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Background: It has been reported that the S1P (sphingosine 1-phosphate) receptor modulator fingolimod reduces infarction in rodent models of stroke. Recent studies have suggested that circadian rhythms affect stroke and neuroprotection. Therefore, this study revisited the use of fingolimod in mouse focal cerebral ischemia to test the hypothesis that efficacy might depend on whether experiments were performed during the inactive sleep or active wake phases of the circadian cycle. Methods: Two different stroke models were implemented in male C57Bl/6 mice—transient middle cerebral artery occlusion and permanent distal middle cerebral artery occlusion. Occlusion occurred either during inactive or active circadian phases. Mice were treated with 1 mg/kg fingolimod at 30- or 60-minute postocclusion and 1 day later for permanent and transient middle cerebral artery occlusion, respectively. Infarct volume, brain swelling, hemorrhagic transformation, and behavioral outcome were assessed at 2 or 3 days poststroke. Three independent experiments were performed in 2 different laboratories. Results: Fingolimod decreased peripheral lymphocyte number in naive mice, as expected. However, it did not significantly affect infarct volume, brain swelling, hemorrhagic transformation, or behavioral outcome at 2 or 3 days after transient or permanent focal cerebral ischemia during inactive or active circadian phases of stroke onset. Conclusions: Outcomes were not improved by fingolimod in either transient or permanent focal cerebral ischemia during both active and inactive circadian phases. These negative findings suggest that further testing of fingolimod in clinical trials may not be warranted unless translational studies can identify factors associated with fingolimod’s efficacy or lack thereof.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

We report a novel forward-model implementation of the full reference tissue model (fFTRM) that addresses the fast-exchange approximation employed by the simplified reference tissue model (SRTM) by incorporating a non-zero dissociation time constant from the specifically bound compartment. The forward computational approach avoided errors associated with noisy and nonorthogonal basis functions using an inverse linear model. Compared to analysis by a multilinear single-compartment reference tissue model (MRTM), fFTRM provided improved accuracy for estimation of binding potentials at early times in the scan, with no worse reproducibility across sessions. To test the model's ability to identify small focal changes in binding potential using a within-scan challenge, we employed a nonhuman primate model of focal dopamine release elicited by deep brain microstimulation remote to ventral striatum (VST) during imaging by simultaneous PET and fMRI. The new model reported an unambiguously lateralized response in VST consistent with fMRI, whereas the MRTM-derived response was not lateralized and was consistent with simulations of model bias. The proposed model enabled better accuracy in PET [11C]raclopride displacement studies and may also facilitate challenges sooner after injection, thereby recovering some sensitivity lost to radioactive decay of the PET tracer. ispartof: JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM vol:42 issue:6 pages:1007-1019 ispartof: location:United States status: published

The dopamine system plays a significant role in drug reward and the pathogenesis of addiction. Psychostimulant drugs acutely increase dopamine levels, triggering receptor internalization. In vitro data suggest that dopamine D1 receptors (D1R) recycle, whereas D2 receptors (D2R) degrade in response to activation. Yet, receptor fates in vivo remain unclear. This study bridges in vitro mechanisms and in vivo measurements of stimulant-induced modulation of receptor states using longitudinal multi-modal imaging combined with neuropharmacology. We demonstrate how repeated amphetamine administration differentially modulates D1R vs. D2R signaling in nonhuman primates over 24 hours using simultaneous positron emission tomography and functional magnetic resonance imaging. In contrast to predominantly inhibitory D2R signaling due to an initial amphetamine challenge, excitatory D1R functional signaling prevails three hours later, while D2Rs stay internalized. These results demonstrate differential externalization mechanisms of the D1R and D2R in vivo and a shift in receptor subtype activation after a dopamine surge.

Compartmental modeling analysis of 11C-raclopride (RAC) PET data can be used to measure the dopaminergic response to intra-scan behavioral tasks. Bias in estimates of binding potential (BPND) and its dynamic changes (ΔBPND) can arise both when head motion is present and when the compartmental model used for parameter estimation deviates from the underlying biology. The purpose of this study was to characterize the effects of motion and model bias within the context of a behavioral task challenge, examining the impacts of different mitigation strategies. Seventy healthy adults were administered bolus plus constant infusion RAC during a simultaneous PET/magnetic resonance (MR) scan with a reward task experiment. BPND and ΔBPND were estimated using an extension of the Multilinear Reference Tissue Model (E-MRTM2) and a new method (DE-MRTM2) was proposed to selectively discount the contribution of the initial uptake period. Motion was effectively corrected with a standard frame-based approach, which performed equivalently to a more complex reconstruction-based approach. DE-MRTM2 produced estimates of ΔBPND in putamen and nucleus accumbens that were significantly different from those estimated from E-MRTM2, while also decoupling ΔBPND values from first-pass k2′ estimation and removing skew in the spatial bias distribution of parametric ΔBPND estimates within the striatum.

Background Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. Methods We demonstrated blood oxygenation level–dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level–dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. Results The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. Conclusions This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2–4, but have little effect on the brains of mice5–9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12–14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25–40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of ‘cortical units’ has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12–16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

The anterior limb of the internal capsule (ALIC) carries thalamic and brainstem fibers from prefrontal cortical regions that are associated with different aspects of emotion, motivation, cognition processing, and decision-making. This large fiber bundle is abnormal in several psychiatric illnesses and a major target for deep brain stimulation. Yet, we have very little information about where specific prefrontal fibers travel within the bundle. Using a combination of tracing studies and diffusion MRI in male nonhuman primates, as well as diffusion MRI in male and female human subjects, we segmented the human ALIC into five regions based on the positions of axons from different cortical regions within the capsule. Fractional anisotropy (FA) abnormalities in patients with bipolar disorder were detected when FA was averaged in the ALIC segment that carries ventrolateral prefrontal cortical connections. Together, the results set the stage for linking abnormalities within the ALIC to specific connections and demonstrate the utility of applying connectivity profiles of large white matter bundles based on animal anatomic studies to human connections and associating disease abnormalities in those pathways with specific connections. The ability to functionally segment large white matter bundles into their components begins a new era of refining how we think about white matter organization and use that information in understanding abnormalities.SIGNIFICANCE STATEMENTThe anterior limb of the internal capsule (ALIC) connects prefrontal cortex with the thalamus and brainstem and is abnormal in psychiatric illnesses. However, we know little about the location of specific prefrontal fibers within the bundle. Using a combination of animal tracing studies and diffusion MRI in animals and human subjects, we segmented the human ALIC into five regions based on the positions of axons from different cortical regions. We then demonstrated that differences in FA values between bipolar disorder patients and healthy control subjects were specific to a given segment. Together, the results set the stage for linking abnormalities within the ALIC to specific connections and for refining how we think about white matter organization in general.

The potential effects of changes in blood flow on the delivery and washout of radiotracers has been an ongoing question in PET bolus injection studies. This study provides practical insight into this topic by experimentally measuring cerebral blood flow (CBF) and neuroreceptor binding using simultaneous PET/MRI. Hypercapnic challenges (7% CO2) were administered to non-human primates in order to induce controlled increases in CBF, measured with pseudo-continuous arterial spin labeling. Simultaneously, dopamine D2/D3 receptor binding of [11C]raclopride or [18F]fallypride was monitored with dynamic PET. Experiments showed that neither time activity curves nor quantification of binding through binding potentials ( BPND) were measurably affected by CBF increases, which were larger than two-fold. Simulations of experimental procedures showed that even large changes in CBF should have little effect on the time activity curves of radiotracers, given a set of realistic assumptions. The proposed method can be applied to experimentally assess the flow sensitivity of other radiotracers. Results demonstrate that CBF changes, which often occur due to behavioral tasks or pharmacological challenges, do not affect PET [11C]raclopride or [18F]fallypride binding studies and their quantification. The results from this study suggest flow effects may have limited impact on many PET neuroreceptor tracers with similar properties.

The full reference tissue model (FRTM) is a PET analysis framework that includes both free and specifically bound compartments within tissues, together with rate constants defining association and dissociation from the specifically bound compartment. The simplified reference tissue model (SRTM) assumes instantaneous exchange between tissue compartments, and this “1-tissue” approximation reduces the number of parameters and enables more robust mapping of non-displaceable binding potentials. Simulations based upon FRTM have shown that SRTM exhibits biases that are spatially dependent, because biases depend upon binding potentials. In this work, we describe a regularized model (rFRTM) that employs a global estimate of the dissociation rate constant from the specifically bound compartment (k4). The model provides an internal calibration for optimizing k4 through the reference-region outflow rate k2′, a model parameter that should be a global constant but varies regionally in SRTM. Estimates of k4 by rFRTM are presented for four PET radioligands. We show that SRTM introduces bias in parameter estimates by assuming an infinite value for k4, and that rFRTM ameliorates bias with an appropriate choice of k4. Theoretical considerations and simulations demonstrate that rFRTM reduces bias in non-displaceable binding potentials. A two-parameter reduction of the model (rFRTM2) provides robust mapping at a voxel-wise level. With a structure similar to SRTM, the model is easily implemented and can be applied as a PET reference region analysis that reduces parameter bias without substantially altering parameter variance.

Multiparametric magnetic resonance imaging (MRI) has become a critical clinical tool for diagnosing focal ischemic stroke severity, staging treatment, and predicting outcome. Imaging during the acute phase focuses on tissue viability in the stroke vicinity, while imaging during recovery requires the evaluation of distributed structural and functional connectivity. Preclinical MRI of experimental stroke models provides validation of non-invasive biomarkers in terms of cellular and molecular mechanisms, while also providing a translational platform for evaluation of prospective therapies. This brief review of translational stroke imaging discusses the acute to chronic imaging transition, the principles underlying common MRI methods employed in stroke research, and the experimental results obtained by clinical and preclinical imaging to determine tissue viability, vascular remodeling, structural connectivity of major white matter tracts, and functional connectivity using task-based and resting-state fMRI during the stroke recovery process.

Magnetic Particle Imaging (MPI) is a rapidly developing imaging modality that directly measures and maps the concentration of injected superparamagnetic iron oxide nanoparticles (SPIOs). Since the agent does not cross the blood-brain barrier, cerebral SPIO concentration provides a direct probe of Cerebral Blood Volume (CBV). Here we provide an initial demonstration of the ability of MPI to detect functional CBV changes (fCBV) by monitoring SPIO concentration during hypercapnic manipulation in a rat model. As a tracer detection method, MPI offers a more direct probe of agent concentration and therefore fCBV than MRI measurements in which the agent is indirectly detected through perturbation of water relaxation time constants such as T2∗. We found that MPI detection could measure CBV changes during hypercapnia with high CNR (CNR = 50) and potentially with high temporal resolution. Although the detection process more closely resembles a tracer method, we also identify evidence of physiological noise in the MPI time-series, with higher time-series variance at higher concentration levels. Our findings suggest that CBV-based MPI can provide a detection modality for hemodynamic changes. Further investigation with tomographic imaging is needed to assess tomographic ability of the method and further study the presence of time-series fluctuations which scale with signal level similar to physiological noise in resting fMRI time-courses.

In the present study, we used a simultaneous PET-MR experimental design to investigate the effects of functionally different compounds (agonist, partial agonist, and antagonist) on 5-HT1Breceptor (5-HT1BR) occupancy and the associated hemodynamic responses. In anesthetized male nonhuman primates (n= 3), we used positron emission tomography (PET) imaging with the radioligand [11C]AZ10419369 administered as a bolus followed by constant infusion to measure changes in 5-HT1BR occupancy. Simultaneously, we measured changes in cerebral blood volume (CBV) as a proxy of drug effects on neuronal activity. The 5-HT1BR partial agonist AZ10419369 elicited a dose-dependent biphasic hemodynamic response that was related to the 5-HT1BR occupancy. The magnitude of the response was spatially overlapping with high cerebral 5-HT1BR densities. High doses of AZ10419369 exerted an extracranial tissue vasoconstriction that was comparable to the less blood–brain barrier-permeable 5-HT1BR agonist sumatriptan. By contrast, injection of the antagonist GR127935 did not elicit significant hemodynamic responses, even at a 5-HT1BR cerebral occupancy similar to the one obtained with a high dose of AZ10419369. Given the knowledge we have of the 5-HT1BR and its function and distribution in the brain, the hemodynamic response informs us about the functionality of the given drug: changes in CBV are only produced when the receptor is stimulated by the partial agonist AZ10419369 and not by the antagonist GR127935, consistent with low basal occupancy by endogenous serotonin.SIGNIFICANCE STATEMENTWe here show that combined simultaneous positron emission tomography and magnetic resonance imaging uniquely enables the assessment of CNS active compounds. We conducted a series of pharmacological interventions to interrogate 5-HT1Breceptor binding and function and determined blood–brain barrier passage of drugs and demonstrate target involvement. Importantly, we show how the spatial and temporal effects on brain hemodynamics provide information about pharmacologically driven downstream CNS drug effects; the brain hemodynamic response shows characteristic dose-related effects that differ depending on agonistic or antagonistic drug characteristics and on local 5-HT1Breceptor density. The technique lends itself to a comprehensivein vivoinvestigation and understanding of drugs' effects in the brain.

Glucose is the principal source of energy for the brain and yet the dynamic response of glucose utilization to changes in brain activity is still not fully understood. Positron emission tomography (PET) allows quantitative measurement of glucose metabolism using 2-[(18)F]-fluorodeoxyglucose (FDG). However, FDG PET in its current form provides an integral (or average) of glucose consumption over tens of minutes and lacks the temporal information to capture physiological alterations associated with changes in brain activity induced by tasks or drug challenges. Traditionally, changes in glucose utilization are inferred by comparing two separate scans, which significantly limits the utility of the method. We report a novel method to track changes in FDG metabolism dynamically, with higher temporal resolution than exists to date and within a single session. Using a constant infusion of FDG, we demonstrate that our technique (termed fPET-FDG) can be used in an analysis pipeline similar to fMRI to define within-session differential metabolic responses. We use visual stimulation to demonstrate the feasibility of this method. This new method has a great potential to be used in research protocols and clinical settings since fPET-FDG imaging can be performed with most PET scanners and data acquisition and analysis are straightforward. fPET-FDG is a highly complementary technique to MRI and provides a rich new way to observe functional changes in brain metabolism.

Although functional MRI traditionally has been applied mainly to study changes in task-induced brain function, evolving acquisition methodologies and improved knowledge of signal mechanisms have increased the utility of this method for studying responses to pharmacological stimuli, a technique often dubbed “phMRI”. The proliferation of higher magnetic field strengths and the use of exogenous contrast agent have boosted detection power, a critical factor for successful phMRI due to the restricted ability to average multiple stimuli within subjects. Receptor-based models of neurovascular coupling, including explicit pharmacological models incorporating receptor densities and affinities and data-driven models that incorporate weak biophysical constraints, have demonstrated compelling descriptions of phMRI signal induced by dopaminergic stimuli. This report describes phMRI acquisition and analysis methodologies, with an emphasis on data-driven analyses. As an example application, statistically efficient data-driven regressors were used to describe the biphasic response to the mu-opioid agonist remifentanil, and antagonism using dopaminergic and GABAergic ligands revealed modulation of the mesolimbic pathway. Results illustrate the power of phMRI as well as our incomplete understanding of mechanisms underlying the signal. Future directions are discussed for phMRI acquisitions in human studies, for evolving analysis methodologies, and for interpretative studies using the new generation of simultaneous PET/MRI scanners. This article is part of the Special Issue Section entitled ‘Neuroimaging in Neuropharmacology’.

Background and Purpose— Small subcortical white matter infarcts are a common stroke subtype often associated with cognitive deficits. The lack of relevant models confined to white matter has limited the investigation of its pathophysiology. Here, we examine tissue and functional outcome after an ischemic lesion within corpus callosum in wild-type (WT) mice and in mice null for a gene, NOTCH3 , linked to white matter ischemic injury in patients. Methods— WT and NOTCH3 knockout mice were subjected to stereotactic microinjections of the potent vasoconstrictor endothelin-1 at the level of periventricular white matter to induce a focal ischemic lesion. Infarct location was confirmed by MRI, and brains were examined for lesion size and histology; behavioral deficits were assessed ≤1 month in WT mice. Results— Ischemic damage featured an early cerebral blood flow deficit, blood–brain barrier opening, and a lesion largely confined to white matter. At later stages, myelin and axonal degeneration and microglial/macrophage infiltration were found. WT mice displayed prolonged cognitive deficit when tested using a novel object recognition task. NOTCH3 mutants showed larger infarcts and greater cognitive deficit at 7 days post stroke. Conclusions— Taken together, these data show the usefulness of microinjections of endothelin-1 into periventricular white matter to study focal infarcts and cognitive deficit in WT mice. In short-term studies, stroke outcome was worse in NOTCH3 null mice, consistent with the notion that the lack of the NOTCH3 receptor affects white matter stroke susceptibility.

Background: Therapeutic hypothermia (TH) improves neurological outcomes after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Although nitric oxide prevents organ injury induced by ischemia and reperfusion, role of nitric oxide during TH after CPR remains unclear. In this article, the authors examined the impact of endogenous nitric oxide synthesis on the beneficial effects of hypothermia after CA/CPR. The authors also examined whether or not inhaled nitric oxide during hypothermia further improves outcomes after CA/CPR in mice treated with TH. Methods: Wild-type mice and mice deficient for nitric oxide synthase 3 (NOS3−/−) were subjected to CA at 37°C and then resuscitated with chest compression. Body temperature was maintained at 37°C (normothermia) or reduced to 33°C (TH) for 24 h after resuscitation. Mice breathed air or air mixed with nitric oxide at 10, 20, 40, 60, or 80 ppm during hypothermia. To evaluate brain injury and cerebral blood flow, magnetic resonance imaging was performed in wild-type mice after CA/CPR. Results: Hypothermia up-regulated the NOS3-dependent signaling in the brain (n = 6 to 7). Deficiency of NOS3 abolished the beneficial effects of hypothermia after CA/CPR (n = 5 to 6). Breathing nitric oxide at 40 ppm improved survival rate in hypothermia-treated NOS3−/− mice (n = 6) after CA/CPR compared with NOS3−/− mice that were treated with hypothermia alone (n = 6; P < 0.05). Breathing nitric oxide at 40 (n = 9) or 60 (n = 9) ppm markedly improved survival rates in TH-treated wild-type mice (n = 51) (both P < 0.05 vs. TH-treated wild-type mice). Inhaled nitric oxide during TH (n = 7) prevented brain injury compared with TH alone (n = 7) without affecting cerebral blood flow after CA/CPR (n = 6). Conclusions: NOS3 is required for the beneficial effects of TH. Inhaled nitric oxide during TH remains beneficial and further improves outcomes after CA/CPR. Nitric oxide breathing exerts protective effects after CA/CPR even when TH is ineffective due to impaired endogenous nitric oxide production.

Background and Purpose— White matter injury caused by cerebral hypoperfusion may contribute to the pathophysiology of vascular dementia and stroke, but the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that oxidative stress interferes with endogenous white matter repair by disrupting renewal processes mediated by oligodendrocyte precursor cells (OPCs). Methods— In vitro, primary rat OPCs were exposed to sublethal CoCl 2 for 7 days to induce prolonged chemical hypoxic stress. Then, OPC proliferation/differentiation was assessed. In vivo, prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in mice. Then, reactive oxygen species production, myelin density, oligodendrocyte versus OPC counts, and cognitive function were evaluated. To block oxidative stress, OPCs and mice were treated with the radical scavenger edaravone. Results— Prolonged chemical hypoxic stress suppressed OPC differentiation in vitro. Radical scavenging with edaravone ameliorated these effects. After 28 days of cerebral hypoperfusion in vivo, reactive oxygen species levels were increased in damaged white matter, along with the suppression of OPC-to-oligodendrocyte differentiation and loss of myelin staining. Concomitantly, mice showed functional deficits in working memory. Radical scavenging with edaravone rescued OPC differentiation, ameliorated myelin loss, and restored working memory function. Conclusions— Our proof-of-concept study demonstrates that after prolonged cerebral hypoperfusion, oxidative stress interferes with white matter repair by disrupting OPC renewal mechanisms. Radical scavengers may provide a potential therapeutic approach for white matter injury in vascular dementia and stroke.

Objective Although there is growing awareness of the long-term cognitive effects of repetitive mild traumatic brain injury (rmTBI; eg, sports concussions), whether repeated concussions cause long-term cognitive deficits remains controversial. Moreover, whether cognitive deficits depend on increased amyloid β deposition and tau phosphorylation or are worsened by the apolipoprotein E4 allele remains unknown. Here, we use an experimental model of rmTBI to address these clinical controversies. Methods A weight drop rmTBI model was used that results in cognitive deficits without loss of consciousness, seizures, or gross or microscopic evidence of brain damage. Cognitive function was assessed using a Morris water maze (MWM) paradigm. Immunostaining and enzyme-linked immunosorbent assay (ELISA) were used to assess amyloid β deposition and tau hyperphosphorylation. Brain volume and white matter integrity were assessed by magnetic resonance imaging (MRI). Results Mice subjected to rmTBI daily or weekly but not biweekly or monthly had persistent cognitive deficits as long as 1 year after injuries. Long-term cognitive deficits were associated with increased astrocytosis but not tau phosphorylation or amyloid β (by ELISA); plaques or tangles (by immunohistochemistry); or brain volume loss or changes in white matter integrity (by MRI). APOE4 was not associated with worse MWM performance after rmTBI. Interpretation Within the vulnerable time period between injuries, rmTBI produces long-term cognitive deficits independent of increased amyloid β or tau phosphorylation. In this model, cognitive outcome is not influenced by APOE4 status. The data have implications for the long-term mental health of athletes who suffer multiple concussions. Ann Neurol 2013;74:65–75

The serotonin 5-HT(2c) receptor is implicated in a number of diseases including obesity, depression, anxiety, and schizophrenia. In order to ascribe the role of 5-HT(2c) in these diseases, a method for measuring 5-HT(2c )density and function in vivo, such as with positron emission tomography (PET), must be developed. Many high-affinity and relatively selective ligands exist for 5-HT(2c) but cannot be accessed with current radiosynthetic methods for use as PET radiotracers. We propose that N-methylation of an arylazepine moiety, a frequent structural feature in 5-HT(2c) ligands, may be a suitable method for producing new radiotracers for 5-HT(2c). The impact of N-methylation has not been previously reported. For the agonists that we selected herein, N-methylation was found to increase affinity up to 8-fold without impairing selectivity. Compound 5, an N-methylated azetidine-derived arylazepine, was found to be brain penetrant and reached a brain/blood ratio of 2.05:1. However, our initial test compound was rapidly metabolized within 20 min of administration and exhibited high nonspecific binding. N-Methylation, with 16 ± 3% isolated radiochemical yield (decay corrected), is robust and may facilitate screening other 5-HT(2c) ligands as radiotracers for PET.

SummaryDistinct brain regions, reproducible from one person to the next, are specialized for processing different kinds of human expertise, such as face recognition and reading. Here, we explore the relationship between age of learning, learning ability, and specialized brain structures. Specifically, we ask whether the existence of reproducible cortical domains necessarily means that certain abilities are innate, or innately easily learned, or whether reproducible domains can be formed, or refined, by interactions between genetic programs and common early experience. Functional MRI showed that intensive early, but not late, experience caused the formation of category-selective regions in macaque temporal lobe for stimuli never naturally encountered by monkeys. And behaviorally, early training produced more fluent processing of these stimuli than the same training in adults. One explanation for these results is that in higher cortical areas, as in early sensory areas, experience drives functional clustering and functional clustering determines how that information is processed.

Chronic use of cocaine is associated with lasting alterations in brain metabolism, circuitry and receptor properties. We used neuroimaging with pharmacologic MRI (phMRI) to assess alterations in response to cocaine (0.5mg/kg) in animals trained to self-administer (SA) cocaine on a fixed-ratio 5 schedule of reinforcement, as well as saline-yoked controls, after 28 days of cocaine abstinence. We fit the cerebral blood volume (CBV) curves for full-width half-maximum (FWHM) as well as peak CBV response. There were significant increases in the FWHM of the response curves in the cocaine-SA animals compared to saline-yoked controls in medial-prefrontal cortex (mPFC) and caudate/putamenm (CPu) and increases in peak CBV in M1 motor cortex, CPu and pedunculopontine tegmental nucleus. Functional connectivity analysis showed increased correlations in the SA rats upon acute cocaine challenge, especially in the S1, mPFC, and thalamus. Since D3 receptors are postulated to increase following chronic cocaine administration we also examined the response to 0.2 mg/kg of the D3 preferring agonist 7-OHDPAT. Cocaine SA animals showed a decreased overall CBV response to this drug, except in the globus pallidus. The hypothalamus showed a negative CBV change in response to cocaine challenge similar to that noted with the D3 agonist and showed a smaller response in the cocaine-SA animals than the controls. Given the good coupling of cerebral hemodynamics with dopamine dynamics previously observed with phMRI, these data suggest that increased persistence of dopamine in prefrontal cortex may be responsible for some of the behavioral alterations observed subsequent to chronic cocaine use.

The aim was to investigate the feasibility of making relative cerebral blood flow (rCBF) maps from MR images acquired with short TR by measuring the initial arrival amount of Gd-DTPA evaluated within a time window before any contrast agent has a chance to leave the tissue. We named this rCBF measurement technique utilizing the early data points of the Gd-DTPA bolus the “early time points” method (ET), based on the hypothesis that early time point signals were proportional to rCBF. Simulation data were used successfully to examine the ideal behavior of ET while monkey's MRI results offered encouraging support to the utility of ET for rCBF calculation. A better brain coverage for ET could be obtained by applying the Simultaneous Echo Refocusing (SER) EPI technique. A recipe to run ET was presented, with attention paid to the noise problem around the time of arrival (TOA) of the contrast agent.

Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with “upstream” propagation of vasodilation toward the cortical surface along the diving arterioles and “downstream” propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the “initial dip” was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I.

Dopamine D3 receptors (D3R) may be important therapeutic targets for both drug abuse and dyskinesias in Parkinson’s disease; however, little is known about their functional circuitry. We wished to determine if D3R antagonists SB-277011 and PG-01037 and D3R-preferring agonist 7-OH-DPAT are D3R selective in vivo. We further wished to characterize the response to D3R drugs using whole brain imaging to identify novel D3R circuitry. We investigated D3R circuitry in rats using pharmacologic MRI and challenge with selective D3R antagonists and agonist at various doses to examine regional changes in cerebral blood volume (CBV). We compared regional activation patterns with D2R/D3R agonists, as well as with prior studies of mRNA expression and autoradiography. D3R antagonists induced positive CBV changes and D3R agonist negative CBV changes in brain regions including nucleus accumbens, infralimbic cortex, thalamus, interpeduncular region, hypothalamus, and hippocampus (strongest in subiculum). All D3R-preferring drugs showed markedly greater responses in nucleus accumbens than in caudate/putamen consistent with D3R selectivity and contrary to what was observed with D2R agonists. At high doses of D3R agonist, functional changes were differentiated across cortical laminae, with layer V–VI yielding positive CBV changes and layer IV yielding negative CBV changes. These results are not inconsistent with differential D1R and D3R innervation in these layers respectively showed previously using post-mortem techniques. MRI provides a new tool for testing the in vivo selectivity of novel D3R dopaminergic ligands where radiolabels may not be available. Further, the functional D3R circuitry strongly involves hypothalamus and subiculum as well as the limbic striatum.

We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) forin vivomagnetic resonance imaging (MRI) of fosB and ΔfosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ΔfosB probes was verified byin vitroreverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled ΔfosB probe signal elevation occurred only in chronically AMPH-exposed mice. Thein vivotarget specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ΔfosB mRNA in living brains.

The macaque visual cortex contains >30 different functional visual areas, yet surprisingly little is known about the underlying organizational principles that structure its components into a complete “visual” unit. A recent model of visual cortical organization in humans suggests that visual field maps are organized as clusters. Clusters minimize axonal connections between individual field maps that represent common visual percepts, with different clusters thought to carry out different functions. Experimental support for this hypothesis, however, is lacking in macaques, leaving open the question of whether it is unique to humans or a more general model for primate vision. Here we show, using high-resolution blood oxygen level-dependent functional magnetic resonance imaging data in the awake monkey at 7 T, that the middle temporal area (area MT/V5) and its neighbors are organized as a cluster with a common foveal representation and a circular eccentricity map. This novel view on the functional topography of area MT/V5 and satellites indicates that field map clusters are evolutionarily preserved and may be a fundamental organizational principle of the Old World primate visual cortex.

Background and Purpose— The use of lithium as a neuroprotective agent has been demonstrated using various models in which improvements in infarct size, DNA damage, and neurological function were reported. We further investigated neurohemodynamic aspects of the treatment-associated recovery by assessing the therapeutic efficacy of delayed chronic lithium treatment using functional MRI. Methods— Ipsilesional functional MRI activations in the somatosensory cortex, acquired 2 weeks after the 90-minute transient middle cerebral artery occlusion, were compared between lithium- and saline-treated rats. Specifically, MRI signal changes based on blood oxygenation level dependence and functional cerebral blood volume responses were examined using electrical stimulation of forelimbs. Additional immunohistochemical assays were performed. Results— The ratio of ipsilesional to contralesional blood oxygenation level dependence response magnitudes significantly improved with lithium treatments. In contrast, the increase of the functional cerebral blood volume response magnitude ratio was not statistically significant. Nonetheless, the lithium treatment induced significant enhancements of total functional MRI activation (defined as a product of activation volume and response magnitude) for both blood oxygenation level dependence and functional cerebral blood volume methods. Increased cerebral blood volume in periinfarct tissues suggests a possible stroke-induced vascular transformation in both saline- and lithium-treated rats; however, other MRI-derived vascular parameters (vascular size index and microvascular volume) and immunohistochemical staining (CD31, glia fibrillary-associated protein, and matrix metalloproteinase-9) may imply that the neoformation of vasculature was differently affected by the lithium treatment. Conclusions— The delayed chronic lithium treatment enhanced the blood oxygenation level dependence functional MRI response magnitude in the absence of neurological improvement and influenced vascular formation in poststroke animal models.

This study investigated the dynamics of dopamine receptor desensitization and internalization, thereby proposing a new technique for non-invasive, in vivo measurements of receptor adaptations. The D2/D3 agonist quinpirole, which induces receptor internalization in vitro, was administered at graded doses in non-human primates while imaging with simultaneous positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). A pronounced temporal divergence between receptor occupancy and fMRI signal was observed: occupancy remained elevated while fMRI responded transiently. Analogous experiments with an antagonist (prochlorperazine) and a lower-affinity agonist (ropinirole) exhibited reduced temporal dissociation between occupancy and function, consistent with a mechanism of desensitization and internalization that depends upon drug efficacy and affinity. We postulated a model that incorporates internalization into a neurovascular-coupling relationship. This model yielded in vivo desensitization/internalization rates (0.2/min for quinpirole) consistent with published in vitro measurements. Overall, these results suggest that simultaneous PET/fMRI enables characterization of dynamic neuroreceptor adaptations in vivo, and may offer a first non-invasive method for assessing receptor desensitization and internalization.

Increased neural activity in brain tissue is accompanied by an array of supporting physiological processes, including increases in blood flow and the rates at which glucose and oxygen are consumed. These responses lead to secondary effects such as alterations in blood oxygenation and blood volume, and are ultimately the primary determinants of the amplitude and temporal signature of the blood oxygenation level-dependent (BOLD) signal used prevalently to map brain function. We have performed experiments using a combination of optical and MRI-based imaging methods to develop a more comprehensive picture of the physiological events accompanying activation of primary motor cortex during a finger apposition task. Temporal profiles for changes in tissue hemoglobin concentrations were qualitatively similar to those observed for MRI-based flow and oxygenation signals. Quantitative analysis of these signals revealed peak changes of +16 +/- 2% for HbO, -13 +/- 2% for HbR, +8 +/- 3% for total Hb, +83 +/- 9% for cerebral blood flow, and +1.4 +/- 0.1% for the BOLD MRI signal. A mass balance model was used to estimate the change in rate of oxidative metabolism implied by the optical and flow measurements, leading to a computed value of +47 +/- 5%. It should be noted that the optical and MRI observations may in general reflect changes over different volumes of tissue. The ratio of fractional changes in oxidative metabolism to fractional change in blood flow was found to be 0.56 +/- 0.08, in general agreement with previous studies of flow-metabolism coupling.

We have measured the changes in oxy-haemoglobin and deoxy-haemoglobin in the adult human brain during a brief finger tapping exercise using near-infrared spectroscopy (NIRS). The cerebral metabolic rate of oxygen (CMRO2) can be estimated from these NIRS data provided certain model assumptions. The change in CMRO2 is related to changes in the total haemoglobin concentration, deoxy-haemoglobin concentration and blood flow. As NIRS does not provide a measure of dynamic changes in blood flow during brain activation, we relied on aW indkessel model that relates dynamic blood volume and flow changes, which has been used previously for estimating CMRO2 from functional magnetic resonance imaging (fMRI) data. Because of the partial volume effect we are unable to quantify the absolute changes in the local brain haemoglobin concentrations with NIRS and thus are unable to obtain an estimate of the absolute CMRO2 change. An absolute estimate is also confounded by uncertainty in the flow–volume relationship. However, the ratio of the flow change to the CMRO2 change is relatively insensitive to these uncertainties. Fo rt he finger tapping task, we estimate a most probable flow–consumption ratio ranging from 1.5 to 3 in agreement with previous findings presented in the literature, although we cannot exclude the possibility that there is no CMRO2 change. The large range in the ratio arises from the large number of model parameters that must be estimated from the data. A more precise estimate of the flow–consumption ratio will require better estimates of the model parameters or flow information, as can be provided by combining NIRS with fMRI.

In order to identify differences in functional activity, we compared the reactivity of glioma vasculature and the native cerebral vasculature to both dilate and constrict in response to altered P(a)CO(2). Gliomas were generated by unilateral implantation of U87MGdEGFR human glioma tumor cells into the striatum of adult female athymic rats. Relative changes in total and microvascular cerebral blood volume were determined by steady state contrast agent-enhanced magnetic resonance imaging for transitions from normocarbia to hypercarbia and hypocarbia. Although hypercarbia induced a significant increase in both total and microvascular blood volume in normal brain and glioma, reactivity of glioma vasculature was significantly blunted in comparison to normal striatum; glioma total +/- CBV increased by 0.6 +/- 0.1%/mm Hg CO(2) whereas normal striatum increased by 1.5 +/- 0.2%/mm Hg CO(2), (P.0001, group t-test). Reactivity of microvascular blood volume was also significantly blunted. In contrast, hypocarbia decreased both total and microvascular blood volumes more in glioma than in normal striatum. These results indicate that cerebral blood vessels derived by tumor-directed angiogenesis do retain reactivity to CO(2). Furthermore, reduced reactivity of tumor vessels to a single physiological perturbation, such as hypercarbia, should not be construed as a generalized reduction of functional activity of the tumor vascular bed.

The time courses of oxyhaemoglobin ([HbO2]), deoxyhaemoglobin ([HbR]) and total haemoglobin ([HbT]) concentration changes following cortical activation in rats by electrical forepaw stimulation were measured using diffuse optical tomography (DOT) and compared to similar measurements performed previously with fMRI at 2.0 T and 4.7 T. We also explored the qualitative effects of varying stimulus parameters on the temporal evolution of the hemodynamic response. DOT images were reconstructed at a depth of 1.5 mm over a 1 cm square area from 2 mm anterior to bregma to 8 mm posterior to bregma. The measurement set included 9 sources and 16 detectors with an imaging frame rate of 10 Hz. Both DOT [HbR] and [HbO2] time courses were compared to the fMRI BOLD time course during stimulation, and the DOT [HbT] time course was compared to the fMRI cerebral plasma volume (CPV) time course. We believe that DOT and fMRI can provide similar temporal information for both blood volume and deoxyhaemoglobin changes, which helps to cross-validate these two techniques and to demonstrate that DOT can be useful as a complementary modality to fMRI for investigating the hemodynamic response to neuronal activity.

The pattern and role of brain plasticity in stroke recovery has been incompletely characterized. Both ipsilesional and contralesional changes have been described, but it remains unclear how these relate to functional recovery. Our goal was to correlate brain activation patterns with tissue damage, hemodynamics, and neurologic status after temporary stroke, using functional magnetic resonance imaging (fMRI). Transverse relaxation time (T(2))-weighted, diffusion-weighted, and perfusion MRI were performed at days 1 (n = 7), 3 (n = 7), and 14 (n = 7) after 2 hr unilateral middle cerebral artery occlusion in rats. Functional activation and cerebrovascular reactivity maps were generated from contrast-enhanced fMRI during forelimb stimulation and hypercapnia, respectively. Before MRI, rats were examined neurologically. We detected loss of activation responses in the ipsilesional sensorimotor cortex, which was related to T(2) lesion size (r = −0.858 on day 3,r = −0.979 on day 14; p < 0.05). Significant activation responses in the contralesional hemisphere were detected at days 1 and 3. The degree of shift in balance of activation between the ipsilesional and contralesional hemispheres, characterized by the laterality index, was linked to the T(2) and apparent diffusion coefficient in the ipsilesional contralesional forelimb region of the primary somatosensory cortex and primary motor cortex at day 1 (r = −0.807 and 0.782, respectively; p < 0.05) and day 14 (r = −0.898 and −0.970, respectively;p < 0.05). There was no correlation between activation parameters and perfusion status or cerebrovascular reactivity. Finally, we found that the laterality index and neurologic status changed in parallel over time after stroke, so that when all time points were grouped together, neurologic status was inversely correlated with the laterality index (r = −0.571;p = 0.016). This study suggests that the degree of shift of activation balance toward the contralesional hemisphere early after stroke increases with the extent of tissue injury and that functional recovery is associated mainly with preservation or restoration of activation in the ipsilesional hemisphere.

We report on the design and performance of compact detector packages currently installed in the four magnetic out-of-plane spectrometers for electron scattering experiments at the MIT-Bates Linear Accelerator Center. The detector packages have been designed to meet the mechanical requirements arising from out-of-plane particle detection. They offer good trajectory and momentum reconstruction, particle identification and time-of-flight measurements for electrons, pions, protons, and deuterons with large momentum bites and in broad kinematical ranges and high luminosities. The detectors have so far been used with great success in out-of-plane measurements of 12 C ( e → , e ′ p ) , 2 H ( e → , e ′ p ) , virtual Compton scattering below pion threshold and in studies of the N → Δ transition in both exclusive reaction channels 1 H ( e → , e ′ p ) π 0 and 1 H ( e → , e ′ π + )n .