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Central or peripheral injury causes reorganization of the brain's connections and functions. A striking change observed after unilateral stroke or amputation is a recruitment of bilateral cortical responses to sensation or movement of the unaffected peripheral area. The mechanisms underlying this phenomenon are described in a mouse model of unilateral whisker deprivation. Stimulation of intact whiskers yields a bilateral blood-oxygen-level-dependent fMRI response in somatosensory barrel cortex. Whole-cell electrophysiology demonstrated that the intact barrel cortex selectively strengthens callosal synapses to layer 5 neurons in the deprived cortex. These synapses have larger AMPA receptor- and NMDA receptor-mediated events. These factors contribute to a maximally potentiated callosal synapse. This potentiation occludes long-term potentiation, which could be rescued, to some extent, with prior long-term depression induction. Excitability and excitation/inhibition balance were altered in a manner consistent with cell-specific callosal changes and support a shift in the overall state of the cortex. This is a demonstration of a cell-specific, synaptic mechanism underlying interhemispheric cortical reorganization., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Spontaneous low-frequency oscillations (LFOs) of blood-oxygen-level-dependent (BOLD) signals are used to map brain functional connectivity with functional MRI, but their source is not well understood. Here we used optical imaging to assess whether LFOs from vascular signals covary with oscillatory intracellular calcium (Ca(2+)i) and with local field potentials in the rat's somatosensory cortex. We observed that the frequency of Ca(2+)i oscillations in tissue (∼0.07 Hz) was similar to the LFOs of deoxyhemoglobin (HbR) and oxyhemoglobin (HbO2) in both large blood vessels and capillaries. The HbR and HbO2 fluctuations within tissue correlated with Ca(2+)i oscillations with a lag time of ∼5-6 s. The Ca(2+)i and hemoglobin oscillations were insensitive to hypercapnia. In contrast, cerebral-blood-flow velocity (CBFv) in arteries and veins fluctuated at a higher frequency (∼0.12 Hz) and was sensitive to hypercapnia. However, in parenchymal tissue, CBFv oscillated with peaks at both ∼0.06 Hz and ∼0.12 Hz. Although the higher-frequency CBFv oscillation (∼0.12 Hz) was decreased by hypercapnia, its lower-frequency component (∼0.06 Hz) was not. The sensitivity of the higher CBFV oscillations to hypercapnia, which triggers blood vessel vasodilation, suggests its dependence on vascular effects that are distinct from the LFOs detected in HbR, HbO2, Ca(2+)i, and the lower-frequency tissue CBFv, which were insensitive to hypercapnia. Hemodynamic LFOs correlated both with Ca(2+)i and neuronal firing (local field potentials), indicating that they directly reflect neuronal activity (perhaps also glial). These findings show that HbR fluctuations (basis of BOLD oscillations) are linked to oscillatory cellular activity and detectable throughout the vascular tree (arteries, capillaries, and veins).

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.

In the weeks following unilateral peripheral nerve injury, the deprived primary somatosensory cortex (SI) responds to stimulation of the ipsilateral intact limb as demonstrated by functional magnetic resonance imaging (fMRI) responses. The neuronal basis of these responses was studied by using high-resolution fMRI, in vivo electrophysiological recordings, and juxtacellular neuronal labeling in rats that underwent an excision of the forepaw radial, median, and ulnar nerves. These nerves were exposed but not severed in control rats. Significant bilateral increases of fMRI responses in SI were observed in denervated rats. In the healthy SI of the denervated rats, increases in fMRI responses were concordant with increases in local field potential (LFP) amplitude and an increased incidence of single units responding compared with control rats. In contrast, in the deprived SI, increases in fMRI responses were associated with a minimal change in LFP amplitude but with increased incidence of single units responding. Based on action potential duration, juxtacellular labeling, and immunostaining results, neurons responding to intact forepaw stimulation in the deprived cortex were identified as interneurons. These results suggest that the increases in fMRI responses in the deprived cortex reflect increased interneuron activity.

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.

The ability to detect brain anatomy and pathophysiology with MRI is limited by the contrast-to-noise ratio (CNR), which depends on the contrast mechanism used and the spatial resolution. In this work, we show that in MRI of the human brain, large improvements in contrast to noise in high-resolution images are possible by exploiting the MRI signal phase at high magnetic field strength. Using gradient-echo MRI at 7.0 tesla and a multichannel detector, a nominal voxel size of 0.24 x 0.24 x 1.0 mm3 (58 nl) was achieved. At this resolution, a strong phase contrast was observed both between as well as within gray matter (GM) and white matter (WM). In gradient-echo phase images obtained on normal volunteers at this high resolution, the CNR between GM and WM ranged from 3:1 to 20:1 over the cortex. This CNR is an almost 10-fold improvement over conventional MRI techniques that do not use image phase, and it is an approximately 100-fold improvement when including the gains in resolution from high-field and multichannel detection. Within WM, phase contrast appeared to be associated with the major fiber bundles, whereas contrast within GM was suggestive of the underlying layer structure. The observed phase contrast is attributed to local variations in magnetic susceptibility, which, at least in part, appeared to originate from iron stores. The ability to detect cortical substructure from MRI phase contrast at high field is expected to greatly enhance the study of human brain anatomy in vivo.

There is rapid growth in the use of MRI for molecular and cellular imaging. Much of this work relies on the high relaxivity of nanometer-sized, ultrasmall dextran-coated iron oxide particles. Typically, millions of dextran-coated ultrasmall iron oxide particles must be loaded into cells for efficient detection. Here we show that single, micrometer-sized iron oxide particles (MPIOs) can be detected by MRI in vitro in agarose samples, in cultured cells, and in mouse embryos. Experiments studying effects of MRI resolution and particle size from 0.76 to 1.63 microm indicated that T(2)* effects can be readily detected from single MPIOs at 50-microm resolution and significant signal effects could be detected at resolutions as low as 200 microm. Cultured cells were labeled with fluorescent MPIOs such that single particles were present in individual cells. These single particles in single cells could be detected both by MRI and fluorescence microscopy. Finally, single particles injected into single-cell-stage mouse embryos could be detected at embryonic day 11.5, demonstrating that even after many cell divisions, daughter cells still carry individual particles. These results demonstrate that MRI can detect single particles and indicate that single-particle detection will be useful for cellular imaging.

The blood oxygenation level-dependent (BOLD) response to somatosensory stimulation was measured in alpha-chloralose-anesthetized rats. BOLD fMRI was obtained at 40-ms temporal resolution and spatial resolution of 200 x 200 x 2,000 microm(3) by using a gated activation paradigm in an 11.7 T MRI. Results show a consistent heterogeneity of fMRI onset times and amplitudes. The earliest onset time (0.59 +/- 0.17 s, n = 9) corresponded anatomically to layer IV, with superficial and deeper layers starting significantly later (1.27 +/- 0.43 s in layers I-III, and 1.11 +/- 0.45 s in layer VI). The amplitude of BOLD signal changes also varied with the cortical depth from the pial surface. Changes in the supragranular layers (8.3%) were 44% bigger than changes in the intermediate layers (5.5%), located only approximately 700 microm below, and 144% larger than the bottom layer (3.5%), located approximately 1.4 mm below the pial surface. The data presented demonstrate that BOLD signal changes have distinct amplitude and temporal characteristics, which vary spatially across cortical layers.

The role of glycolysis vs. respiration in tumor energy metabolism has been studied, to date, primarily in vitro by using single cells, multicellular spheroids, or tissue slices. With the advent of in vivo NMR spectroscopy, several investigators have shown that tumor energy status depends on its blood flow. Since manipulation of blood flow alters both oxygen and glucose delivery to a solid tumor, these studies have not been able to separate the relative contribution of oxygen vs. glucose in energy metabolism in vivo. In the present study, we have overcome this problem by combining two methods: the tissue-isolated R3230AC mammary adenocarcinoma perfused ex vivo and 31P NMR spectroscopy. The isolated tumor permits one to control the perfusion pressure as well as the metabolite concentrations in the perfusate. NMR spectroscopy permits one to measure the ratio of nucleoside triphosphate to inorganic phosphate (NTP/Pi) and pH. Our results show that (i) the NTP/Pi ratio ex vivo is similar to that observed in vivo prior to surgery, (ii) the NTP/Pi ratio is insensitive to flow changes at high flow rates but is proportional to flow rate at flows comparable to those found in vivo, (iii) the NTP/Pi ratio of these tumors is resistant to hypoxia and is not maintained when glucose is removed or replaced with glutamine, and (iv) although both O2 and glucose are consumed by these tumors, the effect of perfusate flow rate appears to be mediated largely through glucose delivery. The current approach not only provides information about the role of glycolysis vs. respiration in a rodent tumor but also is general and versatile enough to provide similar data in human tumors perfused ex vivo.

A technique has been developed for proton magnetic resonance imaging (MRI) of perfusion, using water as a freely diffusable tracer, and its application to the measurement of cerebral blood flow (CBF) in the rat is demonstrated. The method involves labeling the inflowing water proton spins in the arterial blood by inverting them continuously at the neck region and observing the effects of inversion on the intensity of brain MRI. Solution to the Bloch equations, modified to include the effects of flow, allows regional perfusion rates to be measured from an image with spin inversion, a control image, and a T1 image. Continuous spin inversion labeling the arterial blood water was accomplished, using principles of adiabatic fast passage by applying continuous-wave radiofrequency power in the presence of a magnetic field gradient in the direction of arterial flow. In the detection slice used to measure perfusion, whole brain CBF averaged 1.39 +/- 0.19 ml.g-1.min-1 (mean +/- SEM, n = 5). The technique's sensitivity to changes in CBF was measured by using graded hypercarbia, a condition that is known to increase brain perfusion. CBF vs. pCO2 data yield a best-fit straight line described by CBF (ml.g-1.min-1) = 0.052pCO2 (mm Hg) - 0.173, in excellent agreement with values in the literature. Finally, perfusion images of a freeze-injured rat brain have been obtained, demonstrating the technique's ability to detect regional abnormalities in perfusion.

To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 mumol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (t1/2 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 +/- 0.004 mumol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.

A technique for making 31P NMR spectroscopic measurements in rat kidney, heart, and liver in vivo is presented. Two-turn solenoid coils were surgically implanted around the organ sufficiently in advance of NMR experiments to allow recovery of the animal. These chronically implanted coils allowed acquisition of high-resolution spectra at 40.5 and 97.3 MHz. No resolution improvement occurred at the higher field. Spectra were stable for up to 24 hr, during which time a variety of experiments could be performed. By accumulating spectra at 10-min intervals, the effects of intraperitoneal fructose injections were monitored; in kidney and liver, a rapid increase in sugar phosphates at the expense of Pi and ATP resulted. Fructose had no effect on heart metabolite levels. Spectra from the heart in vivo were obtained at systole and diastole by gating the spectrometer to the aortic pressure wave; no differences in phosphate metabolites were detected. Finally, saturation transfer techniques were used to monitor the rate of ATP synthesis in the kidney. The unidirectional rate constant for the conversion of Pi to ATP was 0.12 +/- 0.03 sec-1.