiological recordings have high temporal resolution, while optical imaging allows recording of genetically-defined populations at high spatial resolution. Combining these two modalities for simultaneous large-scale, multimodal sensing of neural activity across multiple brain regions can be very powerful. Here, transparent, inkjet-printed electrode arrays with outstanding optical and electrical properties are seamlessly integrated with morphologically conformant transparent polymer skulls. Implanted on transgenic mice expressing the Calcium (Ca2+ ) indicator GCaMP6f in excitatory neurons, these "eSee-Shells" provide a robust opto-electrophysiological interface for over 100 days. eSee-Shells enable simultaneous mesoscale Ca2+ imaging and electrocorticography (ECoG) acquisition from multiple brain regions covering 45 mm2 of cortex under anesthesia and in awake animals. The clarity and transparency of eSee-Shells allow recording single-cell Ca2+ signals directly below the electrodes and interconnects. Simultaneous multimodal measurement of cortical dynamics reveals changes in both ECoG and Ca2+ signals that depend on the behavioral state.

Wide-Field Calcium Imaging of Dynamic Cortical Networks during Locomotion.

Motor behavior results in complex exchanges of motor and sensory information across cortical regions. Therefore, fully understanding the cerebral cortex's role in motor behavior requires a mesoscopic-level description of the cortical regions engaged, their functional interactions, and how these functional interactions change with behavioral state. Mesoscopic Ca2+ imaging through transparent polymer skulls in mice reveals elevated activation of the dorsal cerebral cortex during locomotion. Using the correlations between the time series of Ca2+ fluorescence from 28 regions (nodes) obtained using spatial independent component analysis (sICA), we examined the changes in functional connectivity of the cortex from rest to locomotion with a goal of understanding the changes to the cortical functional state that facilitate locomotion. Both the transitions from rest to locomotion and from locomotion to rest show marked increases in correlation among most nodes. However, once a steady state of continued locomotion is reached, many nodes, including primary motor and somatosensory nodes, show decreases in correlations, while retrosplenial and the most anterior nodes of the secondary motor cortex show increases. These results highlight the changes in functional connectivity in the cerebral cortex, representing a series of changes in the cortical state from rest to locomotion and on return to rest.

Disparate insults relevant to schizophrenia converge on impaired spike synchrony and weaker synaptic interactions in prefrontal local circuits.

Schizophrenia results from hundreds of known causes, including genetic, environmental, and developmental insults that cooperatively increase risk of developing the disease. In spite of the diversity of causal factors, schizophrenia presents with a core set of symptoms and brain abnormalities (both structural and functional) that particularly impact the prefrontal cortex. This suggests that many different causal factors leading to schizophrenia may cause prefrontal neurons and circuits to fail in fundamentally similar ways. The nature of convergent malfunctions in prefrontal circuits at the cell and synaptic levels leading to schizophrenia are not known. Here, we apply convergence-guided search to identify core pathological changes in the functional properties of prefrontal circuits that lie downstream of mechanistically distinct insults relevant to the disease. We compare the impacts of blocking NMDA receptors in monkeys and deleting a schizophrenia risk gene in mice on activity timing and effective communication in prefrontal local circuits. Although these manipulations operate through distinct molecular pathways and biological mechanisms, we found they produced convergent pathophysiological effects on prefrontal local circuits. Both manipulations reduced the frequency of synchronous (0-lag) spiking between prefrontal neurons and weakened functional interactions between prefrontal neurons at monosynaptic lags as measured by information transfer between the neurons. The two observations may be related, as reduction in synchronous spiking between prefrontal neurons would be expected to weaken synaptic connections between them via spike-timing-dependent synaptic plasticity. These data suggest that the link between spike timing and synaptic connectivity could comprise the functional vulnerability that multiple risk factors exploit to produce disease.

Through the looking glass: A review of cranial window technology for optical access to the brain.

Deciphering neurologic function is a daunting task, requiring understanding the neuronal networks and emergent properties that arise from the interactions among single neurons. Mechanistic insights into neuronal networks require tools that simultaneously assess both single neuron activity and the consequent mesoscale output. The development of cranial window technologies, in which the skull is thinned or replaced with a synthetic optical interface, has enabled monitoring neuronal activity from subcellular to mesoscale resolution in awake, behaving animals when coupled with advanced microscopy techniques. Here we review recent achievements in cranial window technologies, appraise the relative merits of each design and discuss the future research in cranial window design.

Cortex-wide neural interfacing via transparent polymer skulls.

Neural computations occurring simultaneously in multiple cerebral cortical regions are critical for mediating behaviors. Progress has been made in understanding how neural activity in specific cortical regions contributes to behavior. However, there is a lack of tools that allow simultaneous monitoring and perturbing neural activity from multiple cortical regions. We engineered 'See-Shells'-digitally designed, morphologically realistic, transparent polymer skulls that allow long-term (>300 days) optical access to 45 mm2 of the dorsal cerebral cortex in the mouse. We demonstrate the ability to perform mesoscopic imaging, as well as cellular and subcellular resolution two-photon imaging of neural structures up to 600 µm deep. See-Shells allow calcium imaging from multiple, non-contiguous regions across the cortex. Perforated See-Shells enable introducing penetrating neural probes to perturb or record neural activity simultaneously with whole cortex imaging. See-Shells are constructed using common desktop fabrication tools, providing a powerful tool for investigating brain structure and function.

Altered levels of the splicing factor muscleblind modifies cerebral cortical function in mouse models of myotonic dystrophy.

Myotonic dystrophy (DM) is a progressive, multisystem disorder affecting skeletal muscle, heart, and central nervous system. In both DM1 and DM2, microsatellite expansions of CUG and CCUG RNA repeats, respectively, accumulate and disrupt functions of alternative splicing factors, including muscleblind (MBNL) proteins. Grey matter loss and white matter changes, including the corpus callosum, likely underlie cognitive and executive function deficits in DM patients. However, little is known how cerebral cortical circuitry changes in DM. Here, flavoprotein optical imaging was used to assess local and contralateral responses to intracortical motor cortex stimulation in DM-related mouse models. In control mice, brief train stimulation generated ipsilateral and contralateral homotopic fluorescence increases, the latter mediated by the corpus callosum. Single pulse stimulation produced an excitatory response with an inhibitory-like surround response mediated by GABAA receptors. In a mouse model of DM2 (Mbnl2 KO), we observed prolonged and increased responsiveness to train stimulation and loss of the inhibition from single pulse stimulation. Conversely, mice overexpressing human MBNL1 (MBNL1-OE) exhibited decreased contralateral response to train stimulation and reduction of inhibitory-like surround to single pulse stimulation. Therefore, altering levels of two key DM-associated splicing factors modifies functions of local cortical circuits and contralateral responses mediated through the corpus callosum.

Abnormal excitability and episodic low-frequency oscillations in the cerebral cortex of the tottering mouse.

The Ca(2+) channelopathies caused by mutations of the CACNA1A gene that encodes the pore-forming subunit of the human Cav2.1 (P/Q-type) voltage-gated Ca(2+) channel include episodic ataxia type 2 (EA2). Although, in EA2 the emphasis has been on cerebellar dysfunction, patients also exhibit episodic, nonmotoric abnormalities involving the cerebral cortex. This study demonstrates episodic, low-frequency oscillations (LFOs) throughout the cerebral cortex of tottering (tg/tg) mice, a widely used model of EA2. Ranging between 0.035 and 0.11 Hz, the LFOs in tg/tg mice can spontaneously develop very high power, referred to as a high-power state. The LFOs in tg/tg mice are mediated in part by neuronal activity as tetrodotoxin decreases the oscillations and cortical neuron discharge contain the same low frequencies. The high-power state involves compensatory mechanisms because acutely decreasing P/Q-type Ca(2+) channel function in either wild-type (WT) or tg/tg mice does not induce the high-power state. In contrast, blocking l-type Ca(2+) channels, known to be upregulated in tg/tg mice, reduces the high-power state. Intriguingly, basal excitatory glutamatergic neurotransmission constrains the high-power state because blocking ionotropic or metabotropic glutamate receptors results in high-power LFOs in tg/tg but not WT mice. The high-power LFOs are decreased markedly by acetazolamide and 4-aminopyridine, the primary treatments for EA2, suggesting disease relevance. Together, these results demonstrate that the high-power LFOs in the tg/tg cerebral cortex represent a highly abnormal excitability state that may underlie noncerebellar symptoms that characterize CACNA1A mutations.

Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization.

Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn(2+) release and intracellular accumulation occurs with SD, and elevated intracellular Zn(2+) ([Zn(2+) ]i ) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn(2+) ]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn(2+) ]i loading prior to SD, by transient ZnCl2 application with the Zn(2+) ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn(2+) ]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn(2+) release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn(2+) ]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn(2+) ]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn(2+) ]i accumulation in a range of brain injury models.

Vascular, electrophysiological, and metabolic consequences of cortical spreading depression in a mouse model of simulated neurosurgical conditions.

OBJECTIVES: Cortical spreading depression (CSD) is a metabolically taxing wave of cellular depolarization that propagates slowly across the brain. Though CSD is known to occur after brain injury in humans, it is unknown if CSD occurs during neurosurgical procedures. This study evaluates CSD in a mouse model of simulated neurosurgical conditions. METHODS: Mice were intubated and ventilated, maintained at ~37°C, an arterial line placed to monitor mean arterial pressure and maintain pCO(2) ~30 mmHg. Mice were given simulated neuroanesthesia (fentanyl, propofol, and isofluorane). Burrholes and craniotomies were made to record the response to cortical bipolar cauterization. Separate sets of experiments (three animals each) examined electrocorticographic (ECoG) activity, optical measures of blood volume and vascular diameters (540 nm absorbance), and autofluorescence attributed to NADH (750 nm, two-photon excitation). RESULTS: Ipsilateral cauterization invariably resulted in a propagating CSD wave identified by slow DC potential shifts (2·8±0·2 mm/minute, n = 6) and suppression of ECoG activity (range 0·5-7·3 minutes, n = 10). Each CSD was associated with an initial arteriolar constriction and decreased blood volume, followed by a longer-lasting vasodilation and increased blood volume. Tissue oxygenation, assessed indirectly by NADH imaging, was consistent with demand on oxidative metabolism following each CSD. Repetitive SDs resulted in loss of tissue autofluorescence, suggestive of tissue compromise. CONCLUSIONS: CSD is consistently elicited by simulated neurosurgical stimuli under simulated intraoperative conditions in mice. These events caused ECoG depression, transient vasoconstriction, and metabolic demand that propagated from the manipulation site. It is likely that CSD occurs during neurosurgery and may contribute to surgical brain injuries otherwise poorly explained.

Spreading depression and related events are significant sources of neuronal Zn2+ release and accumulation.

Spreading depression (SD) involves coordinated depolarizations of neurons and glia that propagate through the brain tissue. Repetitive SD-like events are common following human ischemic strokes, and are believed to contribute to the enlargement of infarct volume. Accumulation of Zn(2+) is also implicated in ischemic neuronal injury. Synaptic glutamate release contributes to SD propagation, and because Zn(2+) is costored with glutamate in some synaptic vesicles, we examined whether Zn(2+) is released by SD and may therefore provide a significant source of Zn(2+) in the postischemic period. Spreading depression-like events were generated in acutely prepared murine hippocampal slices by deprivation of oxygen and glucose (OGD), and Zn(2+) release was detected extracellularly by a Zn(2+)-selective indicator FluoZin-3. Deprivation of oxygen and glucose-SD produced large FluoZin-3 increases that propagated with the event, and signals were abolished in tissues from ZnT3 knockout animals lacking synaptic Zn(2+). Synaptic Zn(2+) release was also maintained with repetitive SDs generated by microinjections of KCl under normoxic conditions. Intracellular Zn(2+) accumulation in CA1 neurons, assessed using microinjection of FluoZin-3, showed significant increases following SD that was attributed to synaptic Zn(2+) release. These results suggest that Zn(2+) is released during SDs and could provide a significant source of Zn(2+) that contributes to neurodegeneration in the postischemic period.

Zn²+ chelation improves recovery by delaying spreading depression-like events.

We earlier reported that Zn²+ chelation improved recovery of synaptic potentials after transient oxygen and glucose deprivation in brain slices. Such an effect could be because of reduced accumulation of Zn²+ in postsynaptic neurons, or could also be due to prevention of the onset of spreading depression-like events. A combination of optical and electrical recording was used here to show that Zn²+ chelation is effective because it delays spreading depression-like events. If the duration of oxygen/glucose deprivation was sufficient to generate a spreading depression-like event, irrecoverable Ca²+-dependent loss of synaptic potentials occurred, regardless of Zn²+ availability. These results identify a key mechanism underlying protective effects of Zn²+ chelation, and emphasize the importance of evaluating spreading depression-like events in studies of neuroprotection.

Descriptors of American Physical Therapy Association physical therapist members' reading of professional publications.

One of the components of evidence-based practice (EBP) is reading the literature. The purpose of this investigation was 1) to determine which publications are read most frequently by physical therapists (PTs), 2) to identify employment and education characteristics related to reading patterns, and 3) to determine how PTs use information gained from reading professional publications. A survey was constructed, pretested, revised, and then mailed to a random sample of 1,000 physical therapists, 500 from the general American Physical Therapy Association (APTA) membership and 500 from the combined Geriatric and Orthopedic section memberships. The data were analyzed with descriptive statistics. After all mailings, 43.3% of the sample responded. Of those responding, 66.3% were employed as clinicians, 14.7% as clinician-administrators, 6% as educators, and 4.9% as administrators. Overall, and for clinicians, the two most frequently read professional publications were non peer-reviewed. For clinician groups "patient management" was among the top two most selected uses of information from reading professional publications. Educators and those with an advanced doctorate indicated "class lectures" as either their second most frequently selected use or tied for first with "keeping current." Only educators and those with or pursuing an advanced doctorate reported using information from reading professional publications for "research ideas" or "research methods" among their top five uses. Slightly more than 10% of the respondents cited a peer-reviewed published article as having been most influential on their practice. Non peer-reviewed professional publications appear to serve as a more frequent source of information for "patient management" than do peer-reviewed publications. Efforts to increase the use of EBP need to be explored and evaluated for impact on physical therapists' practice.