High-resolution Functional Mapping Research Articles

Investigating Intra-Individual Networks of Response Inhibition and Interference Resolution using 7T MRI

Response inhibition and interference resolution are often considered subcomponents of an overarching inhibition system that utilizes the so-called cortico-basal-ganglia loop. Up until now, most previous functional magnetic resonance imaging (fMRI) literature has compared the two using between-subject designs, pooling data in the form of a meta-analysis or comparing different groups. Here, we investigate the overlap of activation patterns underlying response inhibition and interference resolution on a within-subject level, using ultra-high field MRI. In this model-based study, we furthered the functional analysis with cognitive modelling techniques to provide a more in-depth understanding of behaviour. We applied the stop-signal task and multi-source interference task to measure response inhibition and interference resolution, respectively. Our results lead us to conclude that these constructs are rooted in anatomically distinct brain areas and provide little evidence for spatial overlap. Across the two tasks, common BOLD responses were observed in the inferior frontal gyrus and anterior insula. Interference resolution relied more heavily on subcortical components, specifically nodes of the commonly referred to indirect and hyperdirect pathways, as well as the anterior cingulate cortex, and pre-supplementary motor area. Our data indicated that orbitofrontal cortex activation is specific to response inhibition. Our model-based approach provided evidence for the dissimilarity in behavioural dynamics between the two tasks. The current work exemplifies the importance of reducing inter-individual variance when comparing network patterns and the value of UHF-MRI for high resolution functional mapping.

Combining mGRASP and Optogenetics Enables High-Resolution Functional Mapping of Descending Cortical Projections

SummaryWe have applied optogenetics and mGRASP, a light microscopy technique that labels synaptic contacts, to map the number and strength of defined corticocollicular (CC) connections. Using mGRASP, we show that CC projections form small, medium, and large synapses, and both the number and the distribution of synapse size vary among the IC regions. Using optogenetics, we show that low-frequency stimulation of CC axons expressing channelrhodopsin produces prolonged elevations of the CC miniature EPSC (mEPSC) rate. Functional analysis of CC mEPSCs reveals small-, medium-, and large-amplitude events that mirror the synaptic distributions observed with mGRASP. Our results reveal that descending ipsilateral projections dominate CC feedback via an increased number of large synaptic contacts, especially onto the soma of IC neurons. This study highlights the feasibility of combining microscopy (i.e., mGRASP) and optogenetics to reveal synaptic weighting of defined projections at the level of single neurons, enabling functional connectomic mapping in diverse neural circuits.

A transparent epidural electrode array for use in conjunction with optical imaging

BackgroundCombining optical imaging and direct cortical stimulation can be a powerful technique for high-resolution functional mapping of the cortex. However, stimulating electrodes often obstruct the field of view, resulting in a lack of functional images behind the electrodes. New methodTo overcome this problem, we developed a transparent electrode array with 16-contact grids for epidural cortical stimulation. Using a commercially available indium tin oxide (ITO)-coated polyethylene terephthalate (PET) sheet, the electrode array was fabricated using a photolithography process. Because a complete circuit pattern, including the electrode contact itself, was formed in the transparent metal ITO layer, our electrode array was completely transparent and therefore could be used with optical imaging. ResultsCortical stimulation was performed using the transparent electrode array. Evoked neural activity was successfully monitored through the array using a voltage-sensitive dye and optical imaging. The newly developed electrode array made it possible to detect optical signals from directly below the stimulating electrode. The electrode array could also be used for epidural recording of somatosensory evoked potentials. Comparison with existing methodsA variety of surface electrodes for cortical recording and stimulation exist. However, this study aimed to make electrodes as transparent as possible. We provided a simple and low-cost fabrication process for producing the transparent electrode arrays. ConclusionsOur transparent epidural electrode can be used for both stimulation and recordings without interfering with the detection of optical signals from the underlying cortex.

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

ABSTRACTPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.

Single-cell imaging tools for brain energy metabolism: a review.

Neurophotonics comes to light at a time in which advances in microscopy and improved calcium reporters are paving the way toward high-resolution functional mapping of the brain. This review relates to a parallel revolution in metabolism. We argue that metabolism needs to be approached both in vitro and in vivo, and that it does not just exist as a low-level platform but is also a relevant player in information processing. In recent years, genetically encoded fluorescent nanosensors have been introduced to measure glucose, glutamate, ATP, NADH, lactate, and pyruvate in mammalian cells. Reporting relative metabolite levels, absolute concentrations, and metabolic fluxes, these sensors are instrumental for the discovery of new molecular mechanisms. Sensors continue to be developed, which together with a continued improvement in protein expression strategies and new imaging technologies, herald an exciting era of high-resolution characterization of metabolism in the brain and other organs.

Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

High resolution T2*-weighted Magnetic Resonance Imaging at 3 Tesla using PROPELLER-EPI

We report the application of PROPELLER-EPI for high resolution T2*-weighted imaging with sub-millimeter in-plane resolution on a clinical 3 Tesla scanner. Periodically rotated blades of a long-axis PROPELLER-EPI sequence were acquired with fast gradient echo readout and acquisition matrix of 320×50 per blade. Images were reconstructed by using 2D-gridding, phase and geometric distortion correction and compensation of resonance frequency drifts that occurred during extended measurements. To characterize these resonance frequency offsets, short FID calibration measurements were added to the PROPELLER-EPI sequence. Functional PROPELLER-EPI was performed with volunteers using a simple block design of right handed finger tapping. Results indicate that PROPELLER-EPI can be employed for fast, high resolution T2*-weighted imaging provided geometric distortions and possible resonance frequency drifts are properly corrected. Even small resonance frequency drifts below 10Hz as well as non-corrected geometric distortions degraded image quality substantially. In the initial fMRI experiment image quality and signal-to-noise ratio was sufficient for obtaining high resolution functional activation maps.

Spatiotemporal characteristics and vascular sources of neural-specific and -nonspecific fMRI signals at submillimeter columnar resolution

The neural specificity of hemodynamic-based functional magnetic resonance imaging (fMRI) signals is dependent on both the vascular regulation and the sensitivity of the applied fMRI technique to different types and sizes of blood vessels. In order to examine the specificity of MRI-detectable hemodynamic responses, submillimeter blood oxygenation level-dependent (BOLD) and cerebral blood volume (CBV) fMRI studies were performed in a well-established cat orientation column model at 9.4 T. Neural-nonspecific and -specific signals were separated by comparing the fMRI responses of orthogonal orientation stimuli. The BOLD response was dominantly neural-nonspecific, mostly originating from pial and intracortical emerging veins, and thus was highly correlated with baseline blood volume. Uneven baseline CBV may displace or distort small functional domains in high-resolution BOLD maps. The CBV response in the parenchyma exhibited dual spatiotemporal characteristics, a fast and early neural-nonspecific response (with 4.3-s time constant) and a slightly slower and delayed neural-specific response (with 9.4-s time constant). The nonspecific CBV signal originates from early-responding arteries and arterioles, while the specific CBV response, which is not correlated with baseline blood volume, arises from late-responding microvessels including small pre-capillary arterioles and capillaries. Our data indicate that although the neural specificity of CBV fMRI signals is dependent on stimulation duration, high-resolution functional maps can be obtained from steady-state CBV studies.

Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription.

High-resolution functional magnetic resonance imaging mapping of noxious heat and tactile activations along the central sulcus in New World monkeys

This study mapped the fine-scale functional representation of tactile and noxious heat stimuli in cortical areas around the central sulcus of anesthetized squirrel monkeys by using high-resolution blood oxygen level-dependent (BOLD) fMRI at 9.4T. Noxious heat (47.5°C) stimulation of digits evoked multiple spatially distinct and focal BOLD activations. Consistent activations were observed in areas 3a, 3b, 1, and 2, whereas less frequent activation was present in M1. Compared with tactile activations, thermal nociceptive activations covered more area and formed multiple foci within each functional area. In general, noxious heat activations in area 3b did not colocalize with tactile responses. The spatial relationships of heat and tactile activations in areas 3a and 1/2 varied across animals. Subsequent electrophysiological mapping confirmed that the evoked heat and tactile BOLD signals were somatotopically appropriate. The magnitude and temporal profiles of the BOLD signals to noxious heat stimuli differed across cortical areas. Comparatively late-peaking but stronger signals were observed in areas 3b and 2, whereas earlier-peaking but weaker signals were observed in areas 3a, 1, and M1. In sum, this study not only confirmed the involvement of somatosensory areas of 3a, 3b, and 1, but also identified the engagements of area 2 and M1 in the processing of heat nociceptive inputs. Differential BOLD response profiles of the individual cortical areas along the central sulcus suggest that these areas play different roles in the encoding of nociceptive inputs. Thermal nociceptive and tactile inputs may be processed by different clusters of neurons in different areas.To critically bridge animal and human pain studies, human fMRI was related to primate fMRI and electrophysiology of nociceptive processing, examining the functional role of the primary somatosensory cortex in heat nociception and demonstrating that subregion areas 3a, 3b, 1, 2, and M1 are responsive to noxious heat stimuli.

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.

Two-photon uncaging of γ-aminobutyric acid in intact brain tissue

We have synthesized a photosensitive (or caged) 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI) derivative of gamma-aminobutyric acid (GABA). Two-photon excitation of CDNI-GABA produced rapid activation of GABAergic currents in neurons in brain slices with an axial resolution of approximately 2 mum and enabled high-resolution functional mapping of GABA-A receptors. Two-photon uncaging of GABA, the main inhibitory neurotransmitter, should allow detailed studies of receptor function and synaptic integration with subcellular precision.

Removal of spatial biological artifacts in functional maps by local similarity minimization

Functional maps obtained by various technologies, including optical imaging techniques, f-MRI, PET, and others, may be contaminated with biological artifacts such as vascular patterns or large patches of parenchyma. These artifacts originate mostly from changes in the microcirculation that result from either activity-dependent changes in volume or from oximetric changes that do not co-localize with neuronal activity per se. Standard methods do not always suffice to reduce such artifacts, in which case conspicuous spatial artifacts mask details of the underlying activity patterns. Here we propose a simple algorithm that efficiently removes spatial biological artifacts contaminating high-resolution functional maps. We validated this procedure by applying it to cortical maps resulting from optical imaging, based either on voltage-sensitive dye signals or on intrinsic signals. To remove vascular spatial patterns we first constructed a template of typical artifacts (vascular/cardiac pulsation/vasomotion), using principle components derived from baseline information obtained in the absence of stimulation. Next, we modified this template by means of local similarity minimization (LSM), achieved by measuring neighborhood similarity between contaminated data and the artifact template and then abolishing the similarity. LSM thus removed spatial patterns originating from the cortical vasculature components, including large fields of capillary parenchyma, helping to unveil details of neuronal activity patterns that were otherwise masked by these vascular artifacts. Examples obtained from our imaging experiments with anaesthetized cats and behaving monkeys showed that the LSM method is both general and reproducible, and is often superior to other available procedures.

The brain in the age of old: The hippocampal formation is targeted differentially by diseases of late life

To rely on the anatomical organization of the hippocampal formation in understanding whether and how late-life diseases such as diabetes and stroke contribute to age-related cognitive decline. Magnetic resonance imaging (MRI) was used to document brain infarcts and to generate high-resolution functional maps of the hippocampal formation in 240 community-based nondemented elders (mean age, 79.7 years) who received a comprehensive medical evaluation. Sixty participants had type 2 diabetes mellitus, whereas 74 had MRI-documented brain infarcts, and the first analysis was designed to pinpoint hippocampal subregions differentially linked to each disorder. Then, guided by the results, additional functional MRI studies in aging rhesus monkeys and mice were used to test proposed mechanisms of dysfunction. Although both diabetes and brain infarcts were associated with hippocampal dysfunction, each was linked to separate hippocampal subregions, suggesting distinct underlying mechanisms. The hippocampal subregion linked to diabetes implicated blood glucose as a pathogenic mechanism, a hypothesis confirmed by imaging aging rhesus monkeys and a mouse model of diabetes. The hippocampal subregion linked to infarcts suggested transient hypoperfusion as a pathogenic mechanism, a hypothesis provisionally confirmed by comparing anatomical patterns across subjects with infarcts in different vascular territories. Taken together with previous findings, these results clarify how diseases of late life differentially target the hippocampal formation, identify elevations in blood glucose as a contributing cause of age-related memory decline, and suggest specific interventions that can preserve cognitive health.

High-resolution functional brain mapping with near infrared spectroscopy (NIRS)

High-resolution functional brain mapping with near infrared spectroscopy (NIRS)

Nonnvasive assessment of vascular architecture and function during modulated blood oxygenation using susceptibility weighted magnetic resonance imaging

Susceptibility weighted imaging (SWI) is a BOLD-sensitive method for visualizing anatomical features such as small cerebral veins in high detail. The purpose of this study was to evaluate high-resolution SWI in combination with a modulation of blood oxygenation by breathing of air, carbogen, and oxygen and to directly visualize the effects of changing blood oxygenation on the magnetic field inside and around venous blood vessels. Signal changes associated with the response to carbogen and oxygen breathing were evaluated in different anatomic regions in healthy volunteers and in two patients with brain tumors. In the magnitude images inhalation of carbogen led to significant signal intensity changes ranging from +4.4 +/- 1.9% to +9.5 +/- 1.4% in gray matter and no significant changes in thalamus, putamen, and white matter. During oxygen breathing mean signal changes were smaller than during carbogen breathing. The method is capable of producing high-resolution functional maps of BOLD response to carbogen and oxygen breathing as well as high-resolution images of venous vasculature. Its sensitivity to changes in blood oxygenation was demonstrated by in vivo visualization of the BOLD effect via phase imaging.

What Has Intrinsic Signal Optical Imaging Taught Us About NGF-Induced Rapid Plasticity in Adult Cortex and Its Relationship to the Cholinergic System?

Intrinsic signal optical imaging (ISI) is a high-resolution functional brain mapping technique that is being used to further our understanding of the neocortex and its interaction with drugs. Recent studies using combination ISI and in vivo pharmacology have advanced our insight into the actions of both acetylcholine and neurotrophins on inducing rapid and large-scale cortical plasticity. In particular, it appears that acetylcholine (ACh), nicotinic ACh receptors, nerve growth factor (NGF), and NGF receptors (TrkA and p75) are involved in an important feedback loop between the basal forebrain cholinergic system (BFCS) and the neocortex. Specifically, recent data suggest that NGF expressed in the cortex may act on multiple time scales on the BFCS: acutely to increase BFCS release of acetylcholine, intermediately to induce sprouting of BFCS axons, and long-term to change gene expression of BFCS neurons. In this article, advances in understanding the links in vivo between the BFCS, neocortex, nicotinic ACh receptors, and NGF are reviewed.

Special Report: Noninvasive Multi-Parameter Functional Optical Imaging of the Eye

<p>Advancement in the treatment of blindness depends on the development of new technologies that enable early detection, follow-up, and treatment of disease. The authors describe direct, noninvasive imaging of four parameters: blood flow, blood oximetry, metabolic state, and hidden vasculature, particularly capillaries. These are functional parameters of the retina known to be degraded by retinal disease. The new Retinal Function Imager (Optical Imaging, Ltd., Rehovot, Israel) can image all four parameters as intrinsic reflectance intensity differences over the retina's surface. During the past 2 decades, imaging of small optical signals has been a powerful tool for high-resolution functional mapping in the neocortex. In this article, this technology is applied to the retina and demonstrates a general tool for noninvasively probing retinal function in many modalities. Imaging functional changes before anatomic consequences arise holds promise as a powerful tool for early diagnosis and treatment of retinal disease.</p><p><b>[Ophthalmic Surg Lasers Imaging 2005;36:57–66.]</b></p>

Columnar Resolution of Blood Volume and Oximetry Functional Maps in the Behaving Monkey: Implications for fMRI

The ultimate goal of high-resolution functional brain mapping is single-condition (stimulus versus no-stimulus maps) rather than differential imaging (comparing two “stimulus maps”), because the appropriate (“orthogonal”) stimuli are rarely available. This requires some component(s) of activity-dependent hemodynamic signals to closely colocalize with electrical activity, like the early increase in deoxyhemoglobin, shown previously to yield high-quality functional single-condition maps. Conversely, nonlocal vascular responses dominate in cerebral blood volume (CBV)-based single-condition maps. Differential CBV maps are largely restricted to the parenchyma, implying that part of the CBV response does colocalize with electrical activity at fine spatial scale. By removing surface vascular activation from optical imaging data, we document the existence of a capillary CBV response component, regulated at fine spatial scale and yielding single-condition maps exhibiting ∼100 μm resolution. Blood volume and -flow based single-condition functional mapping at columnar level should thus be feasible, provided that the capillary response component is selectively imaged.

The high-resolution functional map of bacteriophage SPP1 portal protein.

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.