The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity.

OBJECTIVE: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

Orthonasal versus retronasal glomerular activity in rat olfactory bulb by fMRI.

Odorants can reach olfactory receptor neurons (ORNs) by two routes: orthonasally, when volatiles enter the nasal cavity during inhalation/sniffing, and retronasally, when food volatiles released in the mouth pass into the nasal cavity during exhalation/eating. Previous work in humans has shown that both delivery routes of the same odorant can evoke distinct perceptions and patterns of neural responses in the brain. Each delivery route is known to influence specific responses across the dorsal region of the glomerular sheet in the olfactory bulb (OB), but spatial distributions across the entire glomerular sheet throughout the whole OB remain largely unexplored. We used functional MRI (fMRI) to measure and compare activations across the entire glomerular sheet in rat OB resulting from both orthonasal and retronasal stimulations of the same odors. We observed reproducible fMRI activation maps of the whole OB during both orthonasal and retronasal stimuli. However, retronasal stimuli required double the orthonasal odor concentration for similar response amplitudes. Regardless, both the magnitude and spatial extent of activity were larger during orthonasal versus retronasal stimuli for the same odor. Orthonasal and retronasal response patterns show overlap as well as some route-specific dominance. Orthonasal maps were dominant in dorsal-medial regions, whereas retronasal maps were dominant in caudal and lateral regions. These different whole OB encodings likely underlie differences in odor perception between these biologically important routes for odorants among mammals. These results establish the relationships between orthonasal and retronasal odor representations in the rat OB.

APOE genotype-dependent pharmacogenetic responses to rapamycin for preventing Alzheimer's disease.

The ε4 allele of Apolipoprotein (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), the most common form of dementia. Cognitively normal APOE4 carriers have developed amyloid beta (Aß) plaques and cerebrovascular, metabolic and structural deficits decades before showing the cognitive impairment. Interventions that can inhibit Aß retention and restore the brain functions to normal would be critical to prevent AD for the asymptomatic APOE4 carriers. A major goal of the study was to identify the potential usefulness of rapamycin (Rapa), a pharmacological intervention for extending longevity, for preventing AD in the mice that express human APOE4 gene and overexpress Aß (the E4FAD mice). Another goal of the study was to identify the potential pharmacogenetic differences in response to rapamycin between the E4FAD and E3FAD mice, the mice with human APOE ε3 allele. We used multi-modal MRI to measure in vivo cerebral blood flow (CBF), neurotransmitter levels, white matter integrity, water content, cerebrovascular reactivity (CVR) and somatosensory response; used behavioral assessments to determine cognitive function; used biochemistry assays to determine Aß retention and blood-brain barrier (BBB) functions; and used metabolomics to identify brain metabolic changes. We found that in the E4FAD mice, rapamycin normalized bodyweight, restored CBF (especially in female), BBB activity for Aß transport, neurotransmitter levels, neuronal integrity and free fatty acid level, and reduced Aß retention, which were not observe in the E3FAD-Rapa mice. In contrast, E3FAD-Rapa mice had lower CVR responses, lower anxiety and reduced glycolysis in the brain, which were not seen in the E4FAD-Rapa mice. Further, rapamycin appeared to normalize lipid-associated metabolism in the E4FAD mice, while slowed overall glucose-associated metabolism in the E3FAD mice. Finally, rapamycin enhanced overall water content, water diffusion in white matter, and spatial memory in both E3FAD and E4FAD mice, but did not impact the somatosensory responses under hindpaw stimulation. Our findings indicated that rapamycin was able to restore brain functions and reduce AD risk for young, asymptomatic E4FAD mice, and there were pharmacogenetic differences between the E3FAD and E4FAD mice. As the multi-modal MRI methods used in the study are readily to be used in humans and rapamycin is FDA-approved, our results may pave a way for future clinical testing of the pharmacogenetic responses in humans with different APOE alleles, and potentially using rapamycin to prevent AD for asymptomatic APOE4 carriers.

Spontaneous activity forms a foundation for odor-evoked activation maps in the rat olfactory bulb.

Fluctuations in spontaneous activity have been observed by many neuroimaging techniques, but because these resting-state changes are not evoked by stimuli, it is difficult to determine how they relate to task-evoked activations. We conducted multi-modal neuroimaging scans of the rat olfactory bulb, both with and without odor, to examine interaction between spontaneous and evoked activities. Independent component analysis of spontaneous fluctuations revealed resting-state networks, and odor-evoked changes revealed activation maps. We constructed simulated activation maps using resting-state networks that were highly correlated to evoked activation maps. Simulated activation maps derived by intrinsic optical signal (IOS), which covers the dorsal portion of the glomerular sheet, significantly differentiated one odor's evoked activation map from the other two. To test the hypothesis that spontaneous activity of the entire glomerular sheet is relevant for representing odor-evoked activations, we used functional magnetic resonance imaging (fMRI) to map the entire glomerular sheet. In contrast to the IOS results, the fMRI-derived simulated activation maps significantly differentiated all three odors' evoked activation maps. Importantly, no evoked activation maps could be significantly differentiated using simulated activation maps produced using phase-randomized resting-state networks. Given that some highly organized resting-state networks did not correlate with any odors' evoked activation maps, we posit that these resting-state networks may characterize evoked activation maps associated with odors not studied. These results emphasize that fluctuations in spontaneous activity form a foundation for active processing, signifying the relevance of resting-state mapping to functional neuroimaging.

Quantitative ß mapping for calibrated fMRI.

The metabolic and hemodynamic dependencies of the blood oxygenation level-dependent (BOLD) signal form the basis for calibrated fMRI, where the focus is on oxidative energy demanded by neural activity. An important part of calibrated fMRI is the power-law relationship between the BOLD signal and the deoxyhemoglobin concentration, which in turn is related to the ratio between oxidative demand (CMRO2) and blood flow (CBF). The power-law dependence between BOLD signal and deoxyhemoglobin concentration is signified by a scaling exponent ß. Until recently most studies assumed a ß value of 1.5, which is based on numerical simulations of the extravascular BOLD component. Since the basal value of CMRO2 and CBF can vary from subject-to-subject and/or region-to-region, a method to independently measure ß in vivo should improve the accuracy of calibrated fMRI results. We describe a new method for ß mapping through characterizing R2' - the most sensitive relaxation component of BOLD signal (i.e., the reversible magnetic susceptibility component that is predominantly of extravascular origin at high magnetic field) - as a function of intravascular magnetic susceptibility induced by an FDA-approved superparamagnetic contrast agent. In α-chloralose anesthetized rat brain, at 9.4 T, we measured ß values of ~0.8 uniformly across large neocortical swathes, with lower magnitude and more heterogeneity in subcortical areas. Comparison of ß maps in rats anesthetized with medetomidine and α-chloralose revealed that ß is independent of neural activity levels at these resting states. We anticipate that this method for ß mapping can help facilitate calibrated fMRI for clinical studies.

Comparison of glomerular activity patterns by fMRI and wide-field calcium imaging: Implications for principles underlying odor mapping.

Functional imaging signals arise from distinct metabolic and hemodynamic events at the neuropil, but how these processes are influenced by pre- and post-synaptic activities need to be understood for quantitative interpretation of stimulus-evoked mapping data. The olfactory bulb (OB) glomeruli, spherical neuropil regions with well-defined neuronal circuitry, can provide insights into this issue. Optical calcium-sensitive fluorescent dye imaging (OICa(2+)) reflects dynamics of pre-synaptic input to glomeruli, whereas high-resolution functional magnetic resonance imaging (fMRI) using deoxyhemoglobin contrast reveals neuropil function within the glomerular layer where both pre- and post-synaptic activities contribute. We imaged odor-specific activity patterns of the dorsal OB in the same anesthetized rats with fMRI and OICa(2+) and then co-registered the respective maps to compare patterns in the same space. Maps by each modality were very reproducible as trial-to-trial patterns for a given odor, overlapping by ~80%. Maps evoked by ethyl butyrate and methyl valerate for a given modality overlapped by ~80%, suggesting activation of similar dorsal glomerular networks by these odors. Comparison of maps generated by both methods for a given odor showed ~70% overlap, indicating similar odor-specific maps by each method. These results suggest that odor-specific glomerular patterns by high-resolution fMRI primarily tracks pre-synaptic input to the OB. Thus combining OICa(2+) and fMRI lays the framework for studies of OB processing over a range of spatiotemporal scales, where OICa(2+) can feature the fast dynamics of dorsal glomerular clusters and fMRI can map the entire glomerular sheet in the OB.

Brain region and activity-dependent properties of M for calibrated fMRI.

Calibrated fMRI extracts changes in oxidative energy demanded by neural activity based on hemodynamic and metabolic dependencies of the blood oxygenation level-dependent (BOLD) response. This procedure requires the parameter M, which is determined from the dynamic range of the BOLD signal between deoxyhemoglobin (paramagnetic) and oxyhemoglobin (diamagnetic). Since it is unclear if the range of M-values in human calibrated fMRI is due to regional/state differences, we conducted a 9.4T study to measure M-values across brain regions in deep (α-chloralose) and light (medetomidine) anesthetized rats, as verified by electrophysiology. Because BOLD signal is captured differentially by gradient-echo (R2*) and spin-echo (R2) relaxation rates, we measured M-values by the product of the fMRI echo time and R2' (i.e., the reversible magnetic susceptibility component), which is given by the absolute difference between R2* and R2. While R2' mapping was shown to be dependent on the k-space sampling method used, at nominal spatial resolutions achieved at high magnetic field of 9.4T the M-values were quite homogenous across cortical gray matter. However cortical M-values varied in relation to neural activity between brain states. The findings from this study could improve precision of future calibrated fMRI studies by focusing on the global uniformity of M-values in gray matter across different resting activity levels.

Quantitative basis for neuroimaging of cortical laminae with calibrated functional MRI.

Layer-specific neurophysiologic, hemodynamic, and metabolic measurements are needed to interpret high-resolution functional magnetic resonance imaging (fMRI) data in the cerebral cortex. We examined how neurovascular and neurometabolic couplings vary vertically in the rat's somatosensory cortex. During sensory stimulation we measured dynamic layer-specific responses of local field potential (LFP) and multiunit activity (MUA) as well as blood oxygenation level-dependent (BOLD) signal and cerebral blood volume (CBV) and blood flow (CBF), which in turn were used to calculate changes in oxidative metabolism (CMR(O2)) with calibrated fMRI. Both BOLD signal and CBV decreased from superficial to deep laminae, but these responses were not well correlated with either layer-specific LFP or MUA. However, CBF changes were quite stable across laminae, similar to LFP. However, changes in CMR(O2) and MUA varied across cortex in a correlated manner and both were reduced in superficial lamina. These results lay the framework for quantitative neuroimaging across cortical laminae with calibrated fMRI methods.

Rhythmic 3-4Hz discharge is insufficient to produce cortical BOLD fMRI decreases in generalized seizures.

Absence seizures are transient episodes of impaired consciousness accompanied by 3-4 Hz spike-wave discharge on electroencephalography (EEG). Human functional magnetic resonance imaging (fMRI) studies have demonstrated widespread cortical decreases in the blood oxygen-level dependent (BOLD) signal that may play an important role in the pathophysiology of these seizures. Animal models could provide an opportunity to investigate the fundamental mechanisms of these changes, however they have so far failed to consistently replicate the cortical fMRI decreases observed in human patients. This may be due to important differences between human seizures and animal models, including a lack of cortical development in rodents or differences in the frequencies of rodent (7-8 Hz) and human (3-4 Hz) spike-wave discharges. To examine the possible contributions of these differences, we developed a ferret model that exhibits 3-4 Hz spike-wave seizures in the presence of a sulcated cortex. Measurements of BOLD fMRI and simultaneous EEG demonstrated cortical fMRI increases during and following spike-wave seizures in ferrets. However unlike human patients, significant fMRI decreases were not observed. The lack of fMRI decreases was consistent across seizures of different durations, discharge frequencies, and anesthetic regimes, and using fMRI analysis models similar to human patients. In contrast, generalized tonic-clonic seizures under the same conditions elicited sustained postictal fMRI decreases, verifying that the lack of fMRI decreases with spike-wave was not due to technical factors. These findings demonstrate that 3-4 Hz spike-wave discharge in a sulcated animal model does not necessarily produce fMRI decreases, leaving the mechanism for this phenomenon open for further investigation.

Role of mitochondrial calcium uptake homeostasis in resting state fMRI brain networks.

Mitochondrial Ca(2+) uptake influences both brain energy metabolism and neural signaling. Given that brain mitochondrial organelles are distributed in relation to vascular density, which varies considerably across brain regions, we hypothesized different physiological impacts of mitochondrial Ca(2+) uptake across brain regions. We tested the hypothesis by monitoring brain "intrinsic activity" derived from the resting state functional MRI (fMRI) blood oxygen level dependent (BOLD) fluctuations in different functional networks spanning the somatosensory cortex, caudate putamen, hippocampus and thalamus, in normal and perturbed mitochondrial Ca(2+) uptake states. In anesthetized rats at 11.7 T, mitochondrial Ca(2+) uptake was inhibited or enhanced respectively by treatments with Ru360 or kaempferol. Surprisingly, mitochondrial Ca(2+) uptake inhibition by Ru360 and enhancement by kaempferol led to similar dose-dependent decreases in brain-wide intrinsic activities in both the frequency domain (spectral amplitude) and temporal domain (resting state functional connectivity; RSFC). The fact that there were similar dose-dependent decreases in the frequency and temporal domains of the resting state fMRI-BOLD fluctuations during mitochondrial Ca(2+) uptake inhibition or enhancement indicated that mitochondrial Ca(2+) uptake and its homeostasis may strongly influence the brain's functional organization at rest. Interestingly, the resting state fMRI-derived intrinsic activities in the caudate putamen and thalamic regions saturated much faster with increasing dosage of either drug treatment than the drug-induced trends observed in cortical and hippocampal regions. Regional differences in how the spectral amplitude and RSFC changed with treatment indicate distinct mitochondrion-mediated spontaneous neuronal activity coupling within the various RSFC networks determined by resting state fMRI.

Distribution of temperature changes and neurovascular coupling in rat brain following 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") exposure.

(+/-)3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is an abused psychostimulant that produces strong monoaminergic stimulation and whole-body hyperthermia. MDMA-induced thermogenesis involves activation of uncoupling proteins (UCPs), primarily a type specific to skeletal muscle (UCP-3) and absent from the brain, although other UCP types are expressed in the brain (e.g. thalamus) and might contribute to thermogenesis. Since neuroimaging of brain temperature could provide insights into MDMA action, we measured spatial distributions of systemically administered MDMA-induced temperature changes and dynamics in rat cortex and subcortex using a novel magnetic resonance method, Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), with an exogenous temperature-sensitive probe (thulium ion and macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetate (DOTMA(4-))). The MDMA-induced temperature rise was greater in the cortex than in the subcortex (1.6 ± 0.4 °C versus 1.3 ± 0.4 °C) and occurred more rapidly (2.0 ± 0.2 °C/h versus 1.5 ± 0.2 °C/h). MDMA-induced temperature changes and dynamics in the cortex and body were correlated, although the body temperature exceeded the cortex temperature before and after MDMA. Temperature, neuronal activity, and blood flow (CBF) were measured simultaneously in the cortex and subcortex (i.e. thalamus) to investigate possible differences of MDMA-induced warming across brain regions. MDMA-induced warming correlated with increases in neuronal activity and blood flow in the cortex, suggesting that the normal neurovascular response to increased neural activity was maintained. In contrast to the cortex, a biphasic relationship was seen in the subcortex (i.e. thalamus), with a decline in CBF as temperature and neural activity rose, transitioning to a rise in CBF for temperature above 37 °C, suggesting that MDMA affected CBF and neurovascular coupling differently in subcortical regions. Considering that MDMA effects on CBF and heat dissipation (as well as potential heat generation) may vary regionally, neuroprotection may require different cooling strategies.

DYNAmic Multi-coIl TEchnique (DYNAMITE) shimming of the rat brain at 11.7 T.

The in vivo rat model is a workhorse in neuroscience research, preclinical studies and drug development. A repertoire of MR tools has been developed for its investigation; however, high levels of B0 magnetic field homogeneity are required for meaningful results. The homogenization of magnetic fields in the rat brain, i.e. shimming, is a difficult task because of a multitude of complex, susceptibility-induced field distortions. Conventional shimming with spherical harmonic (SH) functions is capable of compensating for shallow field distortions in limited areas, e.g. in the cortex, but performs poorly in difficult-to-shim subcortical structures or for the entire brain. Based on the recently introduced multi-coil approach for magnetic field modeling, the DYNAmic Multi-coIl TEchnique (DYNAMITE) is introduced for magnetic field shimming of the in vivo rat brain and its benefits for gradient-echo echo-planar imaging (EPI) are demonstrated. An integrated multi-coil/radiofrequency (MC/RF) system comprising 48 individual localized DC coils for B0 shimming and a surface transceive RF coil has been developed that allows MR investigations of the anesthetized rat brain in vivo. DYNAMITE shimming with this MC/RF set-up is shown to reduce the B0 standard deviation to a third of that achieved with current shim technology employing static first- through third-order SH shapes. The EPI signal over the rat brain increased by 31%, and a 24% gain in usable EPI voxels could be realized. DYNAMITE shimming is expected to critically benefit a wide range of preclinical and neuroscientific MR research. Improved magnetic field homogeneity, together with the achievable large brain coverage of this method, will be crucial when signal pathways, cortical circuitry or the brain's default network are studied. Together with the efficiency gains of MC-based shimming compared with SH approaches demonstrated recently, DYNAMITE shimming has the potential to replace conventional SH shim systems in small-bore animal scanners.

Analysis of time and space invariance of BOLD responses in the rat visual system.

Neuroimaging studies of functional magnetic resonance imaging (fMRI) and electrophysiology provide the linkage between neural activity and the blood oxygenation level-dependent (BOLD) response. Here, BOLD responses to light flashes were imaged at 11.7T and compared with neural recordings from superior colliculus (SC) and primary visual cortex (V1) in rat brain--regions with different basal blood flow and energy demand. Our goal was to assess neurovascular coupling in V1 and SC as reflected by temporal/spatial variances of impulse response functions (IRFs) and assess, if any, implications for general linear modeling (GLM) of BOLD responses. Light flashes induced high magnitude neural/BOLD responses reproducibly from both regions. However, neural/BOLD responses from SC and V1 were markedly different. SC signals followed the boxcar shape of the stimulation paradigm at all flash rates, whereas V1 signals were characterized by onset/offset transients that exhibited different flash rate dependencies. We find that IRF(SC) is generally time-invariant across wider flash rate range compared with IRF(V1), whereas IRF(SC) and IRF(V1) are both space invariant. These results illustrate the importance of measured neural signals for interpretation of fMRI by showing that GLM of BOLD responses may lead to misinterpretation of neural activity in some cases.

Cross-hemicord spinal fiber reorganization associates with cortical sensory and motor network expansion in the rat model of hemicontusion cervical spinal cord injury.

Magnetic resonance imaging plays an important role in characterizing microstructural changes and reorganization after traumatic injuries to the nervous system. In this study, we tested the feasibility of ex-vivo spinal cord diffusion tensor imaging (DTI) in combination with in vivo brain functional MRI to characterize spinal reorganization and its supraspinal association after a hemicontusion cervical spinal cord injury (SCI). DTI parameters (fractional anisotropy [FA], mean diffusion [MD]) and fiber orientation changes related to reorganization in the contused cervical spinal cord were compared to sham specimens. Altered fiber density and fiber directions occurred across the ipsilateral and contralateral hemicords but with only ipsilateral FA and MD changes. The hemicontusion SCI resulted in ipsilateral fiber breaks, voids and vivid fiber reorientations along the injury epicenter. Fiber directional changes below the injury level were primarily inter-hemispheric, indicating prominent below-level cross-hemispheric reorganization. In vivo resting state functional connectivity of the brain from the respective rats before obtaining the spinal cord samples indicated spatial expansion and increased connectivity strength across both the sensory and motor networks after SCI. The consistency of the neuroplastic changes along the neuraxis (both brain and spinal cord) at the single-subject level, indicates that distinctive reorganizational relationships exist between the spinal cord and the brain post-SCI.

Transient Impairment in Microglial Function Causes Sex-Specific Deficits in Synaptic and Hippocampal Function in Mice Exposed to Early Adversity.

Abnormal development and function of the hippocampus are two of the most consistent findings in humans and rodents exposed to early life adversity, with males often being more affected than females. Using the limited bedding (LB) paradigm as a rodent model of early life adversity, we found that male adolescent mice that had been exposed to LB exhibit significant deficits in contextual fear conditioning and synaptic connectivity in the hippocampus, which are not observed in females. This is linked to altered developmental refinement of connectivity, with LB severely impairing microglial-mediated synaptic pruning in the hippocampus of male and female pups on postnatal day 17 (P17), but not in adolescent P33 mice when levels of synaptic engulfment by microglia are substantially lower. Since the hippocampus undergoes intense synaptic pruning during the second and third weeks of life, we investigated whether microglia are required for the synaptic and behavioral aberrations observed in adolescent LB mice. Indeed, transient ablation of microglia from P13-21, in normally developing mice caused sex-specific behavioral and synaptic abnormalities similar to those observed in adolescent LB mice. Furthermore, chemogenetic activation of microglia during the same period reversed the microglial-mediated phagocytic deficits at P17 and restored normal contextual fear conditioning and synaptic connectivity in adolescent LB male mice. Our data support an additional contribution of astrocytes in the sex-specific effects of LB, with increased expression of the membrane receptor MEGF10 and enhanced synaptic engulfment in hippocampal astrocytes of 17-day-old LB females, but not in LB male littermates. This finding suggests a potential compensatory mechanism that may explain the relative resilience of LB females. Collectively, these studies highlight a novel role for glial cells in mediating sex-specific hippocampal deficits in a mouse model of early-life adversity.

mTOR inhibition enhances synaptic and mitochondrial function in Alzheimer's disease in an APOE genotype-dependent manner.

Apolipoprotein ε4 (APOE4) carriers develop brain metabolic dysfunctions decades before the onset of Alzheimer's disease (AD). A goal of the study is to identify if rapamycin, an inhibitor for the mammalian target of rapamycin (mTOR) inhibitor, would enhance synaptic and mitochondrial function in asymptomatic mice with human APOE4 gene (E4FAD) before they showed metabolic deficits. A second goal is to determine whether there may be genetic-dependent responses to rapamycin when compared to mice with human APOE3 alleles (E3FAD), a neutral AD genetic risk factor. We fed asymptomatic E4FAD and E3FAD mice with control or rapamycin diets for 16 weeks from starting from 3 months of age. Neuronal mitochondrial oxidative metabolism and excitatory neurotransmission rates were measured using in vivo 1H-[13C] proton-observed carbon-edited magnetic resonance spectroscopy, and isolated mitochondrial bioenergetic measurements using Seahorse. We found that rapamycin enhanced neuronal mitochondrial function, glutamate-glutamine cycling, and TCA cycle rates in the asymptomatic E4FAD mice. In contrast, rapamycin enhances glycolysis, non-neuronal activities, and inhibitory neurotransmission of the E3FAD mice. These findings indicate that rapamycin might be able to mitigate the risk for AD by enhancing brain metabolic functions for cognitively intact APOE4 carriers, and the responses to rapamycin are varied by APOE genotypes. Consideration of precision medicine may be needed for future rapamycin therapeutics.

VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Functional MRI and neural responses in a rat model of Alzheimer's disease.

Based on the hypothesis that brain plaques and tangles can affect cortical function in Alzheimer's disease (AD), we investigated functional responses in an AD rat model (called the Samaritan Alzheimer's rat achieved by ventricular infusion of amyloid peptide) and age-matched healthy control. High-field functional magnetic resonance imaging (fMRI) and extracellular neural activity measurements were applied to characterize sensory-evoked responses. Electrical stimulation of the forepaw led to BOLD and neural responses in the contralateral somatosensory cortex and thalamus. In AD brain we noted much smaller BOLD activation patterns in the somatosensory cortex (i.e., about 50% less activated voxels compared to normal brain). While magnitudes of BOLD and neural responses in the cerebral cortex were markedly attenuated in AD rats compared to normal rats (by about 50%), the dynamic coupling between the BOLD and neural responses in the cerebral cortex, as assessed by transfer function analysis, remained unaltered between the groups. However thalamic BOLD and neural responses were unaltered in AD brain compared to controls. Thus cortical responses in the AD model were indeed diminished compared to controls, but the thalamic responses in the AD and control rats were quite similar. Therefore these results suggest that Alzheimer's disease may affect cortical function more than subcortical function, which may have implications for interpreting altered human brain functional responses in fMRI studies of Alzheimer's disease.

Increased resting functional connectivity in spike-wave epilepsy in WAG/Rij rats.

PURPOSE: Functional magnetic resonance imaging (fMRI)-based resting functional connectivity is well suited for measuring slow correlated activity throughout brain networks. Epilepsy involves chronic changes in normal brain networks, and recent work demonstrated enhanced resting fMRI connectivity between the hemispheres in childhood absence epilepsy. An animal model of this phenomenon would be valuable for investigating fundamental mechanisms and testing therapeutic interventions. METHODS: We used fMRI-based resting functional connectivity for studying brain networks involved in absence epilepsy. Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) exhibit spontaneous episodes of staring and unresponsiveness accompanied by spike-wave discharges (SWDs) resembling human absence seizures in behavior and electroencephalography (EEG). Simultaneous EEG-fMRI data in epileptic WAG/Rij rats in comparison to nonepileptic Wistar controls were acquired at 9.4 T. Regions showing cortical fMRI increases during SWDs were used to define reference regions for connectivity analysis to investigate whether chronic seizure activity is associated with changes in network resting functional connectivity. KEY FINDINGS: We observed high degrees of cortical-cortical correlations in all WAG/Rij rats at rest (when no SWDs were present), but not in nonepileptic controls. Strongest connectivity was seen between regions most intensely involved in seizures, mainly in the bilateral somatosensory and adjacent cortices. Group statistics revealed that resting interhemispheric cortical-cortical correlations were significantly higher in WAG/Rij rats compared to nonepileptic controls. SIGNIFICANCE: These findings suggest that activity-dependent plasticity may lead to long-term changes in epileptic networks even at rest. The results show a marked difference between the epileptic and nonepileptic animals in cortical-cortical connectivity, indicating that this may be a useful interictal biomarker associated with the epileptic state.