The first step in understanding the expression mechanisms of brain functions and pathophysiological mechanisms of neurological disorders is to understand which brain regions are associated with those functions and diseases. To address this issue, we need methods to measure and analyze neural activities within the entire brain volume. Activation-induced manganese-enhanced MRI (AIM-MRI) is one such method for mapping whole brain activities (Lin and Koretsky, 1997; Aoki et al., 2004; Duyn and Koretsky, 2008; Silva and Bock, 2008; Kikuta et al., 2015). Manganese ion (Mn²⁺) can pass through voltage-dependent calcium channels (VDCCs), is taken up by mitochondria and other organelles, binds to proteins, and is extruded very slowly from the cell (Nelson, 1986; Rasagado-Flores et al., 1987; Narita et al., 1990; Low et al., 1991; Nordhøy et al., 2003; Chen et al., 2015). VDCCs open more frequently in highly active neurons; hence, in the presence of Mn²⁺ in the extracellular solution, highly active neurons accumulate larger amounts of Mn²⁺ than weakly active neurons (Kikuta et al., 2015). Therefore, Mn²⁺ is a surrogate marker of Ca²⁺ influx in excitable tissues. A paramagnetic ion, Mn²⁺ shortens the longitudinal relaxation time (T1) of protons (H⁺), and the longitudinal relaxation rate R1 (=1/T1) is proportional to Mn²⁺ concentration ([Mn²⁺]; Nordhøy et al., 2003; Aoki et al., 2004; Silva et al., 2004; Tambalo et al., 2009; Kikuta et al., 2015). Thus, [Mn²⁺] can be quantified by the R1 value calculated from the T1 value (Tambalo et al., 2009). Based on these ideas, AIM-MRI can use R1 to measure neural activity changes in a freely-moving subject with the advantage of being insensitive to blood hemodynamics (Tambalo et al., 2009).

Despite these features, AIM-MRI is still not widely used in the field of neuroscience, partially due to insufficient information regarding Mn²⁺ dynamics in the brain, confounding the interpretation of the results. To resolve this issue, we need to know the dynamics of Mn²⁺ in the brain and to thereby determine three time windows after systemic administration. The first is when Mn²⁺ flows into neurons. Without this timing, it is impossible to record changes in neural activities associated with a specific behavior, task, stimuli, operation, or drug administration. The second is when Mn²⁺ in the extracellular space has cleared but is still maintained intracellularly. As MRI cannot discriminate between intracellular and extracellular spaces, the R1 map should be acquired during this time to record neural activities. The third time window is the interval required for repeated measurements. Because MRI is a non-invasive method, one of its merits is repeated measurements within the same subject. To conduct AIM-MRI repeatedly in a single subject, [Mn²⁺] in the brain must have returned to its control level when the next dose of MnCl₂ is administered. Another reason to know when [Mn²⁺] in the brain returns to its normal concentration for repeated measurements is that, although Mn²⁺ is an essential heavy metal (Horning et al., 2015), overexposure has some toxic consequences in neuronal and non-neuronal tissues (Hazell, 2002; Silva and Bock, 2008; Chen et al., 2015; Bouabid et al., 2016). Mn²⁺ binds to many proteins and accumulates in organelles; thus, the clearance of manganese from brain tissue is very slow (Takeda et al., 1995). To determine these three time windows, we conducted a longitudinal study and estimated the time courses of [Mn²⁺] in extra- and intracellular-spaces after systemic administration of MnCl₂ by AIM-MRI with quantitative T1 measurement and microdialysis.

Understanding the behavior of Mn²⁺ in the brain after systemic administration of MnCl₂ for AIM-MRI studies is crucial for optimizing experimental protocols and improving the reliability of the data obtained. In this study, we aimed to determine (1) when Mn²⁺ flows into neurons, (2) when Mn²⁺ in the extracellular space has been cleared while maintaining intracellular Mn²⁺, and (3) what is the appropriate interval required for repeated measurements. Although other studies have evaluated the distribution of Mn²⁺ in the brain after systemic administration of MnCl₂ (Takeda et al., 1995; Kuo et al., 2005; Tambalo et al., 2009; Chan et al., 2017; Topping et al., 2017), the above three time windows have not been previously clarified.

Ventricular R1 rapidly increased and peaked 1 h after administration, and then rapidly decreased. However, while the difference between the pre-administration R1 values and those at 5 h were 40% of those at 1 h, they were still significantly different from control and they remained a little bit higher than controls for at least 24 h (Figures 1, 2B and Table 1). The ventricular R1 time course reported by Kuo et al. (2005) was similar to ours, although they only showed data for 15 min, 45 min, 24 h, and 72 h after administration. The time course of [Mn] in the extracellular fluid obtained by microdialysis was similar to that of the ventricular R1 (Figure 2C), which is consistent with previous studies (Tseng et al., 2003; Chung et al., 2007). Therefore, the time course of the ventricular R1 should reflect the [Mn²⁺] in the extracellular fluid. However, microdialysis showed that the [Mn²⁺] returned to control levels 4 h after administration, but ventricular R1 was significantly different from the control levels until 24 h. This difference may be attributed to the fact that it is difficult to detect Mn bound to macromolecules such as proteins by microdialysis, while MRI can detect this. To summarize, these results suggest that the time window for Mn²⁺ influx into neurons is in the range of 1–3 h after MnCl₂ administration.

The absolute R1 values of the lateral ventricle and the superior and inferior third ventricles differed from one another (Figure 2B and Supplementary Figure 2). The choroid plexus seems to be more abundant in the superior dorsal third ventricle and less abundant in the inferior one (Paxinos and Franklin, 1997). It is known that the absolute value of R1 is lower in free water such as CSF than in bound water such as intracellular fluids and free water is less sensitive to Mn²⁺ than intracellular fluids (Nordhøy et al., 2003). The region of the superior third ventricle contains the intracellular fluid of the choroid plexus, which may have resulted in the higher value of R1. The R1 values of the lateral ventricle were between those of the superior and inferior parts of the third ventricle. This may indicate that the ROI set for the lateral ventricle includes the choroid plexus to some extent. Altogether, although the possibility remains that [Mn²⁺] may vary from place to place, the fact remains that after intraperitoneal administration of MnCl₂, Mn²⁺ in the CSF rises first, followed by that in the brain parenchyma.

Circulating Mn²⁺ dynamics are an important factor for explaining Mn²⁺ dynamics. According to the literature, the time course of R1 in the blood after intraperitoneal administration is similar to the ventricular R1 we observed (Jasmin et al., 2021) and systemic administration of MnCl₂ by intravenous and intraperitoneal routes induced similar time courses of T1 (inverse of R1) changes in the mouse brain (Kuo et al., 2005). The time course of the signal intensity in the gallbladder obtained by T1-weighted images also resembled that of the ventricular R1 (Supplementary Figure 4). Therefore, MnCl₂ administered by intraperitoneal injection is likely transported to the CSF through the circulatory system and excreted from the body via the circulatory system to the bile.

In the brain parenchyma, the R1 values increased more slowly than in the ventricle, reaching peak values 24–48 h after both single and double administrations of MnCl₂ (Figures 1, 3–5, Supplementary Figures 1, 5, and Tables 1, 2). As the R1 values in the ventricles, which reflect extracellular [Mn²⁺], had returned to control levels 24–48 h after MnCl₂ administration, those in the brain parenchyma during this time should reflect intracellular [Mn²⁺]. Taken together, these findings indicate that 24–48 h after MnCl₂ administration is an appropriate time window for MRI examination.

Despite the decrease in [Mn²⁺] in the CSF, manganese concentration in the brain parenchyma continued to increase until 24–48 h after MnCl₂ administration. There are three possible reasons for this. The first is that the R1 of CSF is less sensitive to Mn²⁺ and thus underestimates [Mn²⁺] in the ventricles, the second is that the cells have an active mechanism to take up manganese such as the transferrin transport system (Chen et al., 2015; Horning et al., 2015), and the third is that intracellular manganese binds easily to proteins and thus a small amount of extracellular Mn²⁺ continues to enter to the cells.

We also followed the R1 values until they returned to control levels. After the single and double administration of MnCl₂, it took 5 days and 2 weeks, respectively, for parenchymal R1 values to return to near control values (Figures 3, 5, Supplementary Figures 1, 5, and Tables 1, 2). However, some regions, such as the SC and Thal exhibited slightly higher R1 values compared to the control R1. The residual [Mn²⁺] calculated from the residual R1 values using the relaxivity 5.35 mM^–1 s^–1 obtained from the same MRI scanner (Kikuta et al., 2015) is on the order of several μM. It is reported that the tissue contents of Mn in mammals are several to several tens μM. Therefore, the toxicity of the residual Mn²⁺ is quite small. Nevertheless, because the accumulation of Mn²⁺ through repeated experiments might be toxic, it is desirable to have a longer interval. Therefore, the interval between AIM-MRI experiments should be more than at least 5 days or 2 weeks for single or double administrations, respectively, for repeated AIM-MRI experiments in the same animal, thus avoiding both the effects of residual Mn²⁺ and its toxicity.

The distribution of parenchymal R1 was non-uniform (Figures 1, 4). It is not possible to determine by MRI whether these differences are due to differences in Mn²⁺ accumulation or differences in biochemical environments that may affect the relaxivity of Mn²⁺ (Topping et al., 2017). Therefore, we cannot determine the distribution of neural activity in the brain by the R1 distribution pattern in a single subject. To identify areas where changes in neural activity are seen under certain conditions, comparisons with control animals given the same amount of Mn²⁺ are necessary. It is noted that the non-uniformity of R1 might arise from the scanning quality of the MRI scanner. Thus, it is necessary that the scanning quality should be ensured (see section “Materials and Methods”).

In the case of the double administration, R1 values 24 h after administration were significantly higher in many brain regions than they were following a single administration (Figure 5D). Whereas the cumulative dosage of the double administration is twice as much as that of the single administration, the differences in R1 values between the single and the double administration were small. When considering Mn²⁺ toxicity, a single dose is more suitable for measuring changes in brain activity associated with brief stimuli or tasks using AIM-MRI, while repeated administration is suitable for capturing changes in persistent brain activity, such as in neurological disorders (Kikuta et al., 2015), chronic pain (Inami et al., 2019), and visualization of active polysynaptic circuits (Almeida-Corrêa et al., 2018).

In many AIM-MRI studies, activity-dependent Mn²⁺ accumulation in neurons is assessed by T1-weighted images and quantified by the signal intensities (Lin and Koretsky, 1997; Aoki et al., 2004; Schroeder et al., 2016; Devonshire et al., 2017). However, the signal enhancement can be unreliable in inter-animal comparisons (Tambalo et al., 2009). In addition, the signal intensity does not always correspond to [Mn²⁺], because Mn²⁺ shortens the transverse relaxation time (T2), leading to a decrease in signal intensity associated with [Mn²⁺] (Cory et al., 1987; Pels Rijcken et al., 1994; Silva et al., 2004). Therefore, we measured T1 values instead of the signal intensity of T1-weighted images in the brain and calculated R1 values to quantify the [Mn²⁺].

As mentioned in the introduction, Mn²⁺ is known to be a surrogate marker for Ca²⁺, hence AIM-MRI can measure changes in neural activity by evaluating the R1 map. However, we need to be aware that Mn²⁺ can pass through not only VDCCs but also other Ca²⁺-permeable channels, such as some ligand-gated channels and store-operated calcium channels (Uehara et al., 2002; Itoh et al., 2008; Tu et al., 2009; Chen et al., 2015; Bouabid et al., 2016; Kikuta et al., 2019). The inositol 1,4,5-trisphosphate receptor on the endoplasmic reticulum membrane is also permeable to Mn²⁺ (Striggow and Ehrlich, 1996). Therefore, we should take into consideration that the cellular accumulation of Mn²⁺ may not only depend on neural activity but also on changes in extracellular ligand concentration and intracellular signal transduction. It is known that glial cells also accumulate Mn²⁺ (Tholey et al., 1990; Kikuta et al., 2015). Taken together, these findings suggest that R1 may be affected not only by neural activities but also by other physiological signals such as metabolic changes.

The three time windows of Mn²⁺ dynamics that we have determined should enable neuroscientists to better optimize their experimental protocols for AIM-MRI and to improve the reliability of the data they obtain. Thus, our results pave the way for AIM-MRI to be more widely used in the field of neuroscience.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by The Tohoku University Committee for Animal Experiments and The Kyorin University Animal Care Committee.

HT, TF, SK, and MO conceived and performed experiments. HT, TF, and MO analyzed data. HT, TF, NH, and MO wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.