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The role of astrocytes in neurovascular coupling (NVC) is unclear. Here, we applied a multimodality imaging approach to concomitantly measure synchronized neuronal or astrocytic Ca 2+ and hemodynamic changes in the mouse somatosensory cortex at rest and during sensory electrical stimulation. Strikingly, we found that low-frequency stimulation (0.3-1 Hz), which consistently evokes fast neuronal Ca 2+ transients (6.0 ± 2.7 ms latency) that always precede vascular responses, does not always elicit astrocytic Ca 2+ transients (313 ± 65 ms latency). However, the magnitude of the hemodynamic response is increased when astrocytic transients occur, suggesting a facilitatory role of astrocytes in NVC. High-frequency stimulation (5-10 Hz) consistently evokes a large, delayed astrocytic Ca 2+ accumulation (3.48 ± 0.09 s latency) that is temporarily associated with vasoconstriction, suggesting a role for astrocytes in resetting NVC. At rest, neuronal, but not astrocytic, Ca 2+ fluctuations correlate with hemodynamic low-frequency oscillations. Taken together, these results support a role for astrocytes in modulating, but not triggering, NVC., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent work has shown that thalamocortical (TC) inputs can be plastic after the developmental critical period has closed, but the mechanism that enables re-establishment of plasticity is unclear. Here, we find that long-term potentiation (LTP) at TC inputs is transiently restored in spared barrel cortex following either a unilateral infra-orbital nerve (ION) lesion, unilateral whisker trimming, or unilateral ablation of the rodent barrel cortex. Restoration of LTP is associated with increased potency at TC input and reactivates anatomical map plasticity induced by whisker follicle ablation. The reactivation of TC LTP is accompanied by reappearance of silent synapses. Both LTP and silent synapse formation are preceded by transient re-expression of synaptic GluN2B-containing N-methyl-D-aspartate (NMDA) receptors, which are required for the reappearance of TC plasticity. These results clearly demonstrate that peripheral sensory deprivation reactivates synaptic plasticity in the mature layer 4 barrel cortex with features similar to the developmental critical period., (Published by Elsevier Inc.)

Experience-dependent plasticity in the adult brain has clinical potential for functional rehabilitation following central and peripheral nerve injuries. Here, plasticity induced by unilateral infraorbital (IO) nerve resection in 4-week-old rats was mapped using MRI and synaptic mechanisms were elucidated by slice electrophysiology. Functional MRI demonstrates a cortical potentiation compared to thalamus 2 weeks after IO nerve resection. Tracing thalamocortical (TC) projections with manganese-enhanced MRI revealed circuit changes in the spared layer 4 (L4) barrel cortex. Brain slice electrophysiology revealed TC input strengthening onto L4 stellate cells due to an increase in postsynaptic strength and the number of functional synapses. This work shows that the TC input is a site for robust plasticity after the end of the previously defined critical period for this input. Thus, TC inputs may represent a major site for adult plasticity in contrast to the consensus that adult plasticity mainly occurs at cortico-cortical connections., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Traditional studies of neuroanatomical connections require injection of tracer compounds into living brains, then histology of the postmortem tissue. Here, we describe and validate a compound that reveals neuronal connections in vivo, using MRI. The classic anatomical tracer CTB (cholera-toxin subunit-B) was conjugated with a gadolinium-chelate to form GdDOTA-CTB. GdDOTA-CTB was injected into the primary somatosensory cortex (S1) or the olfactory pathway of rats. High-resolution MR images were collected at a range of time points at 11.7T and 7T. The transported GdDOTA-CTB was visible for at least 1 month post-injection, clearing within 2 months. Control injections of non-conjugated GdDOTA into S1 were not transported and cleared within 1-2 days. Control injections of Gd-Albumin were not transported either, clearing within 7 days. These MR results were verified by classic immunohistochemical staining for CTB, in the same animals. The GdDOTA-CTB neuronal transport was target specific, monosynaptic, stable for several weeks, and reproducible., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ferritin is an iron storage protein expressed in varying concentrations in mammalian cells. The deposition of ferric iron in the core of ferritin makes it a magnetic resonance imaging contrast agent, and ferritin has recently been proposed as a gene expression reporter protein for magnetic resonance imaging. To date, ferritin has been overexpressed in vivo and has been coexpressed with transferrin receptor to increase iron loading in cells. However, ferritin has a relatively low T(2) relaxivity (R(2) approximately 1 mM(-1)s(-1)) at typical magnetic field strengths and so requires high levels of expression to be detected. One way to modulate the transverse relaxivity of a superparamagnetic agent is to cause it to aggregate, thereby manipulating the magnetic field gradients through which water diffuses. In this work, it is demonstrated by computer simulation and in vitro that aggregation of ferritin can alter relaxivity. The effects of aggregate size and intraaggregate perturber spacing on R(2) are studied. Computer modeling indicates that the optimal spacing of the ferritin molecules in aggregate for increasing R(2) is 100-200 nm for a typical range of water diffusion rates. Chemical cross-linking of ferritin at 12 A spacing led to a 70% increase in R(2) compared to uncross-linked ferritin controls. To modulate ferritin aggregation in a potentially biologically relevant manner, ferritin was attached to actin and polymerized in vitro. The polymerization of ferritin-F-actin caused a 20% increase in R(2) compared to unpolymerized ferritin-G-actin. The R(2)-value was increased by another 10% by spacing the ferritin farther apart on the actin filaments. The modulation of ferritin aggregation by binding to cytoskeletal elements may be a useful strategy to make a functional reporter gene for magnetic resonance imaging.

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.

This study reports the detection of single mammalian cells, specifically T cells (T lymphocytes) labeled with dextran-coated superparamagnetic iron oxide particles, using magnetic resonance microscopy. Size amplification due to sequestration of the superparamagnetic particles in vacuoles enhances contrast in localized areas in high-resolution magnetic resonance imaging. Magnetic resonance images of samples containing differing concentrations of T cells embedded in 3% gelatin show a number of dark regions due to the superparamagnetic iron oxide particles, consistent with the number predicted by transmission electron microscopy. Colabeling of T cell samples with a fluorescent dye leads to strong correlations between magnetic resonance and fluorescence microscopic images, showing the presence of the superparamagnetic iron oxide particles at the cell site. This result lays the foundation for our approach to tracking the movement of a specific cell type in live animals and humans.

A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.

39K nuclear magnetic resonance (NMR) spectra were readily obtained, in vivo, from rat muscle, kidney, and brain in 5-10 min with signal-to-noise ratios of approximately 20:1. Quantitation of the K+ signal was achieved by reference to an external standard of KCl/dysprosium nitrate as well as by reference to the proton signal from tissue water. In vitro NMR studies of isolated tissue showed a K+ visibility (NMR K+/total tissue K+) of 96%, 62 +/- 8%, 47 +/- 1.9%, 45 +/- 3.5%, and 43 +/- 2.5% for blood, brain, muscle, kidney, and liver, respectively. Absolute tissue K+ was determined by flame photometry of acid-digested tissue. Changes in tissue K+ status by chronic K+ depletion or acute K+ loading produced changes of 39K NMR signal intensity that were equal to changes of absolute tissue K+. Acidosis, alkalosis, mannitol, or RbCl infusion did not significantly change the NMR K+ signal. These results indicate that the changes in K+ detected by NMR were specifically and accurately detected. To investigate the factors that affect the 39K NMR signal, the effects of liver homogenate on 39K NMR signal intensity were studied. Addition of homogenate produced a 60% loss of signal intensity, suggesting that a large portion of cell K+ may be only 40% visible. Addition of RbCl to undiluted homogenate increased the NMR K+ signal by 11 +/- 2 mumol/g. Addition of H2O or NaCl had no effect, suggesting that Rb+ was replacing K+ in sites of low (less than 40%) NMR visibility. These results demonstrate that 39K NMR experiments can be performed using intact organs. To explain the lack of detectable K+ and changes in K+ NMR visibility, a three compartment model is proposed.