Neuropathological Assessment as an Endpoint in Clinical Trial Design.

Different neurodegenerative conditions can have complex, overlapping clinical presentations that make accurate diagnosis during life very challenging. For this reason, confirmation of the clinical diagnosis still requires postmortem verification. This is particularly relevant for clinical trials of novel therapeutics where it is important to ascertain what disease- and/or pathology-modifying effects the therapeutics have had. Furthermore, it is important to confirm that patients in the trial had the correct clinical diagnosis as this will have a major bearing on the interpretation of trial results. Here we present a simple protocol for pathological assessment of neurodegenerative changes.

Differential methylation analysis in neuropathologically confirmed dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is a common form of dementia in the elderly population. We performed genome-wide DNA methylation mapping of cerebellar tissue from pathologically confirmed DLB cases and controls to study the epigenetic profile of this understudied disease. After quality control filtering, 728,197 CpG-sites in 278 cases and 172 controls were available for the analysis. We undertook an epigenome-wide association study, which found a differential methylation signature in DLB cases. Our analysis identified seven differentially methylated probes and three regions associated with DLB. The most significant CpGs were located in ARSB (cg16086807), LINC00173 (cg18800161), and MGRN1 (cg16250093). Functional enrichment evaluations found widespread epigenetic dysregulation in genes associated with neuron-to-neuron synapse, postsynaptic specialization, postsynaptic density, and CTCF-mediated synaptic plasticity. In conclusion, our study highlights the potential importance of epigenetic alterations in the pathogenesis of DLB and provides insights into the modified genes, regions and pathways that may guide therapeutic developments.

Linking environmental risk factors with epigenetic mechanisms in Parkinson's disease.

Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease, with a complex risk structure thought to be influenced by interactions between genetic variants and environmental exposures, although the full aetiology is unknown. Environmental factors, including pesticides, have been reported to increase the risk of developing the disease. Growing evidence suggests epigenetic changes are key mechanisms by which these environmental factors act upon gene regulation, in disease-relevant cell types. We present a systematic review critically appraising and summarising the current body of evidence of the relationship between epigenetic mechanisms and environmental risk factors in PD to inform future research in this area. Epigenetic studies of relevant environmental risk factors in animal and cell models have yielded promising results, however, research in humans is just emerging. While published studies in humans are currently relatively limited, the importance of the field for the elucidation of molecular mechanisms of pathogenesis opens clear and promising avenues for the future of PD research. Carefully designed epidemiological studies carried out in PD patients hold great potential to uncover disease-relevant gene regulatory mechanisms. Therefore, to advance this burgeoning field, we recommend broadening the scope of investigations to include more environmental exposures, increasing sample sizes, focusing on disease-relevant cell types, and recruiting more diverse cohorts.

Common signatures of differential microRNA expression in Parkinson's and Alzheimer's disease brains.

Dysregulation of microRNA gene expression has been implicated in many neurodegenerative diseases, including Parkinson's disease. However, the individual dysregulated microRNAs remain largely unknown. Previous meta-analyses have highlighted several microRNAs being differentially expressed in post-mortem Parkinson's disease and Alzheimer's disease brains versus controls, but they were based on small sample sizes. In this study, we quantified the expression of the most compelling Parkinson's and Alzheimer's disease microRNAs from these meta-analyses ('candidate miRNAs') in one of the largest Parkinson's/Alzheimer's disease case-control post-mortem brain collections available (n = 451), thereby quadruplicating previously investigated sample sizes. Parkinson's disease candidate microRNA hsa-miR-132-3p was differentially expressed in our Parkinson's (P = 4.89E-06) and Alzheimer's disease samples (P = 3.20E-24) compared with controls. Alzheimer's disease candidate microRNAs hsa-miR-132-5p (P = 4.52E-06) and hsa-miR-129-5p (P = 0.0379) were differentially expressed in our Parkinson's disease samples. Combining these novel data with previously published data substantially improved the statistical support (α = 3.85E-03) of the corresponding meta-analyses, clearly implicating these microRNAs in both Parkinson's and Alzheimer's disease. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with α-synuclein neuropathological Braak staging (P = 3.51E-03/P = 0.0117), suggesting that hsa-miR-132-3p/-5p play a role in α-synuclein aggregation beyond the early disease phase. Our study represents the largest independent assessment of recently highlighted candidate microRNAs in Parkinson's and Alzheimer's disease brains, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both Parkinson's and Alzheimer's disease, pinpointing shared pathogenic mechanisms across these neurodegenerative diseases. Intriguingly, based on publicly available high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation data, hsa-miR-132 may interact with SNCA messenger RNA in the human brain, possibly pinpointing novel therapeutic approaches in fighting Parkinson's disease.

Mitochondrial dysfunction is a key pathological driver of early stage Parkinson's.

BACKGROUND: The molecular drivers of early sporadic Parkinson's disease (PD) remain unclear, and the presence of widespread end stage pathology in late disease masks the distinction between primary or causal disease-specific events and late secondary consequences in stressed or dying cells. However, early and mid-stage Parkinson's brains (Braak stages 3 and 4) exhibit alpha-synuclein inclusions and neuronal loss along a regional gradient of severity, from unaffected-mild-moderate-severe. Here, we exploited this spatial pathological gradient to investigate the molecular drivers of sporadic PD. METHODS: We combined high precision tissue sampling with unbiased large-scale profiling of protein expression across 9 brain regions in Braak stage 3 and 4 PD brains, and controls, and verified these results using targeted proteomic and functional analyses. RESULTS: We demonstrate that the spatio-temporal pathology gradient in early-mid PD brains is mirrored by a biochemical gradient of a changing proteome. Importantly, we identify two key events that occur early in the disease, prior to the occurrence of alpha-synuclein inclusions and neuronal loss: (i) a metabolic switch in the utilisation of energy substrates and energy production in the brain, and (ii) perturbation of the mitochondrial redox state. These changes may contribute to the regional vulnerability of developing alpha-synuclein pathology. Later in the disease, mitochondrial function is affected more severely, whilst mitochondrial metabolism, fatty acid oxidation, and mitochondrial respiration are affected across all brain regions. CONCLUSIONS: Our study provides an in-depth regional profile of the proteome at different stages of PD, and highlights that mitochondrial dysfunction is detectable prior to neuronal loss, and alpha-synuclein fibril deposition, suggesting that mitochondrial dysfunction is one of the key drivers of early disease.

Possible Contribution of Altered Cholinergic Activity in the Visual Cortex in Visual Hallucinations in Parkinson's Disease.

OBJECTIVE: Up to one-third of patients with Parkinson's disease (PD) experience visual hallucinations (VHs). Lewy bodies are sparse in the visual cortices and seem unlikely to explain the hallucinations. Some neuroimaging studies have found that perfusion is reduced in the occipital lobe in individuals with VHs. Recent work has suggested that decreased cholinergic input may directly lead to the decreased perfusion. The investigators hypothesized that individuals with PD and VHs would have biochemical evidence of reduced microvascular perfusion and reduced cholinergic activity in areas of the brain that process visual images. METHODS: Tissue from Brodmann's area (BA) 18 and BA 19 was obtained from a well-characterized cohort matched for age, gender, and postmortem interval in 69 individuals (PD without VHs, N=11; PD without dementia plus VHs N=10, N=10; PD with dementia plus VHs, N=16; and control subjects, N=32). Von Willebrand factor, vascular endothelial growth factor A, and myelin-associated glycoprotein:proteolipid protein-1 (MAG:PLP1) ratio-a measure of tissue oxygenation relative to metabolic demand, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), choline acetyltransferase, and α-synuclein-were quantified by enzyme-linked immunosorbent assay. The primary outcome was the MAG:PLP1 ratio. RESULTS: There was no biochemical evidence of chronic hypoperfusion in PD, although microvessel density was decreased in ventral BA 18 and BA 19. There was no between-group difference in BChE in either dorsal BA 18 or BA 19. AChE concentration was reduced in individuals with PD compared with control subjects in dorsal and ventral BA 18 and dorsal BA 19, and it was increased in ventral BA 19. These changes were most marked in the PD plus VHs group. CONCLUSIONS: These results suggest that changes in cholinergic activity rather than chronic hypoperfusion may underlie VHs in PD.

Locus Coeruleus Pathology Indicates a Continuum of Lewy Body Dementia.

BACKGROUND: Lewy body dementia (LBD) has two main phenotypes: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB), separated by the 'one-year-rule'. They also show different symptom profiles: core DLB features include fluctuating cognition, REM-sleep behaviur disorder, and visual hallucinations. These symptoms are sometimes present in PDD, representing an intermediate 'PDD-DLB' phenotype. OBJECTIVE: DLB-like features may reflect deficits in the functions of the noradrenergic nucleus locus coeruleus (LC). Therefore, we compared the LC in the LBD phenotypes, PD, and controls. METHODS: 38 PD, 56 PDD, 22 DLB, and 11 age-matched control cases from the Parkinson's UK tissue bank were included. LC tissue sections were immunostained for tyrosine-hydroxylase (TH), α-synuclein, tau, and amyloid-ß. TH-neurons were quantified and pathologic burden calculated by %-coverage method. RESULTS: The LC shows a stepwise reduction in neuron count from controls, PD, PDD, to DLB. PDD-DLB cases showed an intermediate clinical phenotype that was reflected pathologically. Cell counts were significantly reduced in DLB compared to PDD after correction for demographic factors. LC degeneration contributed significantly to the onset of all DLB symptoms. While α-synuclein was not significantly different between PDD and DLB cases, DLB exhibited significantly less tau pathology. CONCLUSION: DLB and DLB-like symptoms represent noradrenergic deficits resulting from neuronal loss in the LC. PDD and DLB are likely to represent a clinical continuum based on the presence or absence of DLB-like symptoms mirrored by a pathological continuum in the LC.

The diagonal band of Broca in health and disease.

The diagonal band of Broca (DBB) contains the second largest cholinergic cell group in the human brain, known as the nucleus of the vertical limb of the DBB (nvlDBB). It has major projections to the hippocampus, but it is often underinvestigated, partly due to its ill-defined anatomical boundaries and hence the difficulty of reliable sampling. In this chapter, we have reviewed the historical literature to reestablish the anatomy of the nvlDBB, distinguishing it from neighboring basal forebrain cholinergic nuclei. Although varying degrees of neuronal loss in the nvlDBB have been reported in a range of neurological disorders, and in the aged brain, the significant nvlDBB cholinergic neuronal loss reported in Lewy body dementias is of particular interest. Retrograde tracer study in rodents has demonstrated reciprocal connections between the DBB and the hippocampal CA2 subfield, an area particularly susceptible to Lewy pathologies. Previous functional studies have demonstrated that the nvlDBB is particularly involved in memory retrieval, a cognitive domain severely affected in Lewy body disorders. Based on these observations, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the hippocampal CA2.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

Iron and inflammation: in vivo and post-mortem studies in Parkinson's disease.

In these present studies, in vivo and and post-mortem studies have investigated the association between iron and inflammation. Early-stage Parkinson's disease (PD) patients, of less than 5 years disease duration, showed associations of plasmatic ferritin concentrations with both proinflammatory cytokine interleukin-6 and hepcidin, a regulator of iron metabolism as well as clinical measures. In addition ratios of plasmatic ferritin and iron accumulation in deep grey matter nuclei assessed with relaxometry T2* inversely correlated with disease severity and duration of PD. On the hand, post-mortem material of the substantia nigra compacta (SNc) divided according to Braak and Braak scores, III-IV and V-VI staging, exhibited comparable microgliosis, with a variety of phenotypes present. There was an association between the intensity of microgliosis and iron accumulation as assayed by Perl's staining in the SNc sections. In conclusion, markers of inflammation and iron metabolism in both systemic and brain systems are closely linked in PD, thus offering a potential biomarker for progression of the disease.

Alzheimer's disease pathology explains association between dementia with Lewy bodies and APOE-ε4/TOMM40 long poly-T repeat allele variants.

INTRODUCTION: The role of TOMM40-APOE 19q13.3 region variants is well documented in Alzheimer's disease (AD) but remains contentious in dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). METHODS: We dissected genetic profiles within the TOMM40-APOE region in 451 individuals from four European brain banks, including DLB and PDD cases with/without neuropathological evidence of AD-related pathology and healthy controls. RESULTS: TOMM 40-L/APOE-ε4 alleles were associated with DLB (OR TOMM40 -L = 3.61; P value = 3.23 × 10-9; OR APOE -ε4 = 3.75; P value = 4.90 × 10-10) and earlier age at onset of DLB (HR TOMM40 -L = 1.33, P value = .031; HR APOE -ε4 = 1.46, P value = .004), but not with PDD. The TOMM40-L/APOE-ε4 effect was most pronounced in DLB individuals with concomitant AD pathology (OR TOMM40 -L = 4.40, P value = 1.15 × 10-6; OR APOE - ε 4 = 5.65, P value = 2.97 × 10-8) but was not significant in DLB without AD. Meta-analyses combining all APOE-ε4 data in DLB confirmed our findings (ORDLB = 2.93, P value = 3.78 × 10-99; ORDLB+AD = 5.36, P value = 1.56 × 10-47). DISCUSSION: APOE-ε4/TOMM 40-L alleles increase susceptibility and risk of earlier DLB onset, an effect explained by concomitant AD-related pathology. These findings have important implications in future drug discovery and development efforts in DLB.

Hippocampal CA2 Lewy pathology is associated with cholinergic degeneration in Parkinson's disease with cognitive decline.

Although the precise neuropathological substrates of cognitive decline in Parkinson's disease (PD) remain elusive, it has long been regarded that pathology in the CA2 hippocampal subfield is characteristic of Lewy body dementias, including dementia in PD (PDD). Early non-human primate tracer studies demonstrated connections from the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB, Ch2) to the hippocampus. However, the relationship between Lewy pathology of the CA2 subfield and cholinergic fibres has not been explored. Therefore, in this study, we investigated the burden of pathology in the CA2 subsector of PD cases with varying degrees of cognitive impairment and correlated this with the extent of septohippocampal cholinergic deficit. Hippocampal sections from 67 PD, 34 PD with mild cognitive impairment and 96 PDD cases were immunostained for tau and alpha-synuclein, and the respective pathology burden was assessed semi-quantitatively. In a subset of cases, the degree of CA2 cholinergic depletion was quantified using confocal microscopy and correlated with cholinergic neuronal loss in Ch2. We found that only cases with dementia have a significantly greater Lewy pathology, whereas cholinergic fibre depletion was evident in cases with mild cognitive impairment and this was significantly correlated with loss of cholinergic neurons in Ch2. In addition, multiple antigen immunofluorescence demonstrated colocalisation between cholinergic fibres and alpha-synuclein but not tau pathology. Such specific Lewy pathology targeting the cholinergic system within the CA2 subfield may contribute to the unique memory retrieval deficit seen in patients with Lewy body disorders, as distinct from the memory storage deficit seen in Alzheimer's disease.

Multiple system atrophy prions retain strain specificity after serial propagation in two different Tg(SNCA*A53T) mouse lines.

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

Author Correction: Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues.

In the original version of this Article, the concentration of boric acid buffer for the SDS clearing solution was given incorrectly as '1 M sodium borate' and should have read '0.2 M boric acid'. Also, the composition of PBST incorrectly read '1% Triton X-100 (vol/vol) and 0.1% sodium azide (wt/vol)' and should have read '0.1% Triton X-100 (vol/vol) and 0.01% sodium azide (wt/vol)'. Further, the pH of the OPTIClear solution was not stated, and should have read 'with a pH between 7 to 8 adjusted with hydrochloric acid'. These errors have been corrected in both the PDF and HTML versions of the Article.

Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues.

Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue microstructure have been very effective when used on rodent brain but very few studies have utilised them on human brain material, mainly due to the inherent difficulties in processing post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for fresh and archival human brain tissue, including formalin-fixed paraffin-embedded material. In light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl violet for visualisation of neurons in cleared tissue, with the potential for 3D quantification in regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D. The development of these different strategies for human tissue clearing has wide applicability and, we hope, will provide a baseline for further technique development.

MSA prions exhibit remarkable stability and resistance to inactivation.

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.

Microglial Activation in Traumatic Brain Injury.

Microglia have a variety of functions in the brain, including synaptic pruning, CNS repair and mediating the immune response against peripheral infection. Microglia rapidly become activated in response to CNS damage. Depending on the nature of the stimulus, microglia can take a number of activation states, which correspond to altered microglia morphology, gene expression and function. It has been reported that early microglia activation following traumatic brain injury (TBI) may contribute to the restoration of homeostasis in the brain. On the other hand, if they remain chronically activated, such cells display a classically activated phenotype, releasing pro-inflammatory molecules, resulting in further tissue damage and contributing potentially to neurodegeneration. However, new evidence suggests that this classification is over-simplistic and the balance of activation states can vary at different points. In this article, we review the role of microglia in TBI, analyzing their distribution, morphology and functional phenotype over time in animal models and in humans. Animal studies have allowed genetic and pharmacological manipulations of microglia activation, in order to define their role. In addition, we describe investigations on the in vivo imaging of microglia using translocator protein (TSPO) PET and autoradiography, showing that microglial activation can occur in regions far remote from sites of focal injuries, in humans and animal models of TBI. Finally, we outline some novel potential therapeutic approaches that prime microglia/macrophages toward the beneficial restorative microglial phenotype after TBI.

Chemical Probes for Visualizing Intact Animal and Human Brain Tissue.

Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain.