Acute ischemic stroke induces magnetic resonance susceptibility signs dominated by endothelial nitric oxide synthase activation.

PURPOSE: Acute ischemic stroke induces deoxyhemoglobin accumulation around the ischemic region while activating endothelial nitric oxide synthase (eNOS) coupling and the subsequent release of nitric oxide (NO). Because deoxyhemoglobin is a natural NO spin trap, its interplay with NO could be prominent during acute stroke. Its interaction with NO has been shown to induce overt paramagnetic signals in vitro; our goal was to investigate whether this interplay can be detected using MRI. METHODS: To verify the in vivo image effects using the deoxyhemoglobin-NO interaction during acute stroke, eNOS states were manipulated in an animal model of acute ischemia, and the susceptibility signals, cerebral perfusion, and infarction were assessed noninvasively via MR susceptibility weighted imaging (SWI). RESULTS: Occlusion of the right middle cerebral artery increased eNOS coupling and susceptibility signals in the ischemic cortex while abolishing regional cerebral blood flow. Pharmacological eNOS blockage led to weakened susceptibility signals in the ischemic cortex as well as worsened tissue survival. Consistently, abolishment of eNOS coupling through genetic editing reduced the regional susceptibility signals in the ischemic cortex, causing large infarcts. CONCLUSION: Upregulation of eNOS during acute ischemia sustains tissue viability through the interaction between NO and deoxyhemoglobin. This interplay can be traced in vivo using SWI and can be considered a sensitive marker revealing the delicate oxygenation status of the ischemic tissue, therefore, guiding the management of acute stroke in clinical settings.

Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling.

Postischemic angiogenesis is an important recovery mechanism. Both arteries and veins are upregulated during angiogenesis, but eventually there are more angiogenic veins than arteries in terms of number and length. It is critical to understand how the veins are modulated after ischemia and then transitioned into angiogenic vessels during the proangiogenic stage to finally serve as a restorative strength to the injured area. Using a rat model of transient focal cerebral ischemia, the hypercapnic blood oxygen level-dependent (BOLD) response was used to evaluate vascular reactivity, while the hyperoxic BOLD and tissue oxygen level-dependent (TOLD) responses were used to evaluate the vascular functionality at 1, 3, and 7days after ischemia. Vessel-like venous signals appeared on R2* maps on days 3 and 7, but not on day 1. The large hypercapnic BOLD responses on days 3 and 7 indicated that these areas have high vascular reactivity. The temporal correlation between vascular reactivity and the immunoreactivity to desmin and VEGF further indicates that the integrity of vascular reactivity is associated with the pericyte coverage as regulated by the VEGF level. Vascular functionality remained low on days 1, 3, and 7, as reflected by the small hyperoxic BOLD and large hyperoxic TOLD responses, indicating the low oxygen consumption of the ischemic tissues. These functional changes in proangiogenic veins may be critical for angiogenesis.

White matter tracts for the trafficking of neural progenitor cells characterized by cellular MRI and immunohistology: the role of CXCL12/CXCR4 signaling.

White matter tracts are important for the trafficking of neural progenitor cells (NPCs) in both normal and pathological conditions, but the underlying mechanism is not clear. The directionality of white matter is advantageous for molecules or cells to distribute over a long distance, but this feature is unlikely solely responsible for efficient migration. The present study hypothesizes that the efficient migration of NPCs into white matter is under the influences of neurochemical attraction­CXCL12/CXCR4 signaling, a major mechanism underlying the targeted migration of NPCs. To test this view, the present study investigated the effects of CXCL12 administration into the corpus callosum (CC) on the migratory behavior of transplanted NPCs. A living animal tracking platform based on MRI and a magnetic cell labeling technique was employed. The NPCs were magnetically labeled and then transplanted at the right end of the CC. CXCL12 was infused continuously at the left end. Migration of NPCs was monitored repeatedly over a 7-day course using 3D gradient echo T2*-weighted imaging. It was found that, CXCL12 induced NPCs to migrate up to 1,881 µm from the graft whereas the spontaneous migration was mere 200 µm. CXCL12 induced migration that was nine times as efficient in the speed. The results indicate that the CXCL12/CXCR4 signaling may be a mechanism via which NPCs efficiently migrate along the white matter tracts. The study also presents a potential strategy for facilitating the targeted migration in NPC therapy for brain disorders.

Promoted Growth of Brain Tumor by the Transplantation of Neural Stem/Progenitor Cells Facilitated by CXCL12.

The targeted migration of neural stem/progenitor cells (NSPCs) is a prerequisite for the use of stem cell therapy in the treatment of pathologies. This migration is regulated mainly by C-X-C motif chemokine 12 (CXCL12). Therefore, promotion of the migratory responses of grafted cells by upregulating CXCL12 signaling has been proposed as a strategy for improving the efficacy of such cell therapies. However, the effects of this strategy on brain tumors have not yet been examined in vivo. The aim of the present study was thus to elucidate the effects of grafted rat green fluorescent protein (GFP)-labeled NSPCs (GFP-NSPCs) with CXCL12 enhancement on a model of spontaneous rat brain tumor induced by N-ethyl-N-nitrosourea. T2-weighted magnetic resonance imaging was applied to determine the changes in tumor volume and morphology over time. Postmortem histology was performed to confirm the tumor pathology, expression levels of CXCL12 and C-X-C chemokine receptor type 4, and the fate of GFP-NSPCs. The results showed that the tumor volume and hypointense areas of T2-weighted images were both significantly increased in animals treated with combined NSPC transplantation and CXCL12 induction, but not in control animals or in those with tumors that received only one of the treatments. GFP-NSPCs appear to migrate toward tumors with CXCL12 enhancement and differentiate uniquely into a neuronal lineage. These findings suggest that CXCL12 is an effective chemoattractant that facilitates exogenous NSPC migration toward brain tumors and that CXCL12 and NSPC can act synergistically to promote tumor progression with severe hemorrhage.

Negative cerebral blood volume fMRI response coupled with Ca²âº-dependent brain activity in a dopaminergic road map of nociception.

Decreased cerebral blood volume/flow (CBV/CBF) contributes to negative blood-oxygen-level-dependent (BOLD) functional MRI (fMRI) signals. But it is still strongly debated whether these negative BOLD or CBV/CBF signals are indicative of decreased or increased neuronal activity. The fidelity of Ca(2+) signals in reflecting neuronal excitation is well documented. However, the roles of Ca(2+) signals and Ca(2+)-dependent activity in negative fMRI signals have never been explored; an understanding of this is essential to unraveling the underlying mechanisms and correctly interpreting the hemodynamic response of interest. The present study utilized a nociception-induced negative CBV fMRI response as a model. Ca(2+) signals were investigated in vivo using Mn(2+)-enhanced MRI (MEMRI), and the downstream Ca(2+)-dependent signaling was investigated using phosphorylated cAMP response-element-binding (pCREB) immunohistology. The results showed that nociceptive stimulation led to (1) striatal CBV decreases, (2) Ca(2+) increases via the nigrostriatal pathway, and (3) substantial expression of pCREB in substantia nigra dopaminergic neurons and striatal neurons. Interestingly, the striatal negative fMRI response was abolished by blocking substantia nigra activity but was not affected by blocking the striatal activity. This suggests the importance of input activity other than output in triggering the negative CBV signals. These findings indicate that the striatal negative CBV fMRI signals are associated with Ca(2+) increases and Ca(2+)-dependent signaling along the nigrostriatal pathway. The obtained data reveal a new brain road map in response to nociceptive stimulation of hemodynamic changes in association with Ca(2+) signals within the dopaminergic system.

High-resolution structural and functional assessments of cerebral microvasculature using 3D Gas ΔR2*-mMRA.

The ability to evaluate the cerebral microvascular structure and function is crucial for investigating pathological processes in brain disorders. Previous angiographic methods based on blood oxygen level-dependent (BOLD) contrast offer appropriate visualization of the cerebral vasculature, but these methods remain to be optimized in order to extract more comprehensive information. This study aimed to integrate the advantages of BOLD MRI in both structural and functional vascular assessments. The BOLD contrast was manipulated by a carbogen challenge, and signal changes in gradient-echo images were computed to generate ΔR2* maps. Simultaneously, a functional index representing the regional cerebral blood volume was derived by normalizing the ΔR2* values of a given region to those of vein-filled voxels of the sinus. This method is named 3D gas ΔR2*-mMRA (microscopic MRA). The advantages of using 3D gas ΔR2*-mMRA to observe the microvasculature include the ability to distinguish air-tissue interfaces, a high vessel-to-tissue contrast, and not being affected by damage to the blood-brain barrier. A stroke model was used to demonstrate the ability of 3D gas ΔR2*-mMRA to provide information about poststroke revascularization at 3 days after reperfusion. However, this technique has some limitations that cannot be overcome and hence should be considered when it is applied, such as magnifying vessel sizes and predominantly revealing venous vessels.

In vivo evidence of increased nNOS activity in acute MPTP neurotoxicity: a functional pharmacological MRI study.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin commonly used to produce an animal model of Parkinson's disease. Previous studies have suggested a critical role for neuronal nitric oxide (NO) synthase- (nNOS-) derived NO in the pathogenesis of MPTP. However, NO activity is difficult to assess in vivo due to its extremely short biological half-life, and so in vivo evidence of NO involvement in MPTP neurotoxicity remains scarce. In the present study, we utilized flow-sensitive alternating inversion recovery sequences, in vivo localized proton magnetic resonance spectroscopy, and diffusion-weighted imaging to, respectively, assess the hemodynamics, metabolism, and cytotoxicity induced by MPTP. The role of NO in MPTP toxicity was clarified further by administering a selective nNOS inhibitor, 7-nitroindazole (7-NI), intraperitoneally to some of the experimental animals prior to MPTP challenge. The transient increase in cerebral blood flow (CBF) in the cortex and striatum induced by systemic injection of MPTP was completely prevented by pretreatment with 7-NI. We provide the first in vivo evidence of increased nNOS activity in acute MPTP-induced neurotoxicity. Although the observed CBF change may be independent of the toxicogenesis of MPTP, this transient hyperperfusion state may serve as an early indicator of neuroinflammation.

Neurovascular abnormalities in brain disorders: highlights with angiogenesis and magnetic resonance imaging studies.

The coupling between neuronal activity and vascular responses is controlled by the neurovascular unit (NVU), which comprises multiple cell types. Many different types of dysfunction in these cells may impair the proper control of vascular responses by the NVU. Magnetic resonance imaging, which is the most powerful tool available to investigate neurovascular structures or functions, will be discussed in the present article in relation to its applications and discoveries. Because aberrant angiogenesis and vascular remodeling have been increasingly reported as being implicated in brain pathogenesis, this review article will refer to this hallmark event when suitable.

Hyperglycemia exacerbates intracerebral hemorrhage via the downregulation of aquaporin-4: temporal assessment with magnetic resonance imaging.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is associated with high mortality and neurological deficits, and concurrent hyperglycemia usually worsens clinical outcomes. Aquaporin-4 (AQP-4) is important in cerebral water movement. Our aim was to investigate the role of AQP-4 in hyperglycemic ICH. METHODS: Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) in adult Sprague-Dawley male rats. ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. One set of rats was repeatedly monitored by MRI at 1, 4, and 7 days after ICH induction so as to acquire information on the formation of hematoma and edema. Another set of rats was killed and brains were examined for differences in the degree of hemorrhage and edema, water content, blood-brain barrier destruction, and AQP-4 expression. RESULTS: Hyperglycemia ICH rats exhibited increased brain water content, more severe blood-brain barrier destruction, and greater vasogenic edema as seen on diffusion-weighted MRI. Significant downregulation of AQP-4 was observed in STZ-treated rats after ICH as compared with non-STZ-treated rats. Apoptosis was greater on day 1 after ICH in STZ-treated rats. CONCLUSIONS: The expression of AQP-4 in the brain is downregulated in hyperglycemic rats as compared with normoglycemic rats after ICH. This change is accompanied by increased vasogenic brain edema and more severe blood-brain barrier destruction.

Simple SPION incubation as an efficient intracellular labeling method for tracking neural progenitor cells using MRI.

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.

Manganese-enhanced MRI of rat brain based on slow cerebral delivery of manganese(II) with silica-encapsulated Mn x Fe(1-x) O nanoparticles.

In this work, we report a monodisperse bifunctional nanoparticle system, MIO@SiO2 -RITC, as an MRI contrast agent [core, manganese iron oxide (MIO); shell, amorphous silica conjugated with rhodamine B isothiocyanate (RITC)]. It was prepared by thermal decomposition and modified microemulsion methods. The nanoparticles with varying iron to manganese ratios displayed different saturated magnetizations and relaxivities. In vivo MRI of rats injected intravenously with MIO@SiO2-RITC nanoparticles exhibited enhancement of the T1 contrast in brain tissue, in particular a time-delayed enhancement in the hippocampus, pituitary gland, striatum and cerebellum. This is attributable to the gradual degradation of MIO@SiO2-RITC nanoparticles in the liver, resulting in the slow release of manganese(II) [Mn(II)] into the blood pool and, subsequently, accumulation in the brain tissue. Thus, T1-weighted contrast enhancement was clearly detected in the anatomic structure of the brain as time progressed. In addition, T2*-weighted images of the liver showed a gradual darkening effect. Here, we demonstrate the concept of the slow release of Mn(II) for neuroimaging. This new nanoparticle-based manganese contrast agent allows one simple intravenous injection (rather than multiple infusions) of Mn(II) precursor, and results in delineation of the detailed anatomic neuroarchitecture in MRI; hence, this provides the advantage of the long-term study of neural function.

Dopaminergic imaging of nonmotor manifestations in a rat model of Parkinson's disease by fMRI.

Nonmotor manifestations determine the life quality of patients with Parkinson's disease (PD). Identification of the nonmotor symptoms in PD as definite changes will represent a milestone in its diagnosis. Outcome measures that characterize nonmotor manifestations with specificity for dopaminergic deficiency are essential to that goal. Pain is a prevalent sensory disturbance in PD patients. The prevalence was reported to be up to 83%. Nociceptive stimuli under normal conditions elicit decreases in cerebral blood volume (CBV) in the striatum via dopaminergic neurotransmission. This nociception-induced CBV response is potentially to be defined as a characteristic of the pain symptom of PD. To validate this concept, steady-state CBV-weighted functional magnetic resonance imaging with iron oxide nanoparticles was employed to measure CBV changes in parkinsonian rats. Tyrosine hydroxylase immunohistology was used to identify the dopaminergic integrity to corroborate the imaging findings. Additional experiments that tested pain responses in parkinsonism were also carried out. The results revealed that the lesioned striatum exhibited a weakened CBV decrease in response to the nociceptive stimulus. This weakened CBV response occurred mainly in areas with dopaminergic denervation. A strong correspondence was observed between the distributions of the nociception-induced CBV responses and dopaminergic innervation. The persisting CBV signals in the striatum were abolished by the D2/D3 antagonist eticlopride. The findings of these behavioral, neuroimaging, immunohistological, and pharmacological experiments demonstrate that pain in a rat model of PD can be characterized by nociception induced striatal CBV signal changes with specificity for dopaminergic dysfunction.

MR phase imaging: sensitive and contrast-enhancing visualization in cellular imaging.

The successful translation of stem-cell therapies requires a detailed understanding of the fate of transplanted cells. Magnetic resonance imaging (MRI) has provided a noninvasive means of imaging cell dynamics in vivo by prelabeling cell with T(2) shortening iron oxide particles. However, this approach suffers from a gradual loss of sensitivity since active cell mitosis could decrease the cellular contrast agent (CA) concentration below detection level. In addition, the interpretation of images may be confounded by hypointensities induced by factors other than this CA susceptibility effect (CASE). We therefore examined the feasibility of exploiting the phase information in MRI to increase the sensitivity of cellular imaging and to differentiate the CASE from endogenous image hypointensity. Phase aliasing and the B(0) field inhomogeneity effect were removed by applying a reliable unwrapping algorithm and a high-pass filter, respectively, thus delineating phase variations originating from high spatial frequencies due to the CASE. We found that the filtered phase map detects labeled cells with high sensitivity and can readily differentiate the cell migration track from the white matter, both of which are hypointense in T(2)-weighted magnitude images. Furthermore, an approximate fivefold contrast-to-noise ratio enhancement can be achieved with an MRI phase map over conventional T(2)-weighted magnitude images.

A lifespan MRI evaluation of ventricular enlargement in normal aging mice.

Ventricular enlargement has been proposed as a structural biomarker for the progression of Alzheimer's disease (AD). This biomarker, established in human patients, needs to be translated to animals to facilitate drug development for the disease. However, ventricular enlargement is not exclusive to AD, since the ventricle size increases during normal aging. A longitudinal characterization of ventricular enlargement in normal aging in mice is therefore crucial before further evaluations of mouse models or neurodegenerative diseases associated to brain atrophy. To this end, ventricular enlargement in normal aging mice was characterized over the lifespan (i.e., 2 years). The results showed that the overall ventricle size increased with age, with the expansion beginning during the early life stages and continuing to old age. The reported data represent a biomarker benchmark for normal aging mice under unmodified conditions. This provides a foundation for evaluating the validity of AD mouse models or the effects of potential drugs. The considerable physiological ventricular enlargement during normal aging must be considered in related experiments.

A new scenario for negative functional magnetic resonance imaging signals: endogenous neurotransmission.

Functional magnetic resonance imaging (fMRI) has revolutionized investigations of brain functions. Increases in fMRI signals are usually correlated with neuronal activation, but diverse explanations have been proposed for negative fMRI responses, including decreases in neuronal activity, the vascular-steal effect, and large increases in oxygen consumption. These possible scenarios, although encompassing a wide range of potential neurovascular responses, cannot yet be used to interpret certain types of negative fMRI signals. Recent studies have found that intravenous injection of dopamine D(2) receptor (D2DR) agonist reduced the hemodynamic responses in the caudate-putamen (CPu); however, whether endogenous dopaminergic neurotransmission contributes to fMRI signals remains obscure. Since it has been suggested that the D2DR is involved in pain modulation, and the CPu shows equivocal fMRI signals during noxious stimulation, the present study established an animal model based on graded electrical stimulation to elicit different levels of nociception, and aimed to determine whether nociception-induced endogenous dopaminergic neurotransmission is sufficient to generate negative fMRI responses. Our results from cerebral blood volume (CBV)-weighted fMRI, Fos immunohistochemistry, and electrophysiological recording demonstrated a salient bilateral CBV decreases associated with heightened neuronal activity in the CPu induced by unilateral noxious electrical stimulation. In addition, preinjection of D2DR antagonist reduced the observed CBV decreases. Our findings reveal the role of the D2DR in regulating striatal vascular responses and suggest that endogenous neurotransmission-induced CBV decreases underlie negative fMRI signals. Hence, the influence of endogenous neurotransmission should be considered when interpreting fMRI data, especially in an area involved in strong vasoactive neurotransmission.

Whole-brain functional magnetic resonance imaging mapping of acute nociceptive responses induced by formalin in rats using atlas registration-based event-related analysis.

Nociceptive neuronal activation in subcortical regions has not been well investigated in functional magnetic resonance imaging (fMRI) studies. The present report aimed to use the blood oxygenation level-dependent (BOLD) fMRI technique to map nociceptive responses in both subcortical and cortical regions by employing a refined data processing method, the atlas registration-based event-related (ARBER) analysis technique. During fMRI acquisition, 5% formalin (50 mul) was injected into the left hindpaw to induce nociception. ARBER was then used to normalize the data among rats, and images were analyzed using automatic selection of the atlas-based region of interest. It was found that formalin-induced nociceptive processing increased BOLD signals in both cortical and subcortical regions. The cortical activation was distributed over the cingulate, motor, somatosensory, insular, and visual cortices, and the subcortical activation involved the caudate putamen, hippocampus, periaqueductal gray, superior colliculus, thalamus, and hypothalamus. With the aid of ARBER, the present study revealed a detailed activation pattern that possibly indicated the recruitment of various parts of the nociceptive system. The results also demonstrated the utilization of ARBER in establishing an fMRI-based whole-brain nociceptive map. The formalin induced nociceptive images may serve as a template of central nociceptive responses, which can facilitate the future use of fMRI in evaluation of new drugs and preclinical therapies for pain.

Neuroaxonal ion dyshomeostasis of the normal-appearing corpus callosum in experimental autoimmune encephalomyelitis.

Atrophy of the corpus callosum (CC) is a well-documented observation in clinically definite multiple sclerosis (MS) patients. One recent hypothesis for the neurodegeneration that occurs in MS is that ion dyshomeostasis leads to neuroaxonal damage. To examine whether ion dyshomeostasis occurs in the CC during MS onset, experimental autoimmune encephalomyelitis (EAE) was utilized as an animal MS model to induce autoimmunity-mediated responses. To date, in vivo investigations of neuronal ion homeostasis has not been feasible using traditional neuroscience techniques. Therefore, the current study employed an emerging MRI method, called Mn2+-enhanced MRI (MEMRI). Mn2+ dynamics is closely associated with important neuronal activity events, and is also considered to be a Ca2+ surrogate. Furthermore, when injected intracranially, Mn2+ can be used as a multisynaptic tracer. These features enable MEMRI to detect neuronal ion homeostasis within a multisynaptic circuit that is connected to the injection site. Mn2+ was injected into the visual cortex to trace the CC, and T1-weighted imaging was utilized to observe temporal changes in Mn2+-induced signals in the traced pathways. The results showed that neuroaxonal functional changes associated with ion dyshomeostasis occurred in the CC during an acute EAE attack. In addition, the pathway appeared normal, although EAE-induced immune-cell infiltration was visible around the CC. The findings suggest that ion dyshomeostasis is a major neuronal aberration underlying the deterioration of normal-appearing brain tissues in MS, supporting its involvement in neuroaxonal functioning in MS.

Neuronal dysfunction of a long projecting multisynaptic pathway in response to methamphetamine using manganese-enhanced MRI.

RATIONALE: Manganese (Mn2+)-enhanced magnetic resonance imaging (MEMRI) is an emerging in vivo MR approach for pharmacological research. One new application of MEMRI in this area is to characterize functional changes of a specific neural circuit that is essential to the central effects of a drug challenge. OBJECTIVES: To develop and validate such use of MEMRI in neuropharmacology, the current study applied MEMRI to visualize functional changes within a multisynaptic pathway originating from fasciculus retroflexus (FR) that is central to a commonly abused psychostimulant, methamphetamine (MA). METHODS: Twelve rats were injected intraperitoneally with MA (10 mg/kg) or saline every 2 h for a total of four injections. After 6 days, Mn2+ was injected into the habenular nucleus (FR origin) of all animals, and MEMRI was repeatedly performed at certain points in time over 48 h. The evolution of Mn2+-induced signal enhancement was assessed across the FR tract, the ventral tegmental area (VTA), the striatum, the nucleus accumbens, and the prefrontal cortex (PFC), in both MA-injected animals and controls. RESULTS: MA treatment was found to affect the complexity and efficiency of Mn2+ uptake in the VTA, via the FR tract, with significantly increased Mn2+ accumulation in the VTA, the dorsomedial part of the striatum, and the PFC. CONCLUSIONS: MEMRI successfully visualizes disruptions in the multisynaptic pathway as the consequences of repeated MA exposure. MEMRI is potentially an important method in the future to investigate functional changes within a specific pathway under the influences of pharmacological agents, given its excellent functional, in vivo, spatial, and temporal properties.