Repeated hippocampal seizures lead to brain-wide reorganization of circuits and seizure propagation pathways.

Repeated seizure activity can lead to long-term changes in seizure dynamics and behavior. However, resulting changes in brain-wide dynamics remain poorly understood. This is due partly to technical challenges in precise seizure control and in vivo whole-brain mapping of circuit dynamics. Here, we developed an optogenetic kindling model through repeated stimulation of ventral hippocampal CaMKII neurons in adult rats. We then combined fMRI with electrophysiology to track brain-wide circuit dynamics resulting from non-afterdischarge (AD)-generating stimulations and individual convulsive seizures. Kindling induced widespread increases in non-AD-generating stimulation response and ipsilateral functional connectivity and elevated anxiety. Individual seizures in kindled animals showed more significant increases in brain-wide activity and bilateral functional connectivity. Onset time quantification provided evidence for kindled seizure propagation from the ipsilateral to the contralateral hemisphere. Furthermore, a core of slow-migrating hippocampal activity was identified in both non-kindled and kindled seizures, revealing a novel mechanism of seizure sustainment and propagation.

High-sensitivity detection of optogenetically-induced neural activity with functional ultrasound imaging.

Whole-brain imaging approaches and optogenetic manipulations are powerful tools to map brain-wide neural circuits in vivo. To date, functional magnetic resonance imaging (fMRI) provides the most comprehensive evaluation of such large-scale circuitry. However, functional ultrasound imaging (fUSI) has recently emerged as a complementary imaging modality that can extend such measurements towards the context of diverse behavioral states and tasks. Nevertheless, in order to properly interpret the fUSI signal during these complicated scenarios, it must first be carefully validated against well-established technologies, such as fMRI, in highly controlled experimental settings. Here, to address this need, we compared subsequent fMRI and fUSI recordings in response to direct neuronal activation via optogenetics in the same animals under an identical anesthetic protocol. Specifically, we applied various intensities of light stimulation to the primary motor cortex (M1) of mice and compared the spatiotemporal dynamics of the elicited fMRI and fUSI signals. Overall, our general linear model analysis (t-scores) and time series analysis (z-scores) revealed that fUSI was more sensitive than fMRI for detecting optogenetically-induced neuronal activation. Local field potential recordings in the bilateral M1 and striatum also better co-localized with fUSI activation patterns than those of fMRI. Finally, the fUSI response contained distinct arterial and venous components that provide vascular readouts of neuronal activity with vessel-type specificity.

Brain-wide neural dynamics of poststroke recovery induced by optogenetic stimulation.

Poststroke optogenetic stimulations can promote functional recovery. However, the circuit mechanisms underlying recovery remain unclear. Elucidating key neural circuits involved in recovery will be invaluable for translating neuromodulation strategies after stroke. Here, we used optogenetic functional magnetic resonance imaging to map brain-wide neural circuit dynamics after stroke in mice treated with and without optogenetic excitatory neuronal stimulations in the ipsilesional primary motor cortex (iM1). We identified key sensorimotor circuits affected by stroke. iM1 stimulation treatment restored activation of the ipsilesional corticothalamic and corticocortical circuits, and the extent of activation was correlated with functional recovery. Furthermore, stimulated mice exhibited higher expression of axonal growth-associated protein 43 in the ipsilesional thalamus and showed increased Synaptophysin+/channelrhodopsin+ presynaptic axonal terminals in the corticothalamic circuit. Selective stimulation of the corticothalamic circuit was sufficient to improve functional recovery. Together, these findings suggest early involvement of corticothalamic circuit as an important mediator of poststroke recovery.

Predicting Successful Generation and Inhibition of Seizure-like Afterdischarges and Mapping Their Seizure Networks Using fMRI.

To understand the conditions necessary to initiate and terminate seizures, we investigate optogenetically induced hippocampal seizures with LFP, fMRI, and optogenetic inhibition. During afterdischarge induction using optogenetics, LFP recordings show that stimulations with earlier ictal onset times are more likely to result in afterdischarges and are more difficult to curtail with optogenetic inhibition. These results are generalizable across two initiation sites, the dorsal and ventral hippocampus. fMRI shows that afterdischarges initiated from the dorsal or ventral hippocampus exhibit distinct networks. Short-duration seizures initiated in the dorsal and ventral hippocampus are unilateral and bilateral, respectively, while longer-duration afterdischarges recruit broader, bilateral networks. When optogenetic inhibition is ineffective at stopping seizures, the network activity spreads more extensively but largely overlaps with the network activity associated with seizures that could be curtailed. These results provide insights into how seizures can be inhibited, which has implications for targeted seizure interventions.

Thalamic Input to Orbitofrontal Cortex Drives Brain-wide, Frequency-Dependent Inhibition Mediated by GABA and Zona Incerta.

Anatomical and behavioral data suggest that the ventrolateral orbitofrontal cortex (VLO), which exhibits extensive connectivity and supports diverse sensory and cognitive processes, may exert global influence over brain activity. However, this hypothesis has never been tested directly. We applied optogenetic fMRI to drive various elements of VLO circuitry while visualizing the whole-brain response. Surprisingly, driving excitatory thalamocortical projections to VLO at low frequencies (5-10 Hz) evoked widespread, bilateral decreases in brain activity spanning multiple cortical and subcortical structures. This pattern was unique to thalamocortical projections, with direct stimulations of neither VLO nor thalamus eliciting such a response. High-frequency stimulations (25-40 Hz) of thalamocortical projections evoked dramatically different-though still far-reaching-responses, in the form of widespread ipsilateral activation. Importantly, decreases in brain activity evoked by low-frequency thalamocortical input were mediated by GABA and activity in zona incerta. These findings identify specific circuit mechanisms underlying VLO control of brain-wide neural activities.

Carbon monofilament electrodes for unit recording and functional MRI in same subjects.

Extracellular electrophysiology and functional MRI are complementary techniques that provide information about cellular and network-level neural activity, respectively. However, electrodes for electrophysiology are typically made from metals, which cause significant susceptibility artifacts on MR images. Previous work has demonstrated that insulated carbon fiber bundle electrodes reduce the volume of magnetic susceptibility artifacts and can be used to record local field potentials (LFP), but the relatively large diameter of the probes make them unsuitable for multi- and single-unit recordings. Although single carbon fiber electrodes have recently been used to record single-unit activity, these probes require modifications in order to aid insertion and the use of these probes in fMRI has yet to be validated. Therefore, there is a need for a single-carbon fiber electrode design that (1) minimizes the volume of the susceptibility artifact, (2) can record from a wide frequency band that includes LFP and multi- and single-unit recording, and (3) is practical to insert without additional modifications. Here, we demonstrate that carbon-fiber electrodes made from single carbon monofilaments (35 µm in diameter) meet all of these criteria. Carbon monofilament electrodes modified with the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) have lower impedances and higher signal-to-noise ratio recordings than platinum-iridium electrodes, a current gold standard for chronic single-unit recording. Furthermore, these probes distort a significantly smaller volume of voxels compared to tungsten and platinum-iridium electrodes in agarose phantom and in vivo MR images, leading to higher contrast-to-noise ratio in regions proximal to the electrode implantation site during fMRI. Collectively, this work establishes that carbon monofilaments are a practical choice for combined electrophysiology-fMRI experiments.

Epilepsy-predictive magnetic resonance imaging changes following experimental febrile status epilepticus: Are they translatable to the clinic?

OBJECTIVE: A subset of children with febrile status epilepticus (FSE) are at risk for development of temporal lobe epilepsy later in life. We sought a noninvasive predictive marker of those at risk that can be identified soon after FSE, within a clinically realistic timeframe. METHODS: Longitudinal T2 -weighted magnetic resonance imaging (T2 WI MRI) of rat pups at several time points after experimental FSE (eFSE) was performed on a high-field scanner followed by long-term continuous electroencephalography. In parallel, T2 WI MRI scans were performed on a 3.0-T clinical scanner. Finally, chronic T2 WI MRI signal changes were examined in rats that experienced eFSE and were imaged months later in adulthood. RESULTS: Epilepsy-predicting T2 changes, previously observed at 2 hours after eFSE, persisted for at least 6 hours, enabling translation to the clinic. Repeated scans, creating MRI trajectories of T2 relaxation times following eFSE, provided improved prediction of epileptogenesis compared with a single MRI scan. Predictive signal changes centered on limbic structures, such as the basolateral and medial amygdala. T2 WI MRI changes, originally described on high-field scanners, can also be measured on clinical MRI scanners. Chronically elevated T2 relaxation times in hippocampus were observed months after eFSE in rats, as noted for post-FSE changes in children. SIGNIFICANCE: Early T2 WI MRI changes after eFSE provide a strong predictive measure of epileptogenesis following eFSE, on both high-field and clinical MRI scanners. Importantly, the extension of the acute signal changes to at least 6 hours after the FSE enables its inclusion in clinical studies. Chronic elevations of T2 relaxation times within the hippocampal formation and related structures are common to human and rodent FSE, suggesting that similar processes are involved across species.

Optogenetic study of networks in epilepsy.

Currently, approximately 30% of patients with epilepsy do not have adequate seizure control. A greater understanding of the underlying mechanisms by which seizures start or propagate could lead to new therapeutic strategies. The recent development of optogenetics, because of its unprecedented precision for controlling activity within distinct neuronal populations, has revolutionized neuroscience, including epilepsy research. This Review discusses recent breakthroughs made with optogenetics in epilepsy research. These breakthroughs include new insights into the key roles that different cell types play in mediating seizures as well as in the development of epilepsy. Subsequently, we discuss how targeting different brain regions and cell populations has opened up the possibility of highly specific therapies that can stop seizures on demand. Finally, we illustrate how combining newly available neuroscience tools with whole-brain imaging techniques will allow researchers to understand better the spread of seizures on a network level. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Activation of Direct and Indirect Pathway Medium Spiny Neurons Drives Distinct Brain-wide Responses.

A central theory of basal ganglia function is that striatal neurons expressing the D1 and D2 dopamine receptors exert opposing brain-wide influences. However, the causal influence of each population has never been measured at the whole-brain scale. Here, we selectively stimulated D1 or D2 receptor-expressing neurons while visualizing whole-brain activity with fMRI. Excitation of either inhibitory population evoked robust positive BOLD signals within striatum, while downstream regions exhibited significantly different and generally opposing responses consistent with-though not easily predicted from-contemporary models of basal ganglia function. Importantly, positive and negative signals within the striatum, thalamus, GPi, and STN were all associated with increases and decreases in single-unit activity, respectively. These findings provide direct evidence for the opposing influence of D1 and D2 receptor-expressing striatal neurons on brain-wide circuitry and extend the interpretability of fMRI studies by defining cell-type-specific contributions to the BOLD signal.

High spatial resolution compressed sensing (HSPARSE) functional MRI.

PURPOSE: To propose a novel compressed sensing (CS) high spatial resolution functional MRI (fMRI) method and demonstrate the advantages and limitations of using CS for high spatial resolution fMRI. METHODS: A randomly undersampled variable density spiral trajectory enabling an acceleration factor of 5.3 was designed with a balanced steady state free precession sequence to achieve high spatial resolution data acquisition. A modified k-t SPARSE method was then implemented and applied with a strategy to optimize regularization parameters for consistent, high quality CS reconstruction. RESULTS: The proposed method improves spatial resolution by six-fold with 12 to 47% contrast-to-noise ratio (CNR), 33 to 117% F-value improvement and maintains the same temporal resolution. It also achieves high sensitivity of 69 to 99% compared the original ground-truth, small false positive rate of less than 0.05 and low hemodynamic response function distortion across a wide range of CNRs. The proposed method is robust to physiological noise and enables detection of layer-specific activities in vivo, which cannot be resolved using the highest spatial resolution Nyquist acquisition. CONCLUSION: The proposed method enables high spatial resolution fMRI that can resolve layer-specific brain activity and demonstrates the significant improvement that CS can bring to high spatial resolution fMRI. Magn Reson Med 76:440-455, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Frequency-selective control of cortical and subcortical networks by central thalamus.

Central thalamus plays a critical role in forebrain arousal and organized behavior. However, network-level mechanisms that link its activity to brain state remain enigmatic. Here, we combined optogenetics, fMRI, electrophysiology, and video-EEG monitoring to characterize the central thalamus-driven global brain networks responsible for switching brain state. 40 and 100 Hz stimulations of central thalamus caused widespread activation of forebrain, including frontal cortex, sensorimotor cortex, and striatum, and transitioned the brain to a state of arousal in asleep rats. In contrast, 10 Hz stimulation evoked significantly less activation of forebrain, inhibition of sensory cortex, and behavioral arrest. To investigate possible mechanisms underlying the frequency-dependent cortical inhibition, we performed recordings in zona incerta, where 10, but not 40, Hz stimulation evoked spindle-like oscillations. Importantly, suppressing incertal activity during 10 Hz central thalamus stimulation reduced the evoked cortical inhibition. These findings identify key brain-wide dynamics underlying central thalamus arousal regulation.

MRI compatible optrodes for simultaneous LFP and optogenetic fMRI investigation of seizure-like afterdischarges.

In preclinical studies, implanted electrodes can cause severe degradation of MRI images and hence are seldom used for chronic studies employing functional magnetic resonance imaging. In this study, we developed carbon fiber optrodes (optical fiber and electrode hybrid devices), which can be utilised in chronic longitudinal studies aiming to take advantage of emerging optogenetic technologies, and compared them with the more widely used tungsten optrodes. We find that optrodes constructed using small diameter (~130 µm) carbon fiber electrodes cause significantly reduced artifact on functional MRI images compared to those made with 50 µm diameter tungsten wire and at the same time the carbon electrodes have lower impedance, which leads to higher quality LFP recordings. In order to validate this approach, we use these devices to study optogenetically-induced seizure-like afterdischarges in rats sedated with dexmedetomidine and compare these to sub (seizure) threshold stimulations in the same animals. The results indicate that seizure-like afterdischarges involve several extrahippocampal brain regions that are not recruited by subthreshold optogenetic stimulation of the hippocampus at 20 Hz. Subthreshold stimulation led to activation of the entire ipsilateral hippocampus and septum, whereas afterdischarges additionally produced activations in the contralateral hippocampal formation, neocortex, cerebellum, nucleus accumbens, and thalamus. Although we demonstrate just one application, given the ease of fabrication, we anticipate that carbon fiber optrodes could be utilised in a variety of studies that could benefit from longitudinal optogenetic functional magnetic resonance imaging.

T2 relaxation time post febrile status epilepticus predicts cognitive outcome.

Evidence from animal models and patient data indicates that febrile status epilepticus (FSE) in early development can result in permanently diminished cognitive abilities. To understand the variability in cognitive outcome following FSE, we used MRI to measure dynamic brain metabolic responses to the induction of FSE in juvenile rats. We then compared these measurements to the ability to learn an active avoidance spatial task weeks later. T2 relaxation times were significantly lower in FSE rats that were task learners in comparison to FSE non-learners. While T2 time in whole brain held the greatest predictive power, T2 in hippocampus and basolateral amygdala were also excellent predictors. These signal differences in response to FSE indicate that rats that fail to meet metabolic and oxygen demand are more likely to develop spatial cognition deficits. Place cells from FSE non-learners had significantly larger firing fields and higher in-field firing rate than FSE learners and control animals and imply increased excitability in the pyramidal cells of FSE non-learners. These findings suggest a mechanistic cause for the spatial memory deficits in active avoidance and are relevant to other acute neurological insults in early development where cognitive outcome is a concern.

Optogenetic fMRI reveals distinct, frequency-dependent networks recruited by dorsal and intermediate hippocampus stimulations.

Although the connectivity of hippocampal circuits has been extensively studied, the way in which these connections give rise to large-scale dynamic network activity remains unknown. Here, we used optogenetic fMRI to visualize the brain network dynamics evoked by different frequencies of stimulation of two distinct neuronal populations within dorsal and intermediate hippocampus. Stimulation of excitatory cells in intermediate hippocampus caused widespread cortical and subcortical recruitment at high frequencies, whereas stimulation in dorsal hippocampus led to activity primarily restricted to hippocampus across all frequencies tested. Sustained hippocampal responses evoked during high-frequency stimulation of either location predicted seizure-like afterdischarges in video-EEG experiments, while the widespread activation evoked by high-frequency stimulation of intermediate hippocampus predicted behavioral seizures. A negative BOLD signal observed in dentate gyrus during dorsal, but not intermediate, hippocampus stimulation is proposed to underlie the mechanism for these differences. Collectively, our results provide insight into the dynamic function of hippocampal networks and their role in seizures.

A novel, noninvasive, predictive epilepsy biomarker with clinical potential.

A significant proportion of temporal lobe epilepsy (TLE), a common, intractable brain disorder, arises in children with febrile status epilepticus (FSE). Preventative therapy development is hampered by our inability to identify early the FSE individuals who will develop TLE. In a naturalistic rat model of FSE, we used high-magnetic-field MRI and long-term video EEG to seek clinically relevant noninvasive markers of epileptogenesis and found that reduced amygdala T2 relaxation times in high-magnetic-field MRI hours after FSE predicted experimental TLE. Reduced T2 values likely represented paramagnetic susceptibility effects derived from increased unsaturated venous hemoglobin, suggesting augmented oxygen utilization after FSE termination. Indeed, T2 correlated with energy-demanding intracellular translocation of the injury-sensor high-mobility group box 1 (HMGB1), a trigger of inflammatory cascades implicated in epileptogenesis. Use of deoxyhemoglobin-sensitive MRI sequences enabled visualization of the predictive changes on lower-field, clinically relevant scanners. This novel MRI signature delineates the onset and suggests mechanisms of epileptogenesis that follow experimental FSE.

Inflammatory processes, febrile seizures, and subsequent epileptogenesis.

Febrile seizures (FS) are the most common type of seizures in infants and preschool children. Inflammatory mediators, which are known triggers of fever, have also been implicated as contributors to the onset of these seizures. Evidence that inflammation is present following FS and during established epilepsy suggests that it could also influence epileptogenesis. However, the potential involvement of inflammatory mediators to the epileptogenic process that may follow prolonged FS has yet to be fully determined. This article reviews the current state of our knowledge and major gaps that remain by focusing on four questions: Does inflammation contribute to the generation of FS? Does prolonged FS or febrile status epilepticus (SE) cause temporal lobe epilepsy in the absence of predisposing factors? Does inflammation contribute to the process by which febrile SE causes limbic epilepsy? And finally, can inflammation be a foundation for biomarkers and therapy for FS-induced epileptogenesis?

Imaging seizure-induced inflammation using an antibody targeted iron oxide contrast agent.

Early inflammation following status epilepticus has been implicated in the development of epilepsy and the evolution of brain injury, yet its precise role remains unclear. The development of non-invasive imaging markers of inflammation would enable researchers to test this hypothesis in vivo and study its temporal progression in relation to epileptogenic insults. In this study we have investigated the potential of a targeted magnetic resonance imaging contrast agent--vascular cell adhesion molecule 1 antibody labelled iron oxide--to image the inflammatory process following status epilepticus in the rat lithium-pilocarpine model. Intravascular administration of the targeted contrast agent was performed at approximately 1 day following status epilepticus. The control group received diazepam prior to pilocarpine to prevent status epilepticus. Magnetic resonance imaging of rats was performed before and after contrast administration. Comparison with quantitative T2 measurements was also performed. At the end of the study, brains were removed for ex vivo magnetic resonance imaging and histology. Marked focal hypointensities caused by contrast agent binding were observed on in vivo magnetic resonance images in the post status epilepticus group. In particular these occurred in the periventricular organs, the hippocampus and the cerebral cortex. Relatively little contrast agent binding was observed in the control group. T2 relaxation times were not significantly increased for the hippocampus or the cerebral cortex in post status epilepticus animals. These results demonstrate the feasibility of in vivo imaging of seizure-induced inflammation in an animal model of epilepsy. The antibody targeted MRI contrast agent identified regions of acute inflammation following status epilepticus and may provide an early marker of brain injury. This technique could be used to determine the role of inflammation in models of epileptogenesis and to study the potential for anti-inflammatory therapeutic interventions.

Structural correlates of active-staining following magnetic resonance microscopy in the mouse brain.

Extensive worldwide efforts are underway to produce knockout mice for each of the ~25,000 mouse genes, which may give new insights into the underlying pathophysiology of neurological disease. Microscopic magnetic resonance imaging (µMRI) is a key method for non-invasive morphological phenotyping, capable of producing high-resolution 3D images of ex-vivo brains, after fixation with an MR contrast agent. These agents have been suggested to act as active-stains, enhancing structures not normally visible on MRI. In this study, we investigated the structural correlates of the MRI agent Gd-DTPA, together with the optimal preparation and scan parameters for contrast-enhanced gradient-echo imaging of the mouse brain. We observed that in-situ preparation was preferential to ex-situ due to the degree of extraction damage. In-situ brains scanned with optimised parameters, enabled images with a high signal-to-noise-ratio (SNR ~30) and comprehensive anatomical delineation. Direct correlation of the MR brain structures to histology, detailed fine histoarchitecture in the cortex, cerebellum, olfactory bulb and hippocampus. Neurofilament staining demonstrated that regions of negative MR contrast strongly correlated to myelinated white-matter structures, whilst structures of more positive MR contrast corresponded to areas with high grey matter content. We were able to identify many sub-regions, particularly within the hippocampus, such as the unmyelinated mossy fibres (stratum lucidum) and their region of synapse in the stratum pyramidale, together with the granular layer of the dentate gyrus, an area of densely packed cell bodies, which was clearly visible as a region of hyperintensity. This suggests that cellular structure influences the site-specific distribution of the MR contrast agent, resulting in local variations in T(2)*, which leads to enhanced tissue discrimination. Our findings provide insights not only into the cellular distribution and mechanism of MR active-staining, but also allow for three dimensional analysis, which enables interpretation of magnetic resonance microscopy brain data and highlights cellular structure for investigation of disease processes in development and disease.

Quantitative MRI predicts status epilepticus-induced hippocampal injury in the lithium-pilocarpine rat model.

Convulsive status epilepticus (SE) is a common medical neurological emergency and is associated with hippocampal injury and the subsequent development of epilepsy. However, pathophysiological mechanisms that underlie injury remain unclear, and a clinically useful prognostic biomarker of at-risk patients remains elusive. We hypothesised that non-invasive quantitative multi-parametric MRI characterisation of the early time course in the lithium-pilocarpine rat model would provide insight into pathophysiological processes, and may help to develop a non-invasive prognostic marker of hippocampal injury. T(1), T(2), apparent diffusion coefficient (ADC), and cerebral blood flow (CBF) were measured before and after SE on days 0, 1, 2, 3, 7, 14 and 21. Hippocampal volume measurements were used to assess final structural outcome. MRI changes were found in the parietal cortex, hippocampus, piriform cortex, and thalamus. Each of the regions displayed time-dependent changes, and returned to baseline levels by Day 7. Hippocampal measurements peaked on Day 2, and further analysis revealed that the magnitude of these peak changes was predictive of the hippocampal volumes on Day 21. This time course is consistent with cell death and an inflammatory process. The maximal changes provide a potential clinically useful prognostic marker of final hippocampal volume.

Characterizing the origin of the arterial spin labelling signal in MRI using a multiecho acquisition approach.

Arterial spin labelling (ASL) can noninvasively isolate the MR signal from arterial blood water that has flowed into the brain. In gray matter, the labelled bolus is dispersed within three main compartments during image acquisition: the intravascular compartment; intracellular tissue space; and the extracellular tissue space. Changes in the relative volumes of the extracellular and intracellular tissue space are thought to occur in many pathologic conditions such as stroke and brain tumors. Accurate measurement of the distribution of the ASL signal within these three compartments will yield better understanding of the time course of blood delivery and exchange, and may have particular application in animal models of disease to investigate the relationship between the source of the ASL signal and pathology. In this study, we sample the transverse relaxation of the ASL perfusion weighted and control images acquired with and without vascular crusher gradients at a range of postlabelling delays and tagging durations, to estimate the tricompartmental distribution of labelled water in the rat cortex. Our results provide evidence for rapid exchange of labelled blood water into the intracellular space relative to the transit time through the vascular bed, and provide a more solid foundation for cerebral blood flow quantification using ASL techniques.