In Vivo 3D MRI Staining of Mouse Brain AfterSubcutaneous Application of MnCl2

Takashi Watanabe, Oliver Natt, Susann Boretius, Jens Frahm, and Thomas Michaelis*

Follow-up T1-weighted 3D gradient-echo MRI (2.35 T) of murinebrain in vivo (N � 5) at 120 �m isotropic resolution revealedspatially distinct signal increases 6–48 hr after subcutaneousapplication of MnCl2 (20 mg/kg). The effects result from a short-ening of the water proton T1 relaxation time due to the presenceof unchelated paramagnetic Mn2� ions, which access the brainby systemic circulation and crossing of the blood–brain barrier(BBB). A pronounced Mn2�-induced signal enhancement wasfirst seen in structures without a BBB, such as the choroidplexus, pituitary gland, and pineal gland. Within 24 hr afteradministration, Mn2� contrast highlighted the olfactory bulb,inferior colliculi, cerebellum, and the CA3 subfield of the hip-pocampus. The affinity of Mn2� to various brain systems sug-gests the neuronal uptake of Mn2� ions from the extracellularspace and subsequent axonal transport. Thus, at least part ofthe Mn2� contrast reflects a functional brain response of be-having animals, for example, in the olfactory system. In vivo MRIstaining of the brain by systemic administration of MnCl2 maycontribute to phenotyping mutant mice with morphologic andfunctional alterations of the central nervous system. MagnReson Med 48:852–859, 2002. © 2002 Wiley-Liss, Inc.

Key words: magnetic resonance imaging; mouse; brain; man-ganese; hippocampus

Progress in neurogenetics has renewed interest in imagingneuroscience because 1) it can improve our understandingof the structure and function of the central nervous systemin the context of genetic information, and 2) it can be usedto evaluate novel therapeutic interventions in active ani-mals. In either case, gene technology has led to the use ofan increasing number of mutant mice, which requires theuse of in vivo assessments such as, for example, thoseoffered by noninvasive MRI techniques. Several in vivoMRI studies of mouse brain have demonstrated potentialfor providing detailed morphologic insights (1–6). Never-theless, the anatomic information obtainable is limitedand a considerable number of abnormalities may still haveto be verified by other methods because of their micro-scopic size, sparse distribution, and/or poor MRI detect-ability caused by subtle alterations of MR properties.Therefore, additional means of achieving soft-tissue con-trast enhancement or MRI staining (7) by exogenous com-pounds are highly desirable.

Because common MRI contrast agents consist of che-lated paramagnetic ions, their systemic application doesnot lead to a penetration of the blood-brain barrier (BBB).Such contrast agents are safe for human studies and can be

used clinically to detect disturbances of the BBB. Thesituation differs for free divalent metal ions, which exhibitvariable degrees of neurotoxicity. In particular, high-reso-lution autoradiography has demonstrated that manganeseaccumulates in the olfactory bulb, olfactory nuclei, infe-rior colliculi, amygdala, thalamus, hippocampal forma-tion, and cerebellum (8–12). Upon bolus injection, Mn2�

enters the brain across the capillary endothelium (13–15)and leads to biologic halflives of 51–74 days (9). In con-junction with its well-known capabilities as a paramag-netic MRI contrast agent that effectively reduces the T1

relaxation time of accessible water protons (16), these datasuggest that Mn2� is a good candidate for MRI staining ofanimal brain after systemic administration.

Previous MRI studies of manganese in animal brain ad-dressed its regional distribution and cerebral toxicity dur-ing an acute phase 3–22 hr after Mn2� exposure in ratbrain (17) or in response to chronic poisoning (of severalmonths duration) in rabbits and monkeys (18,19). In con-trast to the autoradiography results, however, the MRIstudies failed to report any signal enhancement in struc-tures such as the olfactory bulb or hippocampal formation.Most likely, however, this observation can be ascribed tothe limited spatial resolution available and the concomi-tant elimination of anatomic contrast by significant partialvolume effects of small (but distinct) neighboring struc-tures. The purpose of this work was to combine the devel-opment of a high-resolution T1-weighted 3D MRI approachto mouse brain in vivo with the subcutaneous applicationof MnCl2. The primary aim was to achieve a more sensitiveaccess to Mn2�-induced soft-tissue contrast and therebyimprove the delineation of specific cerebral microstruc-tures.

MATERIALS AND METHODS

Animals and Anesthesia

Animal studies were performed in accordance with Ger-man animal protection laws after approval was given bythe responsible governmental authority. Female NMRImice (4–6 weeks old, 24–28 g, N � 5) received a 0.4%MnCl2 solution (20 mg/kg) within a glucose solution(4.2%) via subcutaneous injections into the axillary adi-pose tissue of both sides. Animals were fasted for 8 hrprior to MRI examinations.

Anesthesia was induced by i.p. injection of xylazine(12.5 mg/kg), ketamine (125 mg/kg), and atropine(0.08 mg/kg). The animals were intubated with a purpose-built polyethylene endotracheal tube (0.58 mm inner diam-eter, 0.96 mm outer diameter) and artificially ventilated usingan animal respirator (Rhema, Germany) with an inspiratorytime of 0.1 s, a respiratory rate of 120 breaths per minute, andan estimated tidal volume of 0.15–0.25 ml. Anesthesia was

Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut fur bio-physikalische Chemie, Gottingen, Germany.*Correspondence to: T. Michaelis, Ph.D., Biomedizinische NMR ForschungsGmbH, 37070 Gottingen, Germany. E-mail: tmichae@gwdg.deReceived 26 March 2002; revised 12 June 2002; accepted 16 July 2002.DOI 10.1002/mrm.10276Published online in Wiley InterScience (www.interscience.wiley.com).

Magnetic Resonance in Medicine 48:852–859 (2002)

© 2002 Wiley-Liss, Inc. 852

maintained using 0.2–0.6% halothane in a 7:3 mixture ofN2O and O2. For MRI the animals were placed in a proneposition on a purpose-built palate holder equipped with anadjustable nose cone. A heated water blanket and bed wereused to maintain rectal body temperature at 37 � 1°C. Tominimize risks of complications from frequently repeatedanesthesia, T1-weighted 3D MRI data sets were acquired be-fore Mn2� administration (mice A–C) as well as 6 hr (mice A,D, and E), 24 hr (mice B–E), and 48 hr (mice A–D) after Mn2�

administration. After each measurement the animals wererecovered from anesthesia and returned to their cages withfree access to food and water.

MRI

MRI was carried out at 2.35 T using an MRBR 4.7/400-mmmagnet (Magnex Scientific, Abingdon, UK) equipped withB-GA20 gradients (200 mm inner diameter, 100 mT m�1

maximum strength) and driven by a DBX system (BrukerBiospin, Ettlingen, Germany). Radiofrequency excitationand signal reception were accomplished with use of aHelmholtz coil (100 mm) and an elliptical surface coil(20 mm anterior–posterior, 12 mm left–right), respectively.High-resolution data sets were acquired using a T1-weighted 3D gradient-echo MRI sequence (RF-spoiled 3Dfast low-angle shot (FLASH), repetition time (TR) � 22 ms,echo time (TE) � 8.2 ms, receiver bandwidth � 17.9 kHz,flip angle � 30°) at an isotropic voxel resolution of120 �m � 120 �m � 120 �m (field of view (FOV) �30.72 mm � 15.36 mm � 15.36 mm in conjunction with anacquisition matrix of 256 � 106 � 106 zero-filled to 256 �128 � 128 during image reconstruction). Signal averaging(24 accumulations) led to a measuring time of 99 min.

For quantitative evaluations, the signal-to-noise ratio(SNR) of selected brain structures, here defined as the MRIsignal intensity divided by the standard deviation (SD) ofthe noise, was determined using software supplied by themanufacturer. Anatomic cross-sections were obtained bymultiplanar reconstructions from the original 3D MRI datasets. The analysis followed a strategy previously devel-oped for intraindividual comparisons of follow-up MRI

after intraocular Mn2� administration (20). Standardizedregions of interest (ROIs) were selected in murine brain inclose accordance with resolved anatomic structures.

RESULTS AND DISCUSSION

Subcutaneous application of MnCl2 led to spatially dis-tinct signal increases within all of the animal brains. Table1 summarizes the SNR of multiple enhancing structures asa function of time after injection relative to basal values.As demonstrated in Fig. 1 for selected regions, the kineticbehavior leads to a gross separation of two different types

Table 1SNR of Enhanced Structures in Murine Brain After Subcutaneous MnCl2

RegionROI

(mm2)Basal

(N � 3)6 hr

(N � 3)24 hr

(N � 4)48 hr

(N � 4)

Choroid plexus 0.25 28.7 � 0.8 63.3 � 1.3a 43.1 � 6.6a 35.5 � 1.5a

Pineal gland 0.10 32.3 � 5.7 57.6 � 1.0a 36.2 � 2.5 33.1 � 1.6Posterior pituitary 0.13 50.5 � 2.7 59.3 � 3.7a 53.0 � 2.3 54.0 � 1.3Anterior pituitary 0.20 37.3 � 2.1 56.3 � 7.0a 55.1 � 5.5a 58.7 � 5.9a

Frontal cortex 0.26 34.7 � 0.5 36.2 � 1.8 35.3 � 2.9 35.8 � 2.2Basal ganglia 0.40 31.1 � 0.3 33.8 � 2.2 32.5 � 3.0 33.5 � 1.8Olfactory bulb 2.10 34.5 � 0.6 39.1 � 2.2 42.8 � 4.3a 41.5 � 2.7a

Anterior forebrain 0.10 29.5 � 0.6 31.3 � 2.1 36.1 � 3.4a 37.0 � 3.0a

Inferior colliculus 0.70 43.7 � 0.3 43.7 � 2.3 45.8 � 5.0 47.8 � 3.2Cerebellum 0.50 38.8 � 0.3 41.5 � 1.8 42.2 � 4.5 42.9 � 2.6a

Hippocampusb 0.53 27.4 � 2.0 32.4 � 4.5 29.2 � 1.8 30.4 � 1.0CA3 0.15 37.1 � 1.4 39.5 � 2.5 40.7 � 3.7 40.9 � 2.3a

Dentate gyrus 0.10 37.2 � 1.5 38.9 � 2.7 41.0 � 4.2 42.2 � 1.3a

The data represent mean values � SD averaged across animals.aP � 0.05 (unpaired t-test vs. basal).bStratum radiatum and stratum lacunosum-moleculare of the Ammon’s horn.

FIG. 1. Time course of mean SNR values in selected structures ofmurine brain before subcutaneous Mn2� administration (mice A–C),and 6 hr (mice A, D, and E), 24 hr (mice B–E), and 48 hr (mice A–D)after Mn2� administration. Maximum SNR increases are observed inthe choroid plexus (CP) and anterior pituitary (AP) gland, which areaccessible by systemic circulation. In contrast, the need to cross theBBB, and the neuronal uptake of Mn2� causes a slower and weakerenhancement in brain systems such as the olfractory bulb (OB) andthe CA3 region of the hippocampus.

3D MRI of Mouse Brain In Vivo 853

of Mn2�-induced signal enhancement. A pronounced andrelatively fast enhancement, i.e., about a twofold increasein SNR after 6 hr, is seen in the choroid plexus as well asin intracranial endocrine tissue. In contrast, brain struc-tures such as the olfactory bulb and the CA3 region of thehippocampus reveal a weaker and delayed enhancementof about 10–25% SNR increase, which lasts for up to 48 hr.

Rapidly Enhancing Structures

Figure 2 compares MR images of a mouse brain obtainedbefore (left) and 6 hr after (right) MnCl2 injection. The

choroid plexus, pineal gland, and anterior pituitary glandare less well observable without Mn2� contrast, but theposterior lobe of the pituitary gland can be readily identi-fied. After MnCl2 injection, all of these structures show apronounced and rapid enhancement, with a maximumSNR increase at about 6 hr after injection (see also Fig.1 and Table 1). Of note, the elevated signal in the choroidplexus, pineal gland, and posterior pituitary gland recov-ered toward basal values within 1–2 days. In contrast, theanterior lobe of the pituitary gland remained enhancedeven after 48 hr.

FIG. 2. T1-weighted MRI (3D FLASH, TR/TE � 22/8 ms, flip angle � 30°, 120 �m isotropic resolution) of the brain of an anesthetized mouse(left) before and (right) 6 hr after subcutaneous MnCl2 (20 mg/kg). Top: Signal enhancement in the choroid plexus and the pineal and pituitaryglands in a midsagittal section. The pineal and pituitary glands (middle) and the choroid plexus (bottom) within the fourth ventricle incorresponding coronal sections.

854 Watanabe et al.

The observation of a rapid enhancement of the choroidplexus, pineal gland, and pituitary gland is in line withprevious autoradiographic observations (8,13,14) using ra-dioactive Mn2� and MRI (17) after MnCl2 (i.p.). This find-ing may be explained by the direct vascular supply, as thecapillaries traversing these regions are known to be freelypermeable to plasma solutes. The subsequent decrease ofthe SNR supports this explanation, because systemicallyadministered Mn2� has been reported to be rapidly clearedfrom blood and secreted into the bile (21). The prolonged

enhancement of the anterior pituitary gland indicates re-tention and continous accumulation of manganese in thisendocrine tissue. It may reflect a special uptake mecha-nism reported for Ca2� channels of cells of the anteriorpituitary (22).

Slowly Enhancing Structures

In addition to tissue, which exhibits a strong Mn2� con-trast as early as a few hours after injection, Figs. 3 and 4

FIG. 3. T1-weighted MRI of the brain of an anesthetized mouse before (left) and 24 hr after (right) subcutaneous MnCl2 (other parameters as inFig. 2). Top: Signal enhancement in the olfactory bulb and in a tract-like structure along the base of the anterior forebrain (arrowhead) in aparasagittal section 1.5 mm lateral to the midline. Middle: Mn2�-induced contrast in the olfactory bulb reveals four layers with alternating signalenhancement in a coronal section. Bottom: Tract-like structures along the base of the anterior forebrain in a coronal section.

3D MRI of Mouse Brain In Vivo 855

show T1-weighted images of enhanced structures at 24 hrafter subcutaneous MnCl2. The penetration of the BBB andthe distribution of Mn2� in the extracellular space, as wellas a subsequent neuronal uptake and axonal transport,may be responsible for the development of correspond-ingly slower SNR increases. While the former processescould explain the observed tendency for signal increasesas seen, for example, in the frontal cortex and basal ganglia(Table 1), the latter mechanisms cause a specific enhance-ment that, apart from uptake properties, depends on brainfunction.

Figure 3 demonstrates a marked Mn2�-induced en-hancement of the olfactory bulb, which presents as fourconcentrically arranged layers with alternating high andlow signals when passing from superficial to deep layers(Fig. 3, middle right). In comparison with histologic find-ings (23,24), the high signals probably originate from theglomerular layer and the mitral cell layer with adjacentplexiform layers. In addition, tract-like structures becomeenhanced bilaterally at the ventral surface of the anteriorforebrain (Fig. 3, top and bottom right). Rostrally con-

nected to the olfactory bulb, the elevated signal can beobserved about 2.5 mm along the cranial base. In agree-ment with previous tracing studies (23), this structuremost likely represents the lateral olfactory tract, whichprovides further evidence for the uptake and axoplasmictransport of Mn2� by secondary olfactory neurons. Theinterpretation is supported by the observation that thedeeper of the two bright layers within the olfactory bulbappears to include the mitral cell layer, which consists ofcomparatively large somata of the principal secondary ol-factory neurons.

Figure 4 shows the achievable Mn2� contrast in theinferior colliculi and cerebellum. The inferior colliculiyield relatively high signal intensities in the basal statebecause of a correspondingly short T1 relaxation time.Subcutaneous MnCl2 increases the contrast to neighboringtissue even further (Fig. 4, top right). Similarly, MnCl2injection improves the delineation of layered structureswithin the cerebellum (Fig. 4, middle right and bottomright). An outer bright layer circumscribes an interior re-gion without a substantial SNR increase after Mn2� appli-

FIG. 4. T1-weighted MRI of the brain of ananesthetized mouse before (left) and 24 hrafter (right) subcutaneous MnCl2 (other pa-rameters as in Fig. 2). Top: Signal enhance-ment in the inferior colliculi in a coronal sec-tion. Mn2�-induced contrast in the cerebel-lum reveals a laminar structure in amidsagittal (middle) and horizontal (bottom)section.

856 Watanabe et al.

cation. Based on conventional histology (25,26), the brightsignals within the cerebellar cortex probably originatefrom the Purkinje cell layer and adjacent areas, while theinterior non-enhancing areas represent white matter.

Hippocampus

Figure 5 shows the effect of Mn2� on the hippocampalformation. Specific details are readily resolved in sagittal(top) and coronal (bottom) sections due to the high isotro-pic resolution of the 3D gradient-echo images. A C-shapedstructure with relatively high signal intensities in the basalstate (top left) extends from the dentate gyrus to the CA3subfield of the hippocampus. After Mn2� injection, thesestructures are further enhanced. However, such a signalincrease, is not observed within the CA1 subfield. Thesefindings are confirmed in Fig. 6, which compares histo-logic sections of the mouse brain (27) at two differentlevels of the hippocampus with corresponding T1-weighted images before (left) and after (middle) Mn2�

administration. Again, high signals in both the middle andventral sections of the hippocampus are further enhancedafter Mn2� injection. Confocal laser scanning microscopyidentified the stratum pyramidale (SP), stratum lucidum(SL) (containing the mossy fibers), hilus (H), granule celllayer (GL), and isolated neurons (white dots). In compari-son with these histologic data, the microstructures con-trasted by Mn2�-enhanced MRI most likely originate fromthe mossy fibers and adjacent pyramidal cell layer of the

CA3 subfield, as well as from the dentate hilus, with partsof the adjacent granule cell layer.

The relatively bright signal in Ammon’s horn beforeMn2� injection refers to a smaller T1 relaxation time in thecytoplasm and nucleus than in the extracellular space (28).Typically, a high cellular density or tight packing (as inwhite matter) leads to a fast T1 relaxation process. Thisexplanation is in line with morphologic studies of themouse Ammon’s horn (29), which indicate that “the pyra-midal layer is thick and manifests three or four rows ofcells between which there is no more space than thatnecessary to contain the intercellular plexus” (page 17). Inaddition, the mossy fibers are reported to “make up aheavy bundle which travels above the giant pyramids”(page 37) in the CA3 subfield.

Mechanisms

After subcutaneous application of MnCl2 the brain be-comes exposed to Mn2� by systemic circulation. Directaccess is provided to structures without a BBB, whichtherefore exhibit both a rapid and pronounced signal en-hancement in T1-weighted MRI. The effect results from areduction of the T1 relaxation time of water protons due toneighboring paramagnetic Mn2� ions. In addition to tran-sient accumulation in the choroid plexus and the pituitaryand pineal glands, Mn2� ions cross the BBB via the cap-illary endothelium (13–15) and thus enter the brain andCSF. These unspecific mechanisms most likely contribute

FIG. 5. T1-weighted MRI of the brain of ananesthetized mouse before (left) and 24 hrafter (right) subcutaneous MnCl2 (other pa-rameters as in Fig. 2). Top: Signal enhance-ment in the CA3 subfield and dentate gyrusof the dorsal hippocampus in a parasagittalsection 1.5 mm lateral to the midline. Bot-tom: Signal enhancement in the dorsal hip-pocampus in a coronal section.

3D MRI of Mouse Brain In Vivo 857

to the observed tendency for the SNR increase throughoutthe brain.

The specific MRI enhancement of brain structures byMn2� is a consequence of its uptake by neurons throughCa2� channels during depolarization (30,31). This phe-nomenon has been exploited for mapping brain functionsby introducing free Mn2� into the rat brain after pharma-cologic opening of the BBB (32,33). The procedure causesan activation-induced accumulation of Mn2� in relevantbrain systems. Although the present study did not facili-tate the Mn2� transfer through the BBB, the distinct signalincreases in parts of the olfactory and auditory systems, aswell as in the cerebellum and hippocampal formation, arelikely to reflect brain activity of the animals integratedover time after MnCl2 administration. Apart from neuronaluptake, part of the observed enhancement requires axo-plasmic transport of Mn2� ions. Anterograde transport ofradioactive Mn2� after microinjection into the rat basalganglia has been found to result in a region-specific accu-mulation and retention for at least 72 hr (34). Recent MRIadaptations utilized axonal Mn2� transport to delineatethe olfactory and visual pathway in rodents (20,35). In thepresent study, the pronounced Mn2� contrast in the lateralolfactory tract and the olfactory bulb strongly suggests anaxoplasmic transport by active secondary olfactory neu-rons. MRI enhancement in other structures, such as thehippocampal mossy fibers, may also be ascribed to thismechanism.

Finally, contributions to the observed MRI signal en-hancement may also arise from the distribution of Mn2�-

dependent enzymes such as glutamine synthetase and su-peroxide dismutase (15). Glutamine synthetase is an astro-cyte-specific protein that contains eight Mn2� ions andaccounts for approximately 80% of the total manganese inbrain (36). More specifically, manganese superoxide dis-mutase was found to be localized in the mitochondria ofrat hippocampus (37). In agreement with the present ob-servation of a pronounced Mn2� effect in the Ammon’shorn, CA3 pyramidal cells were strongly immunostainedby superoxide dismutase, whereas CA1 pyramidal cellswere only weakly reactive.

CONCLUSIONS

In summary, the present results demonstrate enhancedsoft-tissue contrast in T1-weighted MRI of murine brain invivo after subcutaneous administration of MnCl2. Apartfrom signal increases in structures without a BBB, thespecific enhancement of several brain systems suggests theneuronal uptake and axonal transport of Mn2� in responseto brain function of behaving animals. Although a moredetailed elucidation and assignment of pathways andmechanisms that contribute to the cerebral distribution ofMn2� requires additional studies using conventional his-tology, Mn2�-based MRI staining techniques have the po-tential to become a powerful tool for phenotyping mutantmice with an abnormal morphologic and functional orga-nization of the brain.

FIG. 6. T1-weighted MRI of the brain of an anesthetized mouse before (left) and 48 hr after (middle) subcutaneous MnCl2 (other parametersas in Fig. 2) in comparison with histologic sections (right) (courtesy of Jinno et al. (27)) (GL � granule cell layer, H � hilus, L � stratumlucidum, SP � stratum pyramidale). The section orientation is perpendicular to the dorsoventral axis of the hippocampal formation. Top:At the middle level of the hippocampus, the signal enhancement is most pronounced in the stratum pyramidale in CA3, stratum lucidum,and hilus. Bottom: Corresponding findings in the ventral hippocampus.

858 Watanabe et al.

ACKNOWLEDGMENTS

The authors thank Dr. Shozo Jinno from the Department ofAnatomy and Neurobiology, Faculty of Medicine, KyushuUniversity, for providing the confocal laser microscopicimages used in Fig. 6.

REFERENCES

1. Kornguth S, Anderson M, Markley JL, Shedlovsky A. Near-microscopicmagnetic resonance imaging of the brains of phenylalanine hydroxy-lase-deficient mice, normal littermates, and of normal BALB/c mice at9.4 Tesla. NeuroImage 1994;1:220–229.

2. Munasinghe JP, Gresham GA, Carpenter TA, Hall LD. Magnetic reso-nance imaging of the normal mouse brain. Lab Anim Sci 1995;45:674–679.

3. Bouilleret V, Nehlig A, Marescaux C, Namer IJ. Magnetic resonanceimaging follow-up of progressive hippocampal changes in a mousemodel of mesial temporal lope epilepsy. Epilepsia 2000;41:642–650.

4. Kooy FR, Verhoye M, Lemmon V, Van Der Linden A. Brain mousemodels for neurogenetic disorders using in vivo magnetic resonanceimaging (MRI). Eur J Hum Genet 2001;9:153–159.

5. McDaniel B, Sheng H, Warner DS, Hedlund LW, Benveniste H. Track-ing brain volume changes in C57BL/6J and ApoE-deficient mice in amodel of neurodegeneration: a 5-week longitudinal micro-MRI study.NeuroImage 2001;14:1244–1255.

6. Natt O, Watanabe T, Boretius S, Michaelis T, Frahm J. In vivo high-resolution MRI of mouse brain at low field (2.35 T). In: Proceedings ofThe 10th Annual Meeting of ISMRM, Honolulu, 2002; p 1253.

7. Johnson GA, Cofer GP, Gewalt SL, Hedlund LW. Morphologic pheno-typing with MR microscopy: the visible mouse. Radiology 2002;222:782–793.

8. Takeda A, Akiyama T, Sawashita J, Okada S. Brain uptake of tracemetals, zinc and manganese, in rats. Brain Res 1994;640:341–344.

9. Takeda A, Sawashita J, Okada S. Biological half-lives of zinc andmanganese in rat brain. Brain Res 1995;695:53–58.

10. Takeda A, Ishiwatari S, Okada S. In vivo stimulation-induced release ofmanganese in rat amygdala. Brain Res 1998;811:147–151.

11. Sotogaku N, Oku N, Takeda A. Manganese concentration in mousebrain after intravenous injection. J Neurosci Res 2000;61:350–356.

12. Gallez B, Baudelet C, Geurts M. Regional distribution of manganesefound in the brain after injection of a single dose of manganese-basedcontrast agents. Magn Reson Imaging 1998;16:1211–1215.

13. Murphy VA, Wadhwani KC, Smith QR, Rapoport SI. Saturable trans-port of manganese(II) across the rat blood-brain barrier. J Neurochem1991;57:948–954.

14. Rabin O, Hegedus L, Bourre JM, Smith QR. Rapid brain uptake ofmanganese(II) across the blood-brain barrier. J Neurochem 1993;61:509–517.

15. Aschner M. Manganese homeostasis in the CNS. Environ Res A 1999;80:105–109.

16. Lauterbur PC, Mendonca-Dias MH, Rudin AM. Augmentation of tissuewater proton spin-lattice relaxation rates by in vivo addition of para-magnetic ions. In: Dutton PL, Leigh LS, Scarpaa A, editors. Frontier ofbiological energetics. New York: Academic Press; 1978; p 752–759.

17. London RE, Toney G, Gabel SA, Funk A. Magnetic resonance imagingstudies of the brains of anesthetized rats treated with manganese chlo-ride. Brain Res Bull 1989;23:229–235.

18. Newland MC, Ceckler TL, Kordower JH, Weiss B. Visualizing manga-nese in the primate basal ganglia with magnetic resonance imaging.Exp Neurol 1989;106:251–258.

19. Kim SH, Chang KH, Chi JG, Cheong HK, Kim JY, Kim YM, Han MH.Sequential change of MR signal intensity of the brain after manganeseadministration in rabbits. Invest Radiol 1999;34:383–393.

20. Watanabe T, Michaelis T, Frahm J. Mapping of retinal projections inthe living rat using high-resolution 3D gradient-echo MRI with Mn2�-induced contrast. Magn Reson Med 2001;46:424–429.

21. Bertinchamps AJ, Miller ST, Cotzias GC. Interdependence of routesexcreting manganese. Am J Physiol 1966;211:217–224.

22. Ciu ZJ, Dannies PS. Thyrotropin-releasing hormone mediated Mn2�

entry in perfused rat anterior pituitary cells. Biochem J 1992;283:507–513.

23. Shipley MT, Adamek GD. The connections of the mouse olfactory bulb:a study using orthograde and retrograde transport of wheat germ agglu-tinin conjugated to horseradish peroxidase. Brain Res Bull 1984;12:669–688.

24. Royet JP, Souchier C, Jourdan F, Ploye H. Morphometric study of theglomerular population in the mouse olfactory bulb: numerical densityand size distribution along the rostrocaudal axis. J Comp Neurol 1988;270:559–568.

25. Ozol K, Hayden JM, Oberdick J, Hawkes R. Transverse zones in thevermis of the mouse cerebellum. J Comp Neurol 1999;412:95–111.

26. Yamada K, Fukaya M, Shibata T, Kurihara H, Tanaka K, Inoue Y,Watanabe M. Dynamic transformation of Bergmann glial fibers pro-ceeds in correlation with dendritic outgrowth and synapse formation ofcerebellar Purkinje cells. J Comp Neurol 2000;418:106–120.

27. Jinno S, Aika Y, Fukuda T, Kosaka T. Quantitative analysis of GABAer-gic neurons in the mouse hippocampus, with optical disector usingconfocal laser scanning microscope. Brain Res 1998;814:55–70.

28. Schoeniger JS, Aiken N, Hsu E, Blackband SJ. Relaxation-time anddiffusion NMR microscopy of single neurons. J Magn Reson B 1994;103:261–273.

29. Ramon y Cajal S. The structure of Ammon’s horn. Springfield, IL:Charles C. Thomas; 1968.

30. Drapeau P, Nachshen DA. Manganese fluxes and manganese-depen-dent neurotransmitter release in presynaptic nerve endings isolatedfrom rat brain. J Physiol 1984;348:493–510.

31. Narita K, Kawasaki F, Kita H. Mn and Mg influxes through Ca channelsof motor nerve terminals are prevented by verapamil in frogs. Brain Res1990;510:289–295.

32. Lin YJ, Koretsky AP. Manganese ion enhances T1-weighted MRI duringbrain activation: an approach to direct imaging of brain function. MagnReson Med 1997;38:378–388.

33. Duong TQ, Silva AC, Lee AP, Kim SG. Functional MRI of calcium-dependent synaptic activity: cross correlation with CBF and BOLDmeasurements. Magn Reson Med 2000;43:383–392.

34. Sloot WN, Gramsbergen JP. Axonal transport of manganese and itsrelevance to selective neurotoxicity in the rat basal ganglia. Brain Res1994;657:124–132.

35. Pautler RG, Silva AC, Koretsky AP. In vivo neuronal tract tracing usingmanganese-enhanced magnetic resonance imaging. Magn Reson Med1998;40:740–748.

36. Wedler FC, Denman RB. Glutamine synthetase: the major Mn (II) en-zyme in mammalian brain. Curr Top Cell Regul 1984;24:153–169.

37. Akai F, Maeda M, Suzuki K, Inagaki S, Takagi H, Taniguchi N. Immu-nocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat. Neurosci Lett 1990;115:19–23.

3D MRI of Mouse Brain In Vivo 859