Dissecting cell-type-specific pathways in medial entorhinal cortical-hippocampal network for episodic memory.

Episodic memory, which refers to our ability to encode and recall past events, is essential to our daily lives. Previous research has established that both the entorhinal cortex (EC) and hippocampus (HPC) play a crucial role in the formation and retrieval of episodic memories. However, to understand neural circuit mechanisms behind these processes, it has become necessary to monitor and manipulate the neural activity in a cell-type-specific manner with high temporal precision during memory formation, consolidation, and retrieval in the EC-HPC networks. Recent studies using cell-type-specific labeling, monitoring, and manipulation have demonstrated that medial EC (MEC) contains multiple excitatory neurons that have differential molecular markers, physiological properties, and anatomical features. In this review, we will comprehensively examine the complementary roles of superficial layers of neurons (II and III) and the roles of deeper layers (V and VI) in episodic memory formation and recall based on these recent findings.

Precisely timed theta oscillations are selectively required during the encoding phase of memory.

Brain oscillations have been hypothesized to support cognitive function by coordinating spike timing within and across brain regions, yet it is often not known when timing is either critical for neural computations or an epiphenomenon. The entorhinal cortex and hippocampus are necessary for learning and memory and exhibit prominent theta oscillations (6-9 Hz), which are controlled by pacemaker cells in the medial septal area. Here we show that entorhinal and hippocampal neuronal activity patterns were strongly entrained by rhythmic optical stimulation of parvalbumin-positive medial septal area neurons in mice. Despite strong entrainment, memory impairments in a spatial working memory task were not observed with pacing frequencies at or below the endogenous theta frequency and only emerged at frequencies ≥10 Hz, and specifically when pacing was targeted to maze segments where encoding occurs. Neural computations during the encoding phase were therefore selectively disrupted by perturbations of the timing of neuronal firing patterns.

Memantine ameliorates cognitive deficit in AD mice via enhancement of entorhinal-CA1 projection.

BACKGROUND: Memantine, a low- to moderate-affinity uncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to improve cognitive functions in animal models of Alzheimer's disease (AD). Here we treated APP/PS1 AD mice with a therapeutic dose of memantine (20 mg/kg/day) and examined its underlying mechanisms in ameliorating cognitive defects. METHODS: Using behavioral, electrophysiological, optogenetic and morphology approaches to explore how memantine delay the pathogenesis of AD. RESULTS: Memantine significantly improved the acquisition in Morris water maze (MWM) in APP/PS1 mice without affecting the speed of swimming. Furthermore, memantine enhanced EC to CA1 synaptic neurotransmission and promoted dendritic spine regeneration of EC neurons that projected to CA1. CONCLUSIONS: Our study reveals the underlying mechanism of memantine in the treatment of AD mice.

Mitochondrial complex I abnormalities is associated with tau and clinical symptoms in mild Alzheimer's disease.

BACKGROUND: Mitochondrial electron transport chain abnormalities have been reported in postmortem pathological specimens of Alzheimer's disease (AD). However, it remains unclear how amyloid and tau are associated with mitochondrial dysfunction in vivo. The purpose of this study is to assess the local relationships between mitochondrial dysfunction and AD pathophysiology in mild AD using the novel mitochondrial complex I PET imaging agent [18F]BCPP-EF. METHODS: Thirty-two amyloid and tau positive mild stage AD dementia patients (mean age ± SD: 71.1 ± 8.3 years) underwent a series of PET measurements with [18F]BCPP-EF mitochondrial function, [11C]PBB3 for tau deposition, and [11C] PiB for amyloid deposition. Age-matched normal control subjects were also recruited. Inter and intrasubject comparisons of levels of mitochondrial complex I activity, amyloid and tau deposition were performed. RESULTS: The [18F]BCPP-EF uptake was significantly lower in the medial temporal area, highlighting the importance of the mitochondrial involvement in AD pathology. [11C]PBB3 uptake was greater in the temporo-parietal regions in AD. Region of interest analysis in the Braak stage I-II region showed significant negative correlation between [18F]BCPP-EF SUVR and [11C]PBB3 BPND (R = 0.2679, p = 0.04), but not [11C] PiB SUVR. CONCLUSIONS: Our results indicated that mitochondrial complex I is closely associated with tau load evaluated by [11C]PBB3, which might suffer in the presence of its off-target binding. The absence of association between mitochondrial complex I dysfunction with amyloid load suggests that mitochondrial dysfunction in the trans-entorhinal and entorhinal region is a reflection of neuronal injury occurring in the brain of mild AD.

Maturation of neuronal AD-tau pathology involves site-specific phosphorylation of cytoplasmic and synaptic tau preceding conformational change and fibril formation.

In Alzheimer's disease (AD), tau-protein undergoes a multi-step process involving the transition from a natively unfolded monomer to large, aggregated structures such as neurofibrillary tangles (NFTs). However, it is not yet clear which events initiate the early preclinical phase of AD tauopathy and whether they have impact on the propagation of tau pathology in later disease stages. To address this question, we analyzed the distribution of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope and fibrillary tau detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, we identified phosphorylated-T231-tau diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD [Braak NFT stages < I (including a-1b)] already showed IC- and IN-tau, our findings suggest that these lesions are a prerequisite for the development of AD.

A Comprehensive Analysis Identified Hub Genes and Associated Drugs in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and has become a growing global health problem causing great concern. However, the pathogenesis of AD is unclear and no specific therapeutics are available to provide the sustained remission of the disease. In this study, we used comprehensive bioinformatics to determine 158 potential genes, whose expression levels changed between the entorhinal and temporal lobe cortex samples from cognitively normal individuals and patients with AD. Then, we clustered these genes in the protein-protein interaction analysis and identified six significant genes that had more biological functions. Besides, we conducted a drug-gene interaction analysis of module genes in the drug-gene interaction database and obtained 26 existing drugs that might be applied for the prevention and treatment of AD. In addition, a predictive model was built based on the selected genes using different machine learning algorithms to identify individuals with AD. These findings may provide new insights into AD therapy.

The entorhinal cortex of the monkey: VI. Organization of projections from the hippocampus, subiculum, presubiculum, and parasubiculum.

The organization of projections from the macaque monkey hippocampus, subiculum, presubiculum, and parasubiculum to the entorhinal cortex was analyzed using anterograde and retrograde tracing techniques. Projections exclusively originate in the CA1 field of the hippocampus and in the subiculum, presubiculum, and parasubiculum. The CA1 and subicular projections terminate most densely in Layers V and VI of the entorhinal cortex, with sparser innervation of the deep portion of Layers III and II. Entorhinal projections from CA1 and the subiculum are topographically organized such that a rostrocaudal axis of origin is related to a medial-to-lateral axis of termination. A proximodistal axis of origin in CA1 and distoproximal axis in subiculum are related to a rostrocaudal axis of termination in the entorhinal cortex. The presubiculum sends a dense, bilateral projection to caudal parts of the entorhinal cortex. This projection terminates most densely in Layer III with sparser termination in Layers I, II, and V. The same parts of entorhinal cortex receive a dense projection from the parasubiculum. This projection terminates in Layers III and II. Both presubicular and parasubicular projections demonstrate the same longitudinal topographic organization as the projections from CA1 and the subiculum. These studies demonstrate that: (a) hippocampal and subicular inputs to the entorhinal cortex in the monkey are organized similar to those described in nonprimate species; (b) the topographic organization of the projections from the hippocampus and subicular areas matches that of the reciprocal projections from the entorhinal cortex to the hippocampus and the subicular areas.

Distribution and overlap of entorhinal, premotor, and amygdalar connections in the monkey anterior cingulate cortex.

The anterior cingulate cortex (ACC) is important for decision-making as it integrates motor plans with affective and contextual limbic information. Disruptions in these networks have been observed in depression, bipolar disorder, and post-traumatic stress disorder. Yet, overlap of limbic and motor connections within subdivisions of the ACC is not well understood. Hence, we administered a combination of retrograde and anterograde tracers into structures important for contextual memories (entorhinal cortex), affective processing (amygdala), and motor planning (dorsal premotor cortex) to assess overlap of labeled projection neurons from (outputs) and axon terminals to (inputs) the ACC of adult rhesus monkeys (Macaca mulatta). Our data show that entorhinal and dorsal premotor cortical (dPMC) connections are segregated across ventral (A25, A24a) and dorsal (A24b,c) subregions of the ACC, while amygdalar connections are more evenly distributed across subregions. Among all areas, the rostral ACC (A32) had the lowest relative density of connections with all three regions. In the ventral ACC, entorhinal and amygdalar connections strongly overlap across all layers, especially in A25. In the dorsal ACC, outputs to dPMC and the amygdala strongly overlap in deep layers. However, dPMC input to the dorsal ACC was densest in deep layers, while amygdalar inputs predominantly localized in upper layers. These connection patterns are consistent with diverse roles of the dorsal ACC in motor evaluation and the ventral ACC in affective and contextual memory. Further, distinct laminar circuits suggest unique interactions within specific ACC compartments that are likely important for the temporal integration of motor and limbic information during flexible goal-directed behavior.

Potentially Pathogenic Calcium Oxalate Dihydrate and Titanium Dioxide Crystals in the Alzheimer's Disease Entorhinal Cortex.

Knowing that Alzheimer's disease (AD) nucleates in the entorhinal cortex (EC), samples of 12 EC specimens were probed for crystals by a protocol detecting fewer than 1/5000th of those present. Of the 61 crystals found, 31 were expected and 30 were novel. Twenty-one crystals of iron oxides and 10 atherosclerosis-associated calcium pyrophosphate dihydrate crystals were expected and found. The 30 unexpected crystals were NLRP3-inflammasome activating calcium oxalate dihydrate (12) and titanium dioxide (18). Their unusual distribution raises the possibility that some were of AD origination sites.

Differential expression of secretagogin immunostaining in the hippocampal formation and the entorhinal and perirhinal cortices of humans, rats, and mice.

Secretagogin (SCGN) is a recently discovered calcium-binding protein belonging to the group of EF-hand calcium-binding proteins. SCGN immunostaining has been described in various regions of the human, rat and mouse brain. In these studies, it has been reported that, in general, the patterns of SCGN staining differ between rodents and human brains. These differences have been interpreted as uncovering phylogenetic differences in SCGN expression. Nevertheless, an important aspect that is not usually taken into account is that different methods are used for obtaining and processing brain tissue coming from humans and experimental animals. This is a critical issue since it has been shown that post-mortem time delay and the method of fixation (i.e., perfused vs. nonperfused brains) may influence the results of the immunostaining. Thus, it is not clear whether differences found in comparative studies with the human brain are simply due to technical factors or species-specific differences. In the present study, we analyzed the pattern of SCGN immunostaining in the adult human hippocampal formation (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the entorhinal and perirhinal cortices. This pattern of immunostaining was compared with rat and mouse that were fixed either by perfusion or immersion and with different post-mortem time delays (up to 5 hr) to mimic the way the human brain tissue is usually processed. We found a number of clear similarities and differences in the pattern of labeling among the human, rat, and mouse in these brain regions as well as between the different brain regions examined within each species. These differences were not due to the fixation.

Cytoarchitecture and myeloarchitecture of the entorhinal cortex of the common marmoset monkey (Callithrix jacchus).

The entorhinal cortex (EC) is associated with impaired cognitive function such as in the case of Alzheimer's disease, Parkinson's disease and Huntington's disease. The present study provides a detailed analysis of the cytoarchitectural and myeloarchitectural organization of the EC in the common marmoset Callithrix jacchus. Data were collected using Nissl and fiber stained preparations, supplemented with acetylcholinesterase and parvalbumin immunohistochemistry. The EC layers and subfields in the marmoset seem to be architectonically similar to those that have been proposed in nonhuman primates and humans to date; however, slight differences could be revealed using the present techniques. Throughout its rostrocaudal length, the entorhinal cortex presents a clear six-layered pattern. The entorhinal cortex is divided into six fields, named mainly in accordance to their rostrocaudal and mediolateral positions. At rostral levels, the neurons tend to be organized in patches that are surrounded by large, thick, radially oriented bundles of fibers, and the deep layers are poorly developed. At caudal levels, the divisions are more laminated in appearance. AChE staining at the borders of adjacent fields are consistent with the changes in layering revealed in Nissl-stained sections, of which the lateral regions of the EC display denser AChE staining than that of the medial banks. PV immunoreactivity was found in the labeled somata, dendrites, and axons in all layers and subdivisions. Additionally, we distinguished three subtypes of PV-immunoreactive neurons: multipolar, bipolar and spherical-shaped neurons, based on the shape of the somata and the disposition of the dendrites.

Reduced cognitive performance in aged rats correlates with increased excitation/inhibition ratio in the dentate gyrus in response to lateral entorhinal input.

Aging often impairs cognitive functions associated with the medial temporal lobe (MTL). Anatomical studies identified the layer II pyramidal cells of the lateral entorhinal cortex (LEC) as one of the most vulnerable elements within the MTL. These cells provide a major excitatory input to the dentate gyrus hippocampal subfield through synapses onto granule cells and onto local inhibitory interneurons, and a fraction of these contacts are lost in aged individuals with impaired learning. Using optogenetics, we evaluated the functional status of the remaining inputs in an outbred rat model of aging that distinguishes between learning-impaired and learning-unimpaired individuals. We found that aging affects the presynaptic and postsynaptic strength of the LEC inputs onto granule cells. However, the magnitude of these changes was similar in impaired and unimpaired rats. In contrast, the recruitment of inhibition by LEC activation was selectively reduced in the aged impaired subjects. These findings are consistent with the notion that the preservation of an adequate balance of excitation and inhibition is crucial to maintaining proficient memory performance during aging.

Parallel profiling of DNA methylation and hydroxymethylation highlights neuropathology-associated epigenetic variation in Alzheimer's disease.

BACKGROUND: Alzheimer's disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction. Previous studies of DNA modifications in Alzheimer's disease have been unable to distinguish between DNA methylation and DNA hydroxymethylation. DNA hydroxymethylation has been shown to be enriched in the human brain, although its role in Alzheimer's disease has not yet been fully explored. Here, we utilize oxidative bisulfite conversion, in conjunction with the Illumina Infinium Human Methylation 450K microarray, to identify neuropathology-associated differential DNA methylation and DNA hydroxymethylation in the entorhinal cortex. RESULTS: We identified one experiment-wide significant differentially methylated position residing in the WNT5B gene. Next, we investigated pathology-associated regions consisting of multiple adjacent loci. We identified one significant differentially hydroxymethylated region consisting of four probes spanning 104 bases in the FBXL16 gene. We also identified two significant differentially methylated regions: one consisting of two probes in a 93 base-pair region in the ANK1 gene and the other consisting of six probes in a 99-base pair region in the ARID5B gene. We also highlighted three regions that show alterations in unmodified cytosine: two probes in a 39-base pair region of ALLC, two probes in a 69-base pair region in JAG2, and the same six probes in ARID5B that were differentially methylated. Finally, we replicated significant ANK1 disease-associated hypermethylation and hypohydroxymethylation patterns across eight CpG sites in an extended 118-base pair region in an independent cohort using oxidative-bisulfite pyrosequencing. CONCLUSIONS: Our study represents the first epigenome-wide association study of both DNA methylation and hydroxymethylation in Alzheimer's disease entorhinal cortex. We demonstrate that previous estimates of DNA hypermethylation in ANK1 in Alzheimer's disease were underestimates as it is confounded by hypohydroxymethylation.

Comparison of three gamma oscillations in the mouse entorhinal-hippocampal system.

The entorhinal-hippocampal system is an important circuit in the brain, essential for certain cognitive tasks such as memory and navigation. Different gamma oscillations occur in this circuit, with the medial entorhinal cortex (mEC), CA3 and CA1 all generating gamma oscillations with different properties. These three gamma oscillations converge within CA1, where much work has gone into trying to isolate them from each other. Here, we compared the gamma generators in the mEC, CA3 and CA1 using optogenetically induced theta-gamma oscillations. Expressing channelrhodopsin-2 in principal neurons in each of the three regions allowed for the induction of gamma oscillations via sinusoidal blue light stimulation at theta frequency. Recording the oscillations in CA1 in vivo, we found that CA3 stimulation induced slower gamma oscillations than CA1 stimulation, matching in vivo reports of spontaneous CA3 and CA1 gamma oscillations. In brain slices ex vivo, optogenetic stimulation of CA3 induced slower gamma oscillations than stimulation of either mEC or CA1, whose gamma oscillations were of similar frequency. All three gamma oscillations had a current sink-source pair between the perisomatic and dendritic layers of the same region. Taking advantage of this model to analyse gamma frequency mechanisms in slice, we showed using pharmacology that all three gamma oscillations were dependent on the same types of synaptic receptor, being abolished by blockade of either type A γ-aminobutyric acid receptors or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, and insensitive to blockade of N-methyl-d-aspartate receptors. These results indicate that a fast excitatory-inhibitory feedback loop underlies the generation of gamma oscillations in all three regions.

Organization of the Claustrum-to-Entorhinal Cortical Connection in Mice.

The claustrum, a subcortical structure situated between the insular cortex and striatum, is reciprocally connected with almost all neocortical regions. Based on this connectivity, the claustrum has been postulated to integrate multisensory information and, in turn, coordinate widespread cortical activity. Although studies have identified how sensory information is mapped onto the claustrum, the function of individual topographically arranged claustro-cortical pathways has been little explored. Here, we investigated the organization and function of identified claustro-cortical pathways in mice using multiple anatomical and optogenetic techniques. Retrograde and anterograde tracing demonstrated that the density of anterior claustrum-to-cortical projection differs substantially depending on the target cortical areas. One of the major targets was the medial entorhinal cortex (MEC) and the MEC-projecting claustral neurons were largely segregated from the neurons projecting to primary cortices M1, S1, or V1. Exposure to a novel environment induced c-Fos expression in a substantial number of MEC-projecting claustral neurons and some M1/S1/V1-projecting claustral neurons. Optogenetic silencing of the MEC-projecting claustral neurons during contextual fear conditioning impaired later memory retrieval without affecting basal locomotor activity or anxiety-related behavior. These results suggest that the dense, anterior claustro-MEC pathway that is largely separated from other claustro-cortical pathways is activated by novel context and modulates the MEC function in contextual memory. SIGNIFICANCE STATEMENT: The claustrum is a poorly understood subcortical structure reciprocally connected with widespread neocortical regions. We investigated the organization and function of identified claustro-cortical projections in mice using pathway-specific approaches. Anatomical tracing showed that the density of anterior claustrum-to-cortical projection is dependent on the target cortical areas and that the medial entorhinal cortex (MEC) is one of the major projection targets. Novel context exposure activated multiple claustro-cortical pathways and a large fraction of the activated neurons projected to the MEC. Optogenetic silencing of the claustro-MEC pathway during contextual fear learning suppressed subsequent memory retrieval. These results suggest that the dense claustro-MEC pathway is activated by novel context and modulates MEC function in contextual memory.

Innervation from the entorhinal cortex to the dentate gyrus and the vulnerability to Zn2.

Hippocampal Zn2+ homeostasis is critical for cognitive activity and hippocampus-dependent memory. Extracellular Zn2+ signaling is linked to extracellular glutamate signaling and leads to intracellular Zn2+ signaling, which is involved in cognitive activity. On the other hand, excess intracellular Zn2+ signaling that is induced by excess glutamate signaling is involved in cognitive decline. In the hippocampal formation, the dentate gyrus is the most vulnerable to aging and is thought to contribute to age-related cognitive decline. The layer II of the entorhinal cortex is the most vulnerable to neuronal death in Alzheimer's disease. The perforant pathway provides input from the layer II to the dentate gyrus and is one of the earliest affected pathways in Alzheimer's disease. Medial perforant pathway-dentate granule cell synapses are vulnerable to either excess intracellular Zn2+ or ß-amyloid (Aß)-bound zinc, which induce transient cognitive decline via attenuation of medial perforant pathway LTP. However, it is unknown whether the vulnerability to excess intracellular Zn2+ is involved in region-specific vulnerability to aging and Alzheimer's disease. To discover a strategy to prevent short-term cognitive decline in normal aging process and the pre-dementia stage of Alzheimer's disease, the present paper deals with vulnerability of medial perforant pathway-dentate granule cell synapses to intracellular Zn2+ dyshomeostasis and its possible involvement in differential vulnerability to aging and Alzheimer's disease in the hippocampal formation.

Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment.

Methyl-Seq was recently developed as a targeted approach to assess DNA methylation (DNAm) at a genome-wide level in human. We adapted it for mouse and sought to examine DNAm differences across liver and 2 brain regions: cortex and hippocampus. A custom hybridization array was designed to isolate 99 Mb of CpG islands, shores, shelves, and regulatory elements in the mouse genome. This was followed by bisulfite conversion and sequencing on the Illumina HiSeq2000. The majority of differentially methylated cytosines (DMCs) were present at greater than expected frequency in introns, intergenic regions, near CpG islands, and transcriptional enhancers. Liver-specific enhancers were observed to be methylated in cortex, while cortex specific enhancers were methylated in the liver. Interestingly, commonly shared enhancers were differentially methylated between the liver and cortex. Gene ontology and pathway analysis showed that genes that were hypomethylated in the cortex and hippocampus were enriched for neuronal components and neuronal function. In contrast, genes that were hypomethylated in the liver were enriched for cellular components important for liver function. Bisulfite-pyrosequencing validation of 75 DMCs from 19 different loci showed a correlation of r = 0.87 with Methyl-Seq data. We also identified genes involved in neurodevelopment that were not previously reported to be differentially methylated across brain regions. This platform constitutes a valuable tool for future genome-wide studies involving mouse models of disease.

Laminar and dorsoventral molecular organization of the medial entorhinal cortex revealed by large-scale anatomical analysis of gene expression.

Neural circuits in the medial entorhinal cortex (MEC) encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations.

Reliable non-invasive measurement of human neurochemistry using proton spectroscopy with an anatomically defined amygdala-specific voxel.

Given the central role of the amygdala in fear perception and expression and its likely abnormality in affective disorders and autism, there is great demand for a technique to measure differences in neurochemistry of the human amygdala. Unfortunately, it is also a technically complex target for magnetic resonance spectroscopy (MRS) due to a small volume, high field inhomogeneity and a shared boundary with hippocampus, which can undergo opposite changes in response to stress. We attempted to achieve reliable PRESS-localized single-voxel MRS at 3T of the isolated human amygdala by using anatomy to guide voxel size and location. We present data from 106 amygdala-MRS sessions from 58 volunteers aged 10 to 52 years, including two tests of one-week stability and a feasibility study in an adolescent sample. Our main outcomes were indices of spectral quality, repeated measurement variability (within- and between-subject standard deviations), and sensitivity to stable individual differences measured by intra-class correlation (ICC). We present metrics of amygdala-MRS reliability for n-acetyl-aspartate, creatine, choline, myo-Inositol, and glutamate+glutamine (Glx). We found that scan quality suffers an age-related difference in field homogeneity and modified our protocol to compensate. We further identified an effect of anatomical inclusion near the endorhinal sulcus, a region of high synaptic density, that contributes up to 29% of within-subject variability across 4 sessions (n=14). Remaining variability in line width but not signal-to-noise also detracts from reliability. Statistical correction for partial inclusion of these strong neurochemical gradients decreases n-acetyl-aspartate reliability from an intraclass correlation of 0.84 to 0.56 for 7-minute acquisitions. This suggests that systematic differences in anatomical inclusion can contribute greatly to apparent neurochemical concentrations and could produce false group differences in experimental studies. Precise, anatomically-based prescriptions that avoid age-related sources of inhomogeneity and use longer scan times may permit study of individual differences in neurochemistry throughout development in this late-maturing structure.

Neurochemical alterations of the entorhinal cortex in amnestic mild cognitive impairment (aMCI): a three-year follow-up study.

The neurochemical alterations in the entorhinal cortex have not yet been measured, even though the entorhinal cortex is the earliest involved brain region in aMCI. In this study, we investigated whether brain regions including the entorhinal cortex would show early involvement of neurochemical abnormalities in aMCI, and whether magnetic resonance spectroscopy (MRS) abnormalities might be a predictive marker of conversion of aMCI to Alzheimer's disease (AD). MRS was performed on 13 aMCI patients and 11 patients with no cognitive impairment (NCI). Localizing voxels were placed within the entorhinal cortex, hippocampus, posterior cingulate gyrus, and occipital white matter in the dominant hemisphere. N-acetyl aspartate/creatinine (NAA/Cr) ratios in the entorhinal cortex were significantly lower in aMCI patients than in NCI subjects. After a three-year follow-up, seven aMCI patients converted to AD and six remained stable. Baseline NAA/Cr ratios of entorhinal cortex were decreased in converters, compared to NCI. Our study suggested the entorhinal cortex is the earliest site that is subject to neurochemical alteration in aMCI patients, and baseline MRS metabolite ratios in the entorhinal cortex can be a marker for predicting conversion of aMCI to AD.