In vivo 3D MRI staining of the mouse hippocampal system using intracerebral injection of MnCl2

www.elsevier.com/locate/ynimg NeuroImage 22 (2004) 860 – 867

In vivo 3D MRI staining of the mouse hippocampal system using intracerebral injection of MnCl2 Takashi Watanabe, a,* Jelena Radulovic, b Joachim Spiess, b Oliver Natt, a Susann Boretius, a Jens Frahm, a and Thomas Michaelis a a b

Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut fu¨r biophysikalische Chemie, 37070 Go¨ttingen, Germany Molekulare Neuroendokrinologie, Max-Planck-Institut fu¨r experimentelle Medizin, 37075 Go¨ttingen, Germany

Received 30 October 2003; revised 6 January 2004; accepted 21 January 2004 Available online 15 April 2004 The morphology and function of the hippocampal system of C57BL/6J mice (n = 8) was studied in vivo using T1-weighted 3D magnetic resonance imaging (MRI) (117 Mm isotropic resolution) after bilateral injection of MnCl2 (0.25 Ml, 5 or 200 mM) into the posterior hippocampal formation. The neuronal uptake of the T1-shortening Mn2+ ions resulted in a pronounced MRI signal enhancement within the CA3 subfield and dentate gyrus with milder increases in CA1 and subiculum. This finding is in line with differences in the excitability of hippocampal neurons previously reported using electrophysiologic recordings. The subsequent axonal transport of Mn2+ highlighted the principal extrinsic projections from the posterior hippocampal formation via the fimbria and the precommissural fornix to the dorsal part of the lateral septal nucleus. A strong MRI signal enhancement was also observed in the ventral hippocampal commissure. A timecourse analysis revealed unsaturated conditions of Mn2+ accumulation at about 2 h after injection and optimal contrast-to-noise ratios at about 6 h after injection. The present results using Mn2+-enhanced 3D MRI open new ways for studying the role of the hippocampal system in specific aspects of learning and memory in normal and mutant mice. D 2004 Elsevier Inc. All rights reserved. Keywords: Hippocampus; Efferent pathways; Fornix; Manganese-enhanced MRI; Neuroanatomic tracing

Introduction The hippocampal formation, composed of dentate gyrus, CA1 to CA4, and subiculum, has been identified as central to our capacity for episodic memory (Eichenbaum, 2000; Rosene and Van Hoesen, 1987). Although anatomic connections between the various subregions support the view of a functional circuitry, the exact nature of this network as well as the underlying cellular and molecular mechanisms subserving learning and memory still remain largely unresolved. One way of exploring hippocampal processing is to * Corresponding author. Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut fu¨r biophysikalische Chemie, Am Fassberg 11, 37070 Go¨ttingen, Germany. Fax: +49-551-201-1307. E-mail address: [email protected] (T. Watanabe). Available online on ScienceDirect (www.sciencedirect.com.) 1053-8119/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2004.01.028

examine the full extent of the hippocampal formation while the circuit is activated by a cognitive task (Small et al., 2001). In this respect, magnetic resonance imaging (MRI) techniques are preferable because they allow for noninvasive and repeatable investigations of the same animal at high spatial resolution. In principle, functional MRI studies of animal brain may be based on hemodynamic techniques as commonly employed for humans, that is, the monitoring of task-related MRI signal changes due to perfusion-induced alterations of the focal deoxyhemoglobin level. Extending this concept, however, more recent studies exploited the neuroaxonal uptake of paramagnetic Mn2+ ions which shorten the T1 relaxation properties of affected water protons and thereby enhance the signal of T1-weighted MRI sequences (Duong et al., 2000; Lin and Koretsky, 1997; Pautler et al., 1998; Watanabe et al., 2001, 2002). The approach relies upon the fact that— depending on brain function—Mn2+ ions are taken up by neurons through voltage-gated Ca2+ channels (Drapeau and Nachshen, 1984; Narita et al., 1990) and are axonally transported to respective projection fields (Sloot and Gramsbergen, 1994; Tja¨lve et al., 1995). Accordingly, intracerebral injections of MnCl2 bear the potential for unraveling neuronal connectivities in vivo (Pautler et al., 2003; Saleem et al., 2002). Moreover, a recent MRI study of the song control system of birds (Tindemans et al., 2002) suggested that Mn2+ accumulation in the projection fields most likely reflects the electrophysiologic activity at the level of the injection site (Van der Linden et al., 2002). In contrast to deoxyhemoglobin-sensitive functional MRI sequences such as echo-planar imaging which are prone to magnetic susceptibility artifacts within the head, Mn2+enhanced MRI avoids such problems. Suitable T1-weighted MRI sequences yield robust acquisitions at exquisite three-dimensional spatial resolution several hours after contrast administration. The purpose of the present study was to investigate the use of Mn2+-enhanced MRI to characterize morphologic and functional properties of the hippocampal formation of behaving C57BL/6J mice. The technique used a bilateral injection of MnCl2 into the posterior hippocampal formation and specifically addressed the development of an optimized MRI and injection protocol (i) to determine and visualize the relevant intrahippocampal structures, and (ii) to ‘stain’ the hippocampal axonal projection pathways. The majority of the hippocampal extrinsic

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projection fibers enters the white matter bundle known as fimbria before ascending and joining a midline projection pathway called fornix. Through this fimbria – fornix complex, hippocampal pyramidal cells from all CA subfields project to the septal complex (Chronister and White, 1975; Rosene and Van Hoesen, 1987). Rodents additionally exhibit extensive commissural connections passing through a ventral hippocampal commissure located posterior to the septal area. It is hypothesized that Mn2+-induced MRI signal enhancements in these projection pathways are related to the electrophysiologic activity of the hippocampal formation integrated over the time period between MnCl2 administration and MRI recording.

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over a 15-s period, so that a volume of 0.25 Al was injected in each side. The procedure followed a protocol previously described by Radulovic et al. (1999). After MnCl2 injection, the isoflurane anesthesia was terminated and mice were placed inside a Plexiglas cage (36  21  20 cm) before transportation to the MRI facility. During this period (a few minutes), the animals were active in their novel environment. Magnetic resonance imaging Immediately after removing the animal from the Plexiglas cage, anesthesia was induced by an injection of avertin (i.p., 1.2%, 0.02

Materials and methods Animals Eight male C57BL/6J mice (9 – 12 weeks of age) were obtained from Centre d’Eˆlevage Janvier (France). Animals were individually housed under conventional conditions in macrolon cages according to the recommendations of the Society for Laboratory Animal Science (Germany). Experiments were performed in accordance with the European Council Directive (86/609/EEC) and with permission of the animal protection law enforced by the District Government of Braunschweig, State of Lower Saxony. Three mice were used as controls (nos. 1 – 3), five mice received an injection of MnCl2 (nos. 4 – 8). Bilateral hippocampal cannulation and injection of MnCl2 The bregma, the sagittal suture, and the surface of the skull were used as references for the anterior – posterior (AP), lateral (L), and ventral (V) coordinates, respectively. Double guide cannulae (C235, Plastics One, Roanoke, VA) were placed into the posterior hippocampal formation of both hemispheres at coordinates AP = 2.8 mm, V = 2.0 mm, and L = F 2.5 mm, respectively, in accordance with stereotaxic plates provided by Franklin and Paxinos (1997). The cannulae were implanted under avertin anesthesia (1.2%, 0.02 ml/g body weight injected intraperitoneally) and fixed to the skull by dental cement. A bilateral injection was used to optimize the delineation of the hippocampo-septal projection pathways. According to previous anatomical studies in rodents (Rosene and Van Hoesen, 1987), the fornix and the lateral septal nucleus receive axons from CA3 pyramidal neurons of both sides. Thus, a higher local concentration of manganese and a correspondingly better MRI contrast-to-noise ratio are to be expected after bilateral injections in comparison with an unilateral injection. Intrahippocampal injections of MnCl2 were performed 4 – 5 days after cannula surgery. MnCl2 (Sigma, Taufkirchen, Germany) was dissolved in sterile artificial cerebrospinal fluid (aCSF) on the day of injection and prepared in two different concentrations (200 mM: no. 4; 5 mM: nos. 5 – 8). The aCSF solution contained NaCl (130 mM), NaHCO3 (24 mM), MgSO4 (1.5 mM), CaCl2 (2 mM), KCl (3.5 mM), NaH2PO4 (1.25 mM), and glucose (10 mM) adjusted to pH 7.4 and 300 mOsm/kg H2O. Animals were exposed to a light isoflurane anesthesia (Forene, Abbott, Wiesbaden, Germany) and placed in a prone position. The injection was performed with use of a 28-gauge cannula and a 26-gauge guide cannula connected via plastic tubing to two Hamilton microsyringes. The solution was administered bilaterally by a microinjector (CMA/Microdialysis)

Fig. 1. The mouse hippocampal formation (HF) in (top) a histologic section (cresyl violet, adapted from Rosen et al., 2000) and (bottom) a corresponding horizontal section from a 3D MRI data set of a control animal (no. 1). T1-weighted MRI (2.35 T, 3D FLASH, TR/TE = 17/7.6 ms, flip angle = 25j) of C57BL/6J mice was performed at 117 Am isotropic spatial resolution. The HF (dashed line) is bordered medially by a CSF space, rostrally by the fimbria (Fi) and the lateral ventricle (LV), and laterocaudally by the deep cerebral white matter.

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ml/g body weight) to minimize possible stressful effects during transportation. At the MRI facility, the implanted cannulae were removed from the skull. Subsequently, 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 an inspiratory time of 0.1 s, a respiratory rate of 80 breaths per minute, and an estimated tidal volume of 0.15 – 0.25 ml. Anesthesia was maintained using 1.0 – 1.5% halothane in a 7:3 mixture of N2O and O2. For MRI, the animals were placed in a prone position on a purpose-built palate holder equipped with an adjustable nose cone. A heated water blanket and bed were used to maintain rectal body temperature at 37 F 0.5jC. 3D MRI data sets with a measuring time of 84 min were acquired 2 h (i.e., 1 h 30 min – 2 h 54 min) and 6 h (i.e., 5 h 30 min – 6 h 54 min) after Mn2+ administration. Selection of these periods was based on pilot studies using MnCl2 injections with 200 – 1000 mM concentrations and delays of up to 24 h. After the

first MRI measurement, the skin incisions of the scalp were covered with lidocaine hydrochloride (2% Xylocaine gel). The animals were allowed to recover from anesthesia and placed in their cages in a ventilated cabinet (Iffa Credo, L’Arbresle Cedex, France) with free access to water. The animals were active, drank water, and did not appear to be in any distress. One (no. 8) died during the second MRI investigation after the endotracheal tube was obstructed by sputa. All measurements were carried out at 2.35 T using a MRBR 4.7/400 mm magnet (Magnex Scientific, Abingdon, UK) and a DBX system (Bruker Biospin GmbH, Ettlingen, Germany) equipped with B-GA20 gradients (200 mm inner diameter, 100 mT m 1 maximum gradient strength). Radiofrequency excitation and signal reception were accomplished with use of a Helmholtz coil (100 mm inner diameter) and an elliptical surface coil (20 mm anterior – posterior, 12 mm left – right), respectively. High-resolution 3D MRI data sets were acquired using a T1weighted gradient-echo MRI sequence (rf-spoiled 3D FLASH, TR/

Fig. 2. T1-weighted MR images of C57BL/6J mice 2 h after intrahippocampal injection of (left) 200 mM MnCl2 (no. 4) and (right) 5 mM MnCl2 (no. 7). The parasagittal sections (2.5 mm lateral to the midline) and indicated (arrows) horizontal and coronal sections (top to bottom) reveal a pronounced signal enhancement at and around the injection site. For other parameters, see Fig. 1.

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TE = 17/7.6 ms, flip angle 25j, 32 averages) as described previously (Natt et al., 2002). The use of a 15-mm field-of-view along the rostro-caudal, left-to-right, and dorsal – ventral direction in conjunction with a 128  128  128 data matrix (zero-filled from a 128  96  96 acquisition matrix) resulted in an isotropic voxel resolution of 117  117  117 Am3. Image analysis From the original 3D MRI data sets, anatomically defined crosssections were obtained by multiplanar reconstructions using software supplied by the manufacturer (Paravision, Bruker Biospin

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GmbH). The plane of the anterior commissure – posterior commissure (AC – PC) served as a reference for the selection of standardized sections to facilitate comparisons with minimized intra- and interindividual variability (Natt et al., 2002). MRI volumetry was performed by a region-of-interest (ROI) analysis using a mousedriven cursor. As demonstrated in Fig. 1, the boundaries of the hippocampal formation were readily resolved in native T1-weighted 3D gradientecho images without Mn2+-induced contrast in agreement with histologic sections from a mouse brain atlas (Rosen et al., 2000). The extent of the hippocampal formation is bordered medially by a small CSF space, rostrally by the fimbria and lateral ventricle, and

Fig. 3. The mouse hippocampal formation in (top) a histologic section and corresponding horizontal T1-weighted MR images (left) 2 h and (right) 6 h after intrahippocampal injection of (middle) 200 mM MnCl2 (no. 4) and (bottom) 5 mM MnCl2 (no. 7). Pronounced signal enhancements in the dentate gyrus (DG) and CA3 subfield are complemented by only mild signal increases in the CA1 subfield and subiculum (S). For other parameters, see Fig. 1.

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laterocaudally by the deep cerebral white matter. This area served for an individual anatomic definition of a hippocampal ROI as indicated. The time course of the MRI signal enhancement was determined within the ventral hippocampal commissure and a cortical control region using a 0.50 mm2 ROI in either case. The signalto-noise ratio (SNR) was defined as the mean MRI signal intensity divided by the standard deviation of the noise. The contrast-to-noise ratio (CNR) was obtained by taking the difference of the SNR values between the ROIs in the efferent pathway and the cortex. The analysis followed a strategy previously developed for intra-individual comparisons of MR images obtained after intraocular Mn2+ administration (Watanabe et al., 2001).

Results Mn2+ distribution in the hippocampal formation Two hours after intrahippocampal injection, Mn2+-enhanced MRI demonstrated marked signal increases within and around the posterior hippocampal formation in all C57BL/6J mice investigated (nos. 4 – 8). Fig. 2 shows orthogonal MRI sections cutting through the injection sites of two different animals after receiving either (left) a high concentration (200 mM, no. 4) or (right) a low concentration (5 mM, no. 7) of MnCl2. The uptake of Mn2+ ions spreads from the point of injection into adjacent brain regions such as the visual cortex, laterodorsal part of thalamus, and superior colliculus.

Fig. 4. (Left) Selected coronal T1-weighted MR images along the hippocampo-septal projection pathway of an animal (no. 4) 6 h after intrahippocampal injection of 200 mM MnCl2 and (right) corresponding magnified views. Arrowheads and dashed lines indicate enhanced structures such as the medial part of the fimbria (Fi) bilaterally, the ventral hippocampal commissure (VHC), the dorsal part of the lateral septal nucleus (LSD) bilaterally, the precommissural fornix (Fx), and the triangular septal nucleus (TSN). For other parameters see Fig. 1.

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Quantitative assessments of the volume of the affected MRI signal around the injection site and in the hippocampal ROI confirmed the reliability and reproducibility of the employed injection technique and the availability of Mn2+ ions for neuronal uptake. Because the lower MnCl2 concentration enhanced about 70% of the volume affected by the 40-fold higher concentration in the hippocampal formation, that is, 8.6 F 0.6 Al (nos. 5 – 8) at 2 h after injection, most studies were performed using only 5 mM MnCl2. In comparison with data acquired at 2 h post-injection, Fig. 3 demonstrates that Mn2+-enhanced MRI at 6 h after contrast application revealed a more heterogenous and spatially distinct signal behavior within the hippocampal formation. Whereas mild signal increases are seen in CA1 and the subiculum, the marked signal enhancements in the CA3 subfield and dentate gyrus suggest Mn2+ accumulation in the pyramidal cells. Mn2+ transport along hippocampal projection pathways The application of MnCl2 led to a pronounced signal enhancement of the fimbria – fornix complex at both concentrations in all mice investigated (nos. 4 – 8). At 2 h after injection, Mn2+ transport beyond the level of the ventral hippocampal commissure was still incomplete. In terms of anatomic continuity, an even better contrast enhancement of the efferent pathway was achieved 6 h after injection. As summarized in Fig. 4, coronal MRI sections along the hippocampo-septal projection pathway clearly depict MRI signal increases of the projecting fiber bundles and axonal terminal areas. In fact, the 3D MRI data sets allowed for a visualization of the entire course of fibers through the fimbria up to the septal region. The medial parts of the fimbria were highlighted on both hemispheres (Fig. 4, top row). The enhanced bundles within the fimbria merged rostromedially at the level of the ventral hippocampal commissure (second row) or continued more rostrally into the septal complex (third and bottom row). The projection into the ventral hippocampal commissure showed the most pronounced enhancement. Additional signal increases were observed in the septal complex at the dorsal part of the lateral septal nucleus. Medially, the enhanced band exhibited a V-shaped structure, which identifies a structural complex containing parts of the precommissural fornix and the trigeminal septal nucleus. Although the induced contrast in the efferent pathways was slightly lower for 5 mM MnCl2 injections than for a 200 mM concentration, axonal Mn2+ accumulation at the level of the ventral hippocampal commissure was observable in all animals (nos. 5 – 8). A quantitative analysis of MRI signal intensity time courses confirmed these observations. Table 1 compares the SNR obtained

Table 1 MRI signal and contrast enhancement of the ventral hippocampal commissure in C57BL/6J mice after intrahippocampal injection of 5 mM MnCl2 SNR

Control nos. 1 – 3 2 h post-Mn2+ nos. 5 – 8 6 h post-Mn2+ nos. 5 – 7

CNR

VHC

Cortex

VHC-cortex

24.6 F 1.2 28.9 F 1.7* 32.1 F 1.9**

20.7 F 1.4 22.2 F 1.0 23.8 F 1.3*

3.9 F 0.3 6.7 F 1.0* 8.3 F 0.8**

Values are given as mean F SD; SNR = signal-to-noise ratio; CNR = contrast-to-noise ratio; VHC = ventral hippocampal commissure. * P < 0.05. ** P < 0.01 (unpaired t test vs. control).

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for a cortical control region dorsal to the rhinal fissure with that of the projection pathway at the level of the ventral hippocampal commissure. Already 2 h after Mn2+ application, the ventral hippocampal commissure exhibited a statistically significant SNR increase of about 17% which translates into a CNR increase of 72%. Exploiting the available 3D MRI anatomy, the length of the pathway from the injection site via the fimbria to the ventral hippocampal commissure was determined to be 4.3F0.5 mm and 4.3F0.2 mm for the left and right hemisphere, respectively. Accordingly, the estimated speed of the underlying transport (and partial accumulation) of Mn2+ supports previous studies reporting axonal transport rates of 2 – 6 mm/h (Pautler et al., 1998; Tja¨lve et al., 1995; Van der Linden et al., 2002; Watanabe et al., 2001). MRI at 6 h after injection revealed a general increase of the SNR in the cortex of up to 15% (Table 1) as well as a localized SNR increase of about 30% in the projection pathway which resulted in a more than 2-fold increase of the CNR (113%). Together, these results demonstrate that 2 h are sufficient to transport Mn2+ ions from the posterior hippocampus to the ventral hippocampal commissure, but that—at least for a 5 mM concentration of MnCl2—this process has not led to a saturation of the focal Mn2+ level which still increases at later stages.

Discussion Uptake of Mn2+ by hippocampal neurons Although localized applications of MnCl2 led to a moderate generalized MRI signal increase throughout the entire brain, the observed neuroanatomic specificity of SNR and CNR increases in behaving animals suggests that the Mn2+-enhanced MRI signal and the underlying accumulation of Mn2+ ions reflect the functional involvement of connected neural networks. The unspecific brain enhancement was more pronounced in pilot studies using very high concentrations of MnCl2 of up to 1000 mM. Such conditions led to a fading contrast in late MRI examinations (z 24 h after injection) which may be explained by diffusion processes of free Mn2+ ions within the extracellular space and/or by Mn2+ uptake into blood vessels and a subsequent distribution via the systemic circulation (Watanabe et al., 2001). For these reasons, very high concentrations and 24 h waiting periods were of no advantage for a delineation of hippocampal projection pathways. Mn2+ ions have been shown to pass through Ca2+ channels of excitable cells (Anderson, 1983) and to enter neurons during nerve action potentials (Drapeau and Nachshen, 1984; Narita et al., 1990). Thus, the exposure of brain tissue to Mn2+ and a related region-specific MRI signal enhancement most likely reflect a functionally dependent enrichment of intracellular Mn2+ due to an uptake by active neurons. This understanding has been supported by an activation-induced accumulation of Mn2+ in the rat brain after pharmacologic opening of the blood – brain barrier (Duong et al., 2000; Lin and Koretsky, 1997). The present observation of spatially distinct signal increases after intrahippocampal MnCl2 injection may therefore be ascribed to the specific activity of each subregion over time. The marked difference between the CA1 and CA3 enhancement is in line with previous autoradiographic studies (Takeda et al., 1994) and recent MRI findings after systemic MnCl2 application (Watanabe et al., 2002). Because both subfields contain pyramidal cells, this subregional variations cannot be attributed to gross

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neuroanatomic differences, but more likely represents a functional difference in the electrophysiologic properties of CA1 and CA3 pyramidal cells. In fact, using extracellular recordings (Cohen and Miles, 2000), spontaneous action potentials were invariably detected from the CA3 pyramidal cell population, whereas no such activity could be found in CA1. This suggests that CA3 pyramidal cells are more excitable than those in CA1. Moreover, the results of single-channel recordings indicated that low-voltage-activated Ca2+ channels are particularly abundant on hippocampal CA3 pyramidal neurons, as compared with neurons from area CA1 (Fisher et al., 1990). This higher density is supposed to be responsible for the characteristic endogenous bursting behavior of CA3 neurons (Hablitz and Johnston, 1981). Because lowvoltage-activated Ca2+ channels open in response to even the smallest depolarizations, their high abundance in CA3 may explain the pronounced accumulation of Mn2+ in this subfield. With this understanding, the MRI detection of CA1 and CA3 differences in Mn2+ enhancement seems to reflect the degree of local electric activity in relation to intrahippocampal processing. Neural tract tracing of hippocampal efferent connections The use of Mn2+-enhanced MRI for in vivo neural tract tracing has already been demonstrated for olfactory projections in mice (Pautler et al., 1998), retinal projections in mice and rats (Natt et al., 2002; Pautler et al., 1998; Watanabe et al., 2001), neuronal connections of the basal ganglia in mice and monkeys (Pautler et al., 2003; Saleem et al., 2002), and projections from the high vocal center in birds (Van der Linden et al., 2002). The present work adds the in vivo delineation of efferent fiber pathways from the hippocampal formation in mice. In particular, the enhancement of the fimbria at the medial position because of Mn2+ uptake into ammonic and subicular pyramidal cells of the posterior hippocampus is in agreement with a previous anterograde tracing study (Meibach and Siegel, 1977). After injection and incorporation of 3H-leucine into cells within CA1, CA3, or subiculum at the posterior dorsal level, enhanced fibers were traced into the medial part of the fimbria and to the posterior half of the lateral septal nucleus. The terminal distribution was observed in the dorsolateral quadrant which is consistent with the present finding of Mn2+-induced contrast within the septal region, or more precisely, within the dorsolateral part of the lateral nucleus.Noteworthy, the sensitivity of the chosen approach failed to detect projections into the mammillary complex in the thalamus via the postcommissural fornix. This may be caused by a limited focal availability of Mn2+ resulting from an insufficient accumulation in respective neurons and/or a low axonal fiber density along the target pathway (Watanabe et al., 2001). In addition, it cannot be excluded that the observed unspecific brain enhancement masks a weak focal increase. Toward functional MRI of the hippocampus using Mn2+ contrast To functionally map properties of the hippocampal formation in response to a suitable learning or memory task, Mn2+-enhanced MRI faces several challenges. Specific problems are the true neuronal origin of the Mn2+-induced contrast and its quantitative evaluation as a measure of neural activity. In fact, a general concern with respect to the utility of intraparenchymally injected Mn2+ is that the MRI-detectable SNR and CNR increases may at least partially stem from Mn2+ ions outside neurons. Apart from

ions within the extracellular space, there is some evidence that Mn2+ ions may enter astrocytes (Tiffany-Castiglioni and Qian, 2001). Although a significant glial uptake would certainly compromise the analysis and interpretation of the Mn2+ enhancement within the hippocampal subregions, the present findings strongly support the view that the enhanced hippocampal structures indeed refer to pyramidal neurons. The observed enhancement of the efferent hippocampal projection pathway clearly indicates the predominance of neuronal rather than glial effects because the underlying axonal transport requires the preceding uptake of Mn2+ by hippocampal neurons. Anatomically, the principal hippocampal circuit begins with a projection from the entorhinal cortex to the dentate gyrus, which is relayed to CA3 and subsequently to CA1. Ongoing projections from CA1 to the subiculum and back to the entorhinal cortex close this circuit (Nieuwenhuys et al., 1998). Apart from forming a loop of intrinsic connections, each of the hippocampal subfields, except for the dentate gyrus, also gives rise to extrinsic projections. These anatomic considerations lend further support to the interpretation that the observed Mn2+-enhanced MRI signals in the fimbria and commissural projections reflect the electrophysiologic activity of hippocampal neurons. Depending on the functional activity within this circuitry, the involvement and concurrent electric excitation of the entire hippocampal network is likely to aggravate the uptake of Mn2+ by neurons with extrinsic projections. As a consequence, differences in Mn2+ accumulation rates at a terminal field (or intermediate projection site) would represent differences in Mn2+ influx as a measure of neural activity at the injection site. From this point of view, it is important to note that MRI investigations of the dynamics of focal Mn2+ accumulation should be performed before the Mn2+ process becomes saturated. The present results demonstrate that this condition is met by at least the first 2 h after injection.

Conclusions This high-resolution 3D MRI study of mouse brain in vivo demonstrates the feasibility and reproducibility of ‘staining’ the hippocampal system by a single injection of MnCl2 into the posterior hippocampal formation. The Mn2+-induced signal enhancement was most pronounced within the CA3 subregion in line with differences in the excitability of hippocampal neurons previously seen in electrophysiologic recordings. At 6 h after injection, Mn2+-enhanced MRI delineated the principal hippocampal efferent pathways to the ventral hippocampal commissure and the septal region. A time course analysis of the Mn2+-induced signal enhancement revealed conditions of unsaturated Mn2+ accumulation at 2 h after injection as well as an optimal contrast-to-noise at 6 h after injection. In conjunction with suitable cognitive tasks and behavioral assessments, high-resolution Mn2+-enhanced MRI of normal and mutant mice is expected to become a powerful tool for a further characterization of learning and memory processing in the hippocampal system.

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