Magnetic resonance imaging of brain cell water

In the central nervous system of vertebrates, cell bodies of neurons are often assembled as nuclei or cellular layers that play specific roles as functional units. The purpose of this work was to selectively highlight such cell assemblies by magnetic resonance imaging using signals from water protons that are associated with intracellular paramagnetic ions, while saturating lipid-associated water protons as well as extracellular free water protons. Given the significant correlation between image signal intensity and water proton density, the high signal intensities observed for such cell assemblies must be attributed to their abundant paramagnetic-ion-associated water protons. In the hippocampal formation, the technique visualized cell assemblies that were so far not depicted in human in vivo. In the brainstem, the technique delineated noradrenergic neuron groups such as the locus coeruleus in human and mice in vivo. Their reduced magnetization-transfer ratios together with their prolonged relaxation times compared to other gray matter indicate that the source of their high signal intensity is not the presence of T1-shortening molecules, e.g., neuromelanin, but their high water content. Given the general absence of neuromelanin in noradrenergic neurons of rodents, their high signal intensity in mice in vivo further supports this view.

In the central nervous system of vertebrates, cell bodies of neurons are often assembled as nuclei or cellular layers that play specific roles as functional units, e.g. in the hippocampal formation as shown in Fig. 1 (top left, arrowheads). Magnetic resonance imaging (MRI) can readily differentiate large cell assemblies from surrounding tissue. This is because intracellular water protons exhibit T 1 and T 2 relaxation times which are generally smaller than those of bulk water protons in extracellular spaces 1 (denoted "Free" in Fig. 1), but larger than those of (intraand extra-cellular) water protons that interact with lipid bilayers 2 (denoted "Lipid-associated" in Fig. 1). After administration of exogenous contrast agents 3 , also small cell assemblies in the hippocampal formation ( Fig. 1, top left, arrowheads) can be identified and distinguished from the neuropil (Fig. 1, top left, asterisks) and spaces filled with cerebrospinal fluid (CSF).
Here we attempt to gain signals selectively from the water protons that are associated with intracellular paramagnetic ions by saturating extracellular free water protons by direct on-resonance irradiation, as well as by eliminating signal contributions from lipid-associated protons by off-resonance irradiation of short T 2 lipid protons with broad linewidths. The latter method indirectly saturates lipid-associated water protons through magnetization transfer 4 (MT). The concept neither runs counter to previous studies showing fast water exchange between extra-and intra-cellular spaces relative to T 1 relaxation times 1 nor attempts to differentiate between extra-and intra-cellular pools of water protons. Instead, we propose a new strategy to preferentially image the fraction of intracellular water protons ("Paramagnetic-ion-associated" in Fig. 1). exposed to a lower direct-saturation effect because other molecules increase the R 2 of the water protons more than R 1 . In vitro experiments of solutions with 6 different R 1 and R 2 values confirmed that direct-saturation effects correlate linearly with R 2 /R 1 ratios ( Supplementary Fig. 2a-c).
Mean regional R 1 of GM structures correlate linearly with their water content (Fig. 2d). The upward deviation of the WM R 1 from the linear relationship is again attributable to its large interaction with water protons 2 . This effective cross-relaxation in WM is apparently cancelled out by the more pronounced direct-saturation effect on GM structures, which results in an apparent linear relationship between the MT ratio (α70) and the water content including both GM and WM (Fig. 2e). Accordingly, the even more pronounced direct-saturation effect by a weaker on-resonance irradiation (α15) brings them again out of the apparent linear relationship. The values of the GM structures with significantly higher iron content, e.g., the globus pallidus and red nucleus, and those with significantly higher copper content, e.g., the substantia nigra (SN) and locus coeruleus (LC), are well in line with those of other GM structures. Paramagnetic ion concentrations correlate neither with R 1 nor with MT ratios (Supplementary Fig. 2d-i). These findings indicate that the regional water content determines both MT ratio and R 1 . Accordingly, regional signal intensities in T 1 -weighted MRI with MT (α70 MT) correlate significantly (p < 0.005) with those in M 0 in every subject (Fig. 2f), which also indicates that the regional spin density of intracellular water protons correlate significantly with the regional water content. Figure 2g shows calculated signal intensities for gradient-echo MRI as a function of water content in human brain based on the obtained MT ratios as well as R 1 and R 2 * values ( Supplementary Fig. 3). A strong on-resonance irradiation with short TE and TR (TE2 in Fig. 2g) saturates the free water protons in the extracellular fluid with a water content 6 of 0.99. This results in a typical T 1 -weighted MRI contrast and a high signal intensity for WM. A prolongation of echo time (TE4) reduces the T 1 -weighting and thus the WM signal. The addition of MT (TE4MT) suppresses the MRI signal in proportion to the macromolecular content (i.e., in inverse proportion to the water content), while the saturation of the free water protons is preserved. As a result, the MT effect dominates for brain tissue (water content < 0.9), while the T 1 effect dominates for extracellular fluid (water content > 0.95). When saturating extracellular free water protons, the significant correlation between the signal intensities in MT-MRI and the proton density (Fig. 2f) indicates that the signal intensities reflect the spin density of intracellular water protons with short T 1 values. This relationship between the MT-MRI signal intensity and the water content remains unaffected by a further prolongation of TE (TE13MT) or by a three-dimensional acquisition (3DMT). In contrast,  3,49 ). After Gd-DTPA the signal intensities in the cellular layer are lower than in the neuropil (and higher after Mn 2+ ) indicating that the cellular layer (arrowheads) has a lower content of extracellular water protons than the neuropil (asterisks). (Bottom) Schematic drawing of three major brain water pools that contribute to MRI: "Lipid-associated" (purple) water protons which may be affected by cross-relaxation or magnetization transfer, "paramagnetic-ion-associated" (green) water protons in intracellular spaces, and "free" (red) bulk water protons in extracellular spaces.
The highly significant correlations in pixel-by-pixel plots (Fig. 3) confirm that the water protons whose T 1 is shortened by diamagnetic molecules are proportionally saturated by off-resonance irradiation (Fig. 3b) and that magnetization transfer generates proton-density contrast (Fig. 3c). A pool of pixels with high signal intensity in M 0 as well as in α15MT can be identified in Fig. 3c. With stronger on-resonance irradiation (Fig. 3d,e), a pool of comparable size deviates from the otherwise clearly correlated plots. These pools are supposed to consist of pixels that are dominated by free water protons because the exclusion of CSF from the analysis (see regions-of-interest in Fig. 3a) results in a clear correlation again (Fig. 3f,g).
Magnetization transfer delineates small cell assemblies in the central nervous system of human and mice in vivo. Figure 4 shows images obtained with the parameters given in Fig. 2g as "TE4", "TE4 MT", and "TE13 MT". Repetitive on-resonance irradiation (TE4 in Fig. 4) saturates the CSF water protons while providing the brain tissue with high signal intensities. The magnified image of the hippocampal formation reveals high signal intensities for myelinated layers such as the alveus and the stratum lacunosum-moleculare. There is hardly a structure identifiable within the spinal cord. Magnetization transfer (TE4 MT in Fig. 4) predominantly suppresses the WM signal and shows the H-shaped cell assemblies as bright structures. A prolongation of TE (TE13 MT in Fig. 4) further enhances the image contrast and delineates the small cell assemblies in the hippocampal formation and the spinal cord even more clearly. The MRI pattern of high signal intensity in the hippocampal formation corresponds to the distribution pattern of the acetylcholinesterase reaction product (Fig. 4). This suggests that the MRI signals originate from cytoplasms and proximal dendrites of the neurons in the dentate gyrus as well as in the stratum oriens, pyramidale, and radiatum of the CA1~CA4~prosubiculum 7,8 . The pyramidal and polymorphic neurons that are rich in acetylcholine esterase play a critical role in establishing complicated neural networks 9 .
Similarly, in the brain of mice (Fig. 5a), magnetization transfer suppresses the signal intensities of the myelinated tissue and highlights cell assemblies as bright structures. In the spinal cord, MT distinguishes the unmyelinated cell layers of the Rexed lamina II (white arrows), the myelinated GM, and WM. These observations suggest unmyelinated layers that are tightly packed with nerve cell bodies which are responsible for the highest signal intensity. This assumption was verified by an additional experiment which compares a placebo-treated and a lesioned mouse (Fig. 5b). In agreement with previous observations of this well-established model for nerve cell death 10 , the histologic staining reveals a marked loss of pyramidal cell bodies in the affected animal. The corresponding loss of MRI signal intensity therefore supports the assignment of the high signal intensity to the presence of viable pyramidal cell bodies. As a consequence, the bright structure in T 1 -weighted MRI of the Ammon´s horn of the hippocampus with or without MT (Fig. 5a) must be ascribed to the densely packed layer of pyramidal cell bodies.

Magnetization transfer delineates noradrenergic neuron groups in the brainstem of human and mice in vivo.
In the brainstem, we delineated assemblies of noradrenergic neurons (Fig. 6a, Supplementary   Fig. 4). Noradrenaline, also called norepinephrine, is a catecholamine that acts as hormone and neurotransmitter. Noradrenergic neurons, which produce the neuromodulator and release it from axonal terminals that spread widely throughout the brain, are assembled in the brainstem as A1 to A7 cell groups 11 . The major groups 12 are A2, i.e., the dorsal motor vagus nucleus and nucleus tractus solitarius, in the medulla oblongata and A6, i.e., LC, in the pons. Here, we observed that the noradrenergic cell groups have low MT ratios and high T 1 and T 2 values compared to other GM structures ( Supplementary Fig. 4, Supplementary Table 2). These findings indicate that the noradrenergic cell groups have high water content and that their high concentration of copper is not the source of the high MRI signal intensity.
Similarly, with optimized parameters (Supplementary Tables 1a-h) we also delineated LC and A2 in mice (Fig. 6b,c, and Fig. 7). In T 1 map, neither LC nor A2 group was delineated from surrounding brainstem. The T 1 values (mean ± SD) of the brainstem at 2.35T are 0.93 ± 0.01 s at the level of LC and 0.95 ± 0.03 s at the level of A2 in males (n = 5), or 0.97 ± 0.02 s at the LC level and 1.0 ± 0.03 s at the A2 level in females (n = 4). The absence of T 1 contrast and the T 1 values are in line with data obtained in human brain at 3T (Supplementary Table 2). The lack of delineation does not result from the partial volume effect because T 1 -weighted MRI with MT at the same spatial resolution (117 × 117 × 234 µm 3 ) delineates the LC and A2.
Magnetization-transfer MRI detects signal enhancement of specific cell assemblies after administration of manganese. As mentioned earlier, physiological concentrations of endogenous paramagnetic ions have no significant effect on R 1 or MT ratio ( Supplementary Fig. 2d-i). On the other hand, as acquisition parameters α, TR, and TE, the variables R 1 , R 2 *, and MTR used for the calculation of MRI signal can be described as a function of tissue water content as shown in Supplementary Fig. 3. The R 1 and R 2 * are obtained from the functions given in the legend of Supplementary Fig. 3a and 3d, respectively. The MTRs for "TE4MT", "TE13MT", "3DMT", and "α15TE4MT" are obtained from the functions given in the legend of Supplementary Fig. 3b, 3c, 3e, and 3f, respectively. • = gray matter structure (prefrontal cortex, caudate nucleus, putamen, thalamus, globus pallidus, and substantia nigra for (d) and (e). In addition, subthalamic nucleus, red nucleus, and locus coeruleus for (b), (c), and (f), × = WM = frontal subcortical white matter, • = CSF = cerebrospinal fluid, error bars = standard deviation. (2019) 9:5084 | https://doi.org/10.1038/s41598-019-41587-2 www.nature.com/scientificreports www.nature.com/scientificreports/  Table 3, we observed significant MT ratio reduction as well as significant signal increase in MT-MRI of the brain of mice after administration of Mn 2+ in agreement with a number of previous studies 13 . The Mn 2+ -induced signal-to-noise ratio (SNR) increase for the pyramidal cell layers (81%, p < 0.01) and habenular nuclei (79%, p < 0.05) is significantly greater than for LC (49%). These findings indicate that the uptake and/or storage capacity of LC neurons for Mn 2+ ions is limited and suggest that these mechanisms may be preferentially oriented towards Cu 2+ ions.

Discussion
Generation of image contrast. In order to visualize cell assemblies by in vivo MRI, we exploited differences in spin density, T 1 , and T 2 * of water protons among different tissue compartments because these properties determine the observable magnetization in spoiled gradient-echo MRI. First of all, saturation of extracellular free water protons was accomplished by repetitive strong on-resonance irradiation which provides images where intracellular water protons exhibit high signals. Secondly, we used MT to exploit the spin-density difference between intracellular and lipid-associated water protons as the degree of MT saturation primarily depends on the relative concentrations of water and macromolecules 14 (Fig. 2e). Thirdly, prolongation of TE further adds to the spin-density contrast because T 2 and T 2 * are also largely a function of macromolecule concentration 15 ( Supplementary Fig. 3d). Potentially more pronounced off-resonance artifacts due to TE prolongation may be minimized by increasing the spatial resolution with only mild SNR reductions because the receiver bandwidth may be reduced accordingly. Taken together, we cancelled the T 1 contrast in gradient-echo MRI with the use of www.nature.com/scientificreports www.nature.com/scientificreports/ MT and TE prolongation, which results in an image contrast that primarily reflects proton density (c.f., equations in the Methods and Fig. 2g) and only secondarily T 1 or T 2 * relaxation. The proton-density effect dominates the observable signal for lipid-associated water protons (<0.9), while the T 1 effect applies to the extracellular free water protons (>0.95).
In principle, signal intensity in gradient-echo MRI with strong on-resonance and off-resonance irradiation reflects the density of water protons whose T 1 is shortened by paramagnetic ions. This is because water protons with exposure to diamagnetic molecules become proportionally saturated by off-resonance irradiation (Figs 2b and 3), while the free water protons remain saturated by on-resonance irradiation (Fig. 2g). In other words, the effect of water proton T 1 -shortening on MRI signal intensity (i.e., signal increase) induced by diamagnetic molecules is cancelled by the effect induced by off-resonance irradiation (i.e., signal reduction due to saturation transfer), whereas the effect of water proton T 1 -shortening on MRI signal intensity (i.e., signal increase) induced by paramagnetic ions is not. Accordingly, T 1 -shortening by paramagnetic ions increases the MT-MRI signal intensity. On the other hand, shortening of T 2 or T 2 * by paramagnetic ions reduces the signal intensity not only as a result of the more pronounced dephasing, but also by the more pronounced direct-saturation effect 2 . Therefore, the relaxation enhancement factor 16 of paramagnetic ions as well as their local concentration must be considered, because it has a great influence on T 1 and T 2 relaxation times of affected water protons as well as their ratio. Upon binding to macromolecules, the ions with a higher factor provide more efficient T 1 shortening, more efficient T 1 /T 2 ratio reduction, weaker direct-saturation effect, and thus more efficient signal enhancement in MT-MRI. With regard to the most concentrated paramagnetic ions in brain, the relaxation enhancement factor 16 of Fe 3+ on binding to a macromolecule is only 0.3 while that of Cu 2+ is 10 and that of Mn 2+ is 8. This is because Fe 3+ ion has a much shorter electron relaxation time than Cu 2+ or Mn 2+ ion 17 . Given their high relaxation enhancement factors as well as their high intracellular concentration 5 which meets their high metabolic demand, it is possible that Cu 2+ and Mn 2+ together play a role in high MT-MRI signal intensity of some specific cell assemblies, although we showed that the water proton density plays the central role (Fig. 2d-e, Supplementary Fig. 2). In fact, we demonstrated that administration of Mn 2+ shortens T 1 relaxation times, reduces MT ratio, and increases the signal intensity in MT-MRI 18 .
Within a cellular structure, water proton exchange is fast compared to T 1 so that all water protons have the same T 1 . Moreover, the diffusion is efficient at mixing multiple pools within a cellular structure. However, in www.nature.com/scientificreports www.nature.com/scientificreports/ brain there are regions that restrict the diffusion of the water, that possess less cellular structure (i.e., the neuropil), or that represent CSF spaces. These contributions cause contrast in T 1 maps of the brain (e.g., GM, WM, CSF spaces). If diffusion were efficient at mixing multiple pools of water protons throughout the brain, then it would eliminate all T 1 differences. Instead, the observed T 1 contrast indicates that there are brain regions where the diffusion is not efficient to mix multiple pools. In this regard, the present approach is advantageous because it readily provides higher spatial resolution compared to conventional T 1 mapping and thus is expected to distinguish cellular structures in an unprecedented way. Nevertheless, we do not entirely exclude the contribution of extracellular water protons to the MRI signal by the present method.
In general, the direct-saturation effect alters the image contrast beyond the change caused by "pure MT" alone. This effect occurs during application of strong off-resonance irradiation, e.g., several kHz off the water resonance. In this scenario, samples with shorter T 2 times lead to broader resonance linewidths, more direct radiofrequency saturation, and a larger MTR (Supplementary Fig. 2a-c). The fact that the slope of this line is smaller at higher flip angles is simply due to greater saturation by on-resonance irradiation and thus reduced residual magnetization for absorbing off-resonance irradiation. paramagnetic ions in brain in vivo. Iron, the most concentrated transition metal in the brain, is not sufficiently concentrated to interfere with the interaction between the much more concentrated macromolecules and water protons. Even in the globus pallidus, the structure with the highest iron concentration, the MT ratios and T 1 relaxation times are both within the range (Fig. 2b, Supplementary Fig. 2d,g) expected solely by the macromolecule and water content. In addition, Fe 3+ has a short electron spin relaxation time. Thus, a potential binding interaction with macromolecules in vivo may further reduce its relaxivity 16 by 70%. In fact, the T 1 relaxivity of free Fe 3+ of 7-13 L•mmol −1 • sec −1 is reduced 19 to about 4 L•mmol −1 • sec −1 upon binding to ferritin. Brain iron, 90% of which is bound as Fe 3+ to ferritin 20 , can thus be considered as poor T 1 -shortening ion acting primarily on T 2 relaxation time 21 because of its great magnetic moment (5.9). Accordingly, the globus pallidus and red nucleus as the structure with the highest iron content provide low MT-MRI signal intensity in proportion to their low proton density ( Supplementary Fig. 1).
Copper, a transition metal with a capacity to gain and donate electrons, serves as a cofactor for electron transfer by important enzymes. In the brain of mammals, they include cellular respiration (cytochrome-c oxygenase), anti-oxidant defense (superoxide dismutase), and catecholamine synthesis/metabolism (tyrosine hydroxylase and dopamine β-hydroxylase). In addition, copper is involved in glutamate-dependent activity of hippocampal www.nature.com/scientificreports www.nature.com/scientificreports/ neurons 22,23 . Accordingly, copper is most concentrated in the noradrenergic 5,24 , dopaminergic, and glutamatergic neurons, which reflects their high metabolic demands for the metal 25 . Most copper ions in brain are bound to proteins and thus their distribution among different sub-cellular compartments is tightly regulated 24,25 . Therefore, the T 1 relaxivity of Cu 2+ in brain in vivo may be higher than that of Fe 3+ , because Cu 2+ has a long electron spin relaxation time 17 . At 50 MHz, T 1 relaxivity of free Cu 2+ is 19 about 0.5 L•mmol −1 • sec −1 that may be enhanced upon binding 16 to be about 5 L•mmol −1 • sec −1 in vivo, whereas that of free Fe 3+ is 19 6.9 L•mmol −1 • sec −1 that may be reduced 16 to about 3 L•mmol −1 • sec −1 . T 2 relaxivity of Cu 2+ is much lower than Fe 3+ because of its small magnetic moment 19,26 (1.7-2.2). Given (i) these favourable properties for Cu 2+ to efficiently reduce T 1 and T 1 /T 2 ratio as well as (ii) the high MT-MRI signal intensities of LC, A2, SN, and hippocampal neurons that may correspond to the high metabolic demand for copper 25 in their noradrenergic, dopaminergic, or glutamatergic neurons, it is possible that intracellular copper concentration plays a role in MT-MRI signal intensity, although we showed that it is their high proton density that plays the central role.
Manganese is less concentrated in brain than iron or copper as long as its uptake into the systemic blood circulation is physiologically regulated by the hepatic portal system. Thus, the physiological brain concentration of manganese has no effect on MT ratios or T 1 (Supplementary Fig. 2f,i). After administration of exogenous Mn 2+ ions, however, the T 1 relaxation times, MT ratio, and MRI signal intensity in brain alter significantly 18 . After entering the systemic blood circulation and crossing the capillary endothelium, Mn 2+ ions are supposed to accumulate in the cytosol, mitochondria, lysosomes, and microsomes of brain cells, where they may bind to cytosolic proteins and inner mitochondrial membranes 13 . The resulting immobilization of Mn 2+ ions may increase their T 1 relaxivity 16 in vivo, which reduces the T 1 /T 2 ratio efficiently. www.nature.com/scientificreports www.nature.com/scientificreports/ It is unlikely that the MT contrast of specific cellular assemblies shown in the present study is due to unique lipid or protein structures. If the high signal intensity of those structures were due to the presence of T 1 -shortening molecules, then the T 1 of those structures would have been shorter than other gray matter or surrounding structures. The present study showed that exactly the opposite is the case (Supplementary Table 2). Given the long T 1 and low MTR for those structures compared to other GM, the high MRI signal must be attributed to abundant water protons interacting with any T 1 -shortening paramagnetic ions rather than to abundant T 1 -shortening molecules. It is not the T 1 -relaxation but the proton density that determines the in vivo MT-MRI contrast within the brain (Figs 2, 3). Theoretically (c.f., equations in Methods section), T 1 -shortening plays a role, but the MRI signal intensity is dominated by M 0 . In this regard, low MT ratios and high T 1 and T 2 values of SN, LC, and A2 as well as of caudate nucleus and putamen (Supplementary Table 2) indicate that these structures have high water content and thus yield high signal intensities in MT-MRI (Supplementary Figs 1, 4). Conversely, high MT ratios and low T 1 and T 2 values of thalamus, globus pallidus, red nucleus, and subthalamic nucleus (Supplementary Table 2) indicate that these structures have low water content and thus yield low signal intensities in MT-MRI ( Supplementary Fig. 1).
More specifically, the similar MRI contrast described for SN and LC in human brain so far has been attributed to a T 1 -shortening of intracellular water protons induced by neuromelanin 27,28 . Here we showed that cell assemblies like caudate nucleus and putamen that contain no neuromelanin yield as high intensities as SN and LC. www.nature.com/scientificreports www.nature.com/scientificreports/ Further, we demonstrated high MRI signal intensities in LC and A2 of 3-week-old mice as well (Fig. 7b-g). Given the lack of neuromelanin in rodents 29,30 , the high intensities of the catecholaminergic neurons are not attributable to neuromelanin but reflect their high water content. Even when assuming that paramagnetic ions contributed to their high signal intensities, the neuromelanin is unlikely to play a major role. The concentration of neuromelanin Fe in LC (SN) is only about 3.6 (20) ng/mg wet tissue, whereas the concentration of Fe in LC (SN) is as much as about 25 (150) ng/mg wet tissue 31 . The concentration of neuromelanin Cu in LC (SN) is only about 1.2 (0.4) ng/ mg wet tissue, whereas the concentration of Cu in LC (SN) is as much as 31 (16) ng/mg wet tissue.

Conclusions
To summarize, this is the first report about the in vivo MRI visibility of hippocampal and A2 cell assemblies in human as well as of noradrenergic neurons in animals. The generation of image contrast relies upon a simultaneous saturation of extracellular free water protons and lipid-associated water protons, preserving the signals from the water protons that are associated with intracellular paramagnetic ions. The image signal intensity is thus largely attributable to the density of the paramagnetic-ion-associated intracellular water protons and is shown to be unaffected by physiological concentrations of endogenous paramagnetic ions. Only when exogenous Mn 2+ ions with their high relaxation enhancement factor are delivered to the brain in high concentration, the signal intensity showed its T 1 -dependence. Given that the decline of LC neuron numbers and the resulting noradrenalin shortage in brain are associated with aging, dementia, Aβ plaque load, and the progression of Alzheimer's disease 32 , it is foreseeable that MRI of noradrenergic neuron groups as well as of the hippocampal cell assemblies in both laboratory animals and humans will play an increasing role in translational biomedical research of neurodegenerative diseases and beyond. coil. An on-resonance flip angle α of 70° was used for T 1 -weighted MRI while α of 15° was used for proton-density-weighted MRI. For off-resonance irradiation, magnetization transfer (10 ms Gaussian pulse, frequency offset = 1200 Hz, flip angle = 208.5°, amplitude = 27.9 V) and fat saturation were applied at every interleaved slice (every 41 ms) as provided by the manufacturer. T 1 mapping 33,34 as well as T 2 and M 0 mapping (Model-based Accelerated T2 Mapping, Siemens) were performed at the same spatial resolution. Regions-of-interests are selected in the frontal subcortical white matter, prefrontal cortex, caudate nucleus, putamen, thalamus, globus pallidus, subthalamic nucleus, red nucleus, and substantia nigra. Regional values are compared to each other and to the mean regional R 2 * values [35][36][37] as well as to the mean regional content of water 38,39 , iron 40 , copper 41 , and manganese 42 .

MRI
For magnitization-transfer MRI of LC and A2, the data were accumulated twice (TR/TE = 863/4.4 ms, total acquisition = 9 min 10 s). The slices are positioned perpendicular to the anterior wall of the fourth ventricle. The LC is observable in the 5-7th slices from the top, while A2 is observable in the 16-18th.
Calculation of signal intensity in gradient-echo MRI of the brain. In MRI of the brain in vivo, the signal originates exclusively from water protons. With signal contribution from T 2 coherence being negligible, the observable magnetization in the steady state of spoiled gradient-echo MRI yields: Animals and anesthesia. Mice were housed in groups under standard conditions at a temperature of 22 °C and a 12 h light/dark cycle with ad libitum access to standard food and water. A total of 32 mice were used. Firstly, 24 mice (NMRI, 6 male and 18 female, 3-8 weeks old) were used for the optimization of magnetization transfer. Secondly, to elucidate the nature of MRI contrast in the hippocampus, FVB/N mice (n = 2, male, 9 weeks old) were treated with a kainic acid lesion model 43 . FVB/N mice received a single subcutaneous injection of kainic acid (30 mg/kg, pH 7.3) or placebo (saline). During a subsequent 3 h observation period for behavioral symptoms, the lesioned animal showed signs of epileptic activity, including staring, decreased motility, twitching, rearing, and falling. MRI was performed before administration of kainic acid or placebo as well as 4 days later. Immediately after the second MRI examination, animals were transcardially perfused with neutral phosphate-buffered (2019) 9:5084 | https://doi.org/10.1038/s41598-019-41587-2 www.nature.com/scientificreports www.nature.com/scientificreports/ formalin (10%). Horizontal brain sections were cut at a thickness of 40 μm. Every third section was stained with cresyl violet to determine nerve cell loss. Thirdly, six mice (NMRI, female, 8-12 weeks old) were used for the manganese uptake study.
After induction of anesthesia with 5% isoflurane, animals were intubated with a purpose-built polyethylene endotracheal tube (0.58 mm inner diameter, 0.96 mm outer diameter) and artificially ventilated using an animal respirator (TSE, Bad Homberg, Germany) with a respiratory rate of 25 breaths per minute and an estimated tidal volume of 0.35 ml as previously described [44][45][46] . The animals were then placed in a prone position on a purpose-built palate holder equipped with an adjustable nose cone. The Göttingen animal bed 47 secured a reproducible and reliable fixation of the mouse head and receiver coil in the magnet isocenter. Respiratory movement of the abdomen as well as rectal temperature was monitored by a unit supplied by the manufacturer (Bruker Biospin MRI GmbH, Ettlingen, Germany).
Optimization of magnetization transfer in mouse brain. At  We started with the use of 12 ms off-resonance pulse with mean amplitude of 200 Hz and a frequency offset of 5000 Hz, because this was found to be optimal for magnetization transfer in mouse brain in vivo 48 . With the use of this off-resonance pulse, we first tried to find out an optimal flip angle for the on-resonance pulse in 3D gradient echo MRI in order to delineate the LC and A2. As shown in Supplementary Table 1a, a flip angle of 22° turned out to be optimal. A flip angle of 18°~22° turned out also to be optimal for 12 ms off-resonance pulse with a mean amplitude of 100 Hz and a frequency offset of 2500 Hz (Supplementary Table 1b). Next, we found out that for the off-resonance pulse with a mean amplitude of 200 Hz, a frequency offset turned out to play no major role within the range between 3000 and 7000 Hz (Supplementary Table 1c). For amplitude of 100 Hz (Supplementary  Table 1d), 2500-2800 Hz offset turned out to be optimal. For each respective optimal frequency offsets, the use of a lower amplitude resulted in a lower contrast-to-noise ratio (Supplementary Table 1e,f). The comparison of these two different amplitudes with respective optimal offsets (Supplementary Table 1g) shows that there was no substantial difference between them as far as contrast-to-noise ratio is concerned, because the greater amplitude generally resulted in lower signal-to-noise ratio. The use of the off-resonance pulse with a mean amplitude of 200 Hz and a frequency offset of 5000 Hz yielded the mean signal-to-noise ratio of LC to be 33.9-36.0 and of A2 to be 35.2-36.4, while with a mean amplitude of 100 Hz and a frequency offset of 2500 Hz yielded the mean signal-to-noise ratio of LC to be 36.7-39.2 and of A2 to be 37.8-39.6 (Supplementary Table 1a-g). There was also no difference between males and females (Supplementary Table 1h). t 1 mapping of mouse brain. T 1 relaxation times of LC and A2 were determined using a spin-echo multiple TR saturation recovery method (TE 16 ms, field of view 30 mm × 30 mm, matrix 256 × 256, in-plane resolution 117 µm × 117 µm, slice thickness 234 µm, five slices). TR was varied through 200, 400, 800, 1500, 3000, 5000 ms, six averages, and the total measuring time 209 min. Coronal MRI slices were selected to include LC and A2. The mean MRI signal intensity of each pixel was fitted for each TR value with the standard single exponential function to yield T 1 values.
Manganese uptake study. Each mouse received manganese chloride (0.12 mmol/kg body weight) via subcutaneous injection. The mice were returned to a chamber with unlimited access to food and water. Before and 3 days after Mn 2+ injection, MRI measurements were carried out at 2.35T. An off-resonance RF irradiation with a frequency offset of 5 kHz and a mean amplitude of 200 Hz (flip angle 1045°) was incorporated into a T 1 -weighted gradient-echo MRI sequence (RF-spoiled 3D FLASH, TR/TE 30/7.6 ms, α 25°) at 117 µm isotropic resolution. For evaluation of signal intensities, regions-of-interest were selected in LC, other nerve cell assemblies (hippocampal formation, habenula, cerebellar cortex), cerebral cortex, and in the white matter (corpus callosum, cerebellar white matter). Mouse brain MRI evaluation. For evaluation of signal intensities, anatomically defined cross-sections were obtained from the original 3D MRI data sets by multiplanar reconstructions using software supplied by the manufacturer (Paravision 5.0, Bruker Biospin MRI GmbH, Ettlingen, Germany). The plane of the anterior commissure -posterior commissure served as a reference for the selection of standardized sections to facilitate comparisons with minimized intra-and inter-individual variability. For LC or A2, a rectangular region-of-interest of 6 pixels was taken in the center of the structures. For the brainstem, a circular region-of-interest of 1004 pixels was taken in the brainstem between LC and A2. SNR was defined as the mean MRI signal intensity divided by the standard deviation of the noise. The contrast-to-noise ratio was obtained by taking the difference between the SNR values. The analysis followed a strategy previously developed for intra-individual comparisons of MR images obtained after manganese administration 45 . Statistical evaluation was performed using SPSS ® (version 21.0, IBM ® ) and Microsoft Excel software. Significant differences between two groups of data were determined by the Mann-Whitney's U-test.
www.nature.com/scientificreports www.nature.com/scientificreports/ Compliance with Ethical Standards. All procedures involving human participants in this study were approved by the institutional committee of the Georg-August-Universität Göttingen and performed in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments. All participants gave written informed consent before each examination.
All animal experiments were performed in accordance with German animal protection laws after approval by the responsible governmental authority (Landesamt für Verbraucherschutz und Ernährungssicherheit, Braunschweig, Germany) as well as by the institutional review board for animal protection.
Third party rights. Images and drawings were taken or created by an author of the paper.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.