Segregating the functions of human hippocampus

It is now accepted that hippocampal lesions impair episodic memory. However, the precise functional role of the hippocampus in episodic memory remains elusive. Recent functional imaging data implicate the hippocampus in processing novelty, a finding supported by human in vivo recordings and event-related potential studies. Here we measure hippocampal responses to novelty, using functional MRI (fMRI), during an item-learning paradigm generated from an artificial grammar system. During learning, two distinct types of novelty were periodically introduced: perceptual novelty, pertaining to the physical characteristics of stimuli (in this case visual characteristics), and exemplar novelty, reflecting semantic characteristics of stimuli (in this case grammatical status within a rule system). We demonstrate a left anterior hippocampal response to both types of novelty and adaptation of these responses with stimulus familiarity. By contrast to these novelty effects, we also show bilateral posterior hippocampal responses with increasing exemplar familiarity. These results suggest a functional dissociation within the hippocampus with respect to the relative familiarity of study items. Neural responses in anterior hippocampus index generic novelty, whereas posterior hippocampal responses index familiarity to stimuli that have behavioral relevance (i.e., only exemplar familiarity). These findings add to recent evidence for functional segregation within the human hippocampus during learning.     

Spatial memory and the human hippocampus

The hippocampus and adjacent medial temporal lobe structures are known to support declarative memory, but there is not consensus about what memory functions the hippocampus might support that are distinct from the functions of the adjacent cortex. One idea is that the hippocampus is specifically important for allocentric spatial memory, e.g., the hippocampus is especially needed to remember object locations when there is a shift in viewpoint between study and test. We tested this proposal in two experiments. Patients with damage limited to the hippocampus were given memory tests for object locations in a virtual environment. In the first experiment, participants studied locations of a variable number of images (one to five) and tried to remember the image locations from either the same viewpoint as during study (shift of 0 degrees) or a different viewpoint (shift of 55 degrees, 85 degrees, or 140 degrees). In each viewpoint condition (shifts of 0 degrees, 55 degrees, 85 degrees, and 140 degrees), patients performed normally when remembering one or two image locations. Further, performance declined to a similar degree in each viewpoint condition as patients tried to remember increasing numbers of image locations. In the second experiment, participants tried to remember four images after viewpoint shifts of 0 degrees, 55 degrees, 85 degrees, or 140 degrees. Patients were mildly impaired at all conditions (shifts of 0 degrees, 55 degrees, 85 degrees, and 140 degrees), and the impairment was no greater when viewpoint shifted. We conclude that damage to the hippocampus does not selectively impair viewpoint-independent spatial memory. Rather, hippocampal damage impairs memory as the memory load increases.     

Histochemical detection of esterified cholesterol within human atherosclerotic lesions using the fluorescent probe filipin

The fluorescent cholesterol probe filipin has been used in this study to histochemically examine the morphology and cholesterol composition of intra- and extracellular Sudanophilic lipid deposits which accumulate in human atherosclerotic lesions. Because filipin reacts with unesterified but not esterified cholesterol, detection of cholesteryl esters was carried out by first extracting native unesterified cholesterol with ethanol, and then enzymatically converting esterified to unesterified cholesterol before filipin staining. The size and structure of particles comprising extracellular cholesteryl ester-rich lipid deposits was different in regions of necrosis as detected using filipin compared with the Sudan lipid-soluble dye oil red 0. Whereas oil red 0 staining often indicated that extracellular cholesteryl ester in these regions occurred in amorphous and spherical particles of varying sizes, filipin staining revealed that extracellular cholesteryl ester occurred in spherical particles more uniform in size than indicated by oil red 0 staining. Also, the majority of extracellular cholesteryl ester-rich particles in necrotic regions were smaller than intracellular cholesteryl ester-containing lipid droplets. In addition, the use of filipin to detect intracellular cholesteryl ester allowed distinction of 2 subpopulations of oil red 0-stained cells which did and did not contain detectable cholesteryl ester.     

Memory,scene construction,and the human hippocampus

We evaluated two different perspectives about the function of the human hippocampus–one that emphasizes the importance of memory and another that emphasizes the importance of spatial processing and scene construction. We gave tests of boundary extension, scene construction, and memory to patients with lesions limited to the hippocampus or large lesions of the medial temporal lobe. The patients were intact on all of the spatial tasks and impaired on all of the memory tasks. We discuss earlier studies that associated performance on these spatial tasks to hippocampal function. Our results demonstrate the importance of medial temporal lobe structures for memory and raise doubts about the idea that these structures have a prominent role in spatial cognition.Two traditions of work have influenced discussion about the function of the hippocampus (1). One tradition is based on work with memory-impaired patients and the idea that the hippocampus is important for a particular kind of memory (2, 3). The other tradition is based on work with rodents and the idea that the hippocampus is critical for spatial mapping (4). Its possible role in spatial processing has been recently explored in humans as well (5), and it has been proposed that the human hippocampus is essential for the ability to construct spatially coherent scenes (6, 7).This view of hippocampal function has depended on evidence from two kinds of tasks: boundary extension and scene construction (6, 8). Boundary extension refers to the tendency to reconstruct a scene such that it has a larger background than was actually presented (9). In the Mullally et al. (8) study, memory-impaired patients exhibited boundary extension less strongly than controls. Scene construction refers to the ability to imagine and describe spatially coherent scenes. In two studies, memory-impaired patients made few references to space when visualizing and describing imagined scenes (6, 8).It is unclear how to reconcile such findings with the view that the hippocampus chiefly supports memory functions. In particular, the idea that the construction and visualization of scenes involves the hippocampus seems at odds with the historic distinction between short-term (working) memory and long-term memory and the related idea that short-term memory is independent of the hippocampus (10–12). According to this perspective, hippocampal damage should not impair performance on spatial tasks, so long as testing puts no burden on long-term memory. In an attempt to clarify these issues, we gave tests of boundary extension, scene construction, and memory to patients with well-characterized lesions limited to the hippocampus or large lesions of the medial temporal lobe.     

FoxO3a immunoreactivity is markedly decreased in the dentate gyrus, not the hippocampus proper, of the aged gerbil

Forkhead box O 3a (FoxO3a) has been known to link with aging process and senescence. In this study, we investigated the age-related changes of FoxO3a in the gerbil hippocampus using immunohistochemistry and western blot analysis. In the postnatal month 3 (PM 3) group, FoxO3a immunoreactivity was well detected in pyramidal cells of the hippocampus proper, and granule cells of the dentate gyrus. FoxO3a immunoreactivity in the pyramidal cells of the hippocampus proper was not changed until PM 24. However, in the dentate granule cells, FoxO3a immunoreactivity was much decreased in the dorsal blade, not the ventral blade, of the granule cell layer in the PM 6 and 12 groups compared to the PM 3 group. At PM 24, FoxO3a immunoreactivity in the granule cells was hardly detected. Western blot analysis showed that FoxO3a level was significantly decreased in the PM 24 group. These results indicate that FoxO3a immunoreactivity and levels are markedly decreased in the dentate gyrus of the aged gerbil hippocampus.     

The value of proper sputum collection instruction in detection of acid-fast bacillus

PURPOSE: This study was conducted to assess clinically whether intervention with instruction applying respiratory rehabilitation method for expectorating sputum was useful or not to obtain more suitable sputum for smear examination of acid-fast bacilli. SUBJECT: All specimens examined were sputa obtained from 163 patients without the instruction group and 161 patients with the instruction group, who visited our outpatients clinic during one year from September 1, 2000 to August 31, 2001 and the following one year, respectively. METHOD: Gross appearance of the sputum according to Miller & Jones' classification (M1, M2, P1-P3) and smear positive rate by fluorescence staining method after N-Acetyl-L-cysteine-NaOH treatment and centrifugation were compared between the two groups. RESULTS: M1 and P1 sputa were 21.5% and 21.5% in the no instruction group, while those were 8.1%, 36.6% in the instruction group. Difference in M2, P2 and P3 sputa were not significant between the groups. Smear positive rate was 10.4% in the no instruction group, while it was 21.1% in the instruction group. According to gross appearance of M2, P1 and P2, positive rate was 11.1%, 11.4% and 30.8% in the no instruction group, and 17.7%, 28.8%, and 26.3% in the instruction group. Chest roentogenographic findings judged by type (cavitary and non-cavitary) and extent of the pulmonary lesions of these smear positive cases (17 in the no instruction group and 34 with the instruction group), revealed that the no instruction group consisted of more predominantly severer disease with cavity and moderately or far advanced lesions as compared with the instruction group. CONSIDERATION: We could exclude that the difference in gross appearance and smear-positive rate of the sputum specimen between groups without and with the instruction might be due to differences in disease severity between the two groups with and without the instruction. CONCLUSION: The instruction for sputum expectoration seems to be useful to increase positive rate in the smear examination of acid-fast bacilli.     

Bacterial diversity within the human subgingival crevice

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.     

Cross-frequency coupling supports multi-item working memory in the human hippocampus

Recent findings indicate that the hippocampus supports not only long-term memory encoding but also plays a role in working memory (WM) maintenance of multiple items; however, the neural mechanism underlying multi-item maintenance is still unclear. Theoretical work suggests that multiple items are being maintained by neural assemblies synchronized in the gamma frequency range (25–100 Hz) that are locked to consecutive phase ranges of oscillatory activity in the theta frequency range (4–8 Hz). Indeed, cross-frequency coupling of the amplitude of high-frequency activity to the phase of slower oscillations has been described both in animals and in humans, but has never been linked to a theoretical model of a cognitive process. Here we used intracranial EEG recordings in human epilepsy patients to test pivotal predictions from theoretical work. First, we show that simultaneous maintenance of multiple items in WM is accompanied by cross-frequency coupling of oscillatory activity in the hippocampus, which is recruited during multi-item WM. Second, maintenance of an increasing number of items is associated with modulation of beta/gamma amplitude with theta band activity of lower frequency, consistent with the idea that longer cycles are required for an increased number of representations by gamma cycles. This effect cannot be explained by a difference in theta or beta/gamma power. Third, we describe how the precision of cross-frequency coupling predicts individual WM performance. These data support the idea that working memory in humans depends on a neural code using phase information.Working memory (WM), the ability to maintain information about multiple items over a short time span, is indispensable for goal-directed behavior (1). Precise synchronization of neurons and neural networks results in oscillatory activity patterns in the gamma frequency range (25–100 Hz) and serves to facilitate neural communication and memory processing (2, 3). Data from animals and humans provide evidence that sustained increases of high-frequency activity (4 –7) and theta (4–8 Hz) oscillations (8 –10) are a neural correlate of WM maintenance. However, how multiple items can be simultaneously maintained without interference remains unknown. In animals, action potentials firing with respect to specific phases of ongoing theta oscillations accompany the encoding of sequences of spatial positions (11). In addition, firing rate is modulated by the phase of gamma band activity (12, 13). A related phase code based on interactions of theta phase and gamma oscillations has been suggested to support maintenance of multiple items in WM (14, 15). Such cross-frequency coupling has been described in rodents (16, 17) and recently in the human brain (18, 19), but its link to multi-item WM has not been investigated.Here we address the question of whether multiple items are encoded by modulation of the amplitude of high-frequency oscillations by the phase of oscillations in a lower-frequency range in the human hippocampus. We used a modified Sternberg paradigm in which one, two, or four trial-unique novel faces were presented consecutively (Fig. S1). Oscillatory activity within the hippocampus, which has been shown to be specifically recruited during complex WM tasks involving multiple novel items or relational memory (7, 20, 21), was recorded in 14 epilepsy patients with bilateral hippocampal depth electrodes (Methods). Cross-frequency coupling of high-frequency amplitude to the phase of activity at lower frequency was quantified by calculating Pearson’s cross-correlations between the analytic amplitude of high-frequency oscillations and the real part of wavelet-transformed oscillations at lower frequency, shifted by the average modulation phase Figs. S2–S4.In particular, we addressed three questions derived from theoretical work. First, we studied whether WM maintenance in general was accompanied by increased coupling of gamma amplitude to theta phase as compared to baseline. Second, we tested whether maintenance of an increasing number of items results in a decreased frequency of the lower (modulating) oscillation—corresponding to longer individual theta cycles—and/or in a broader theta phase range during which gamma amplitude was enhanced (22). Finally, we investigated the behavioral relevance of theta-gamma coupling by analyzing the correlation of modulation parameters to individual performance. The analyses presented here are based on an extension (14 instead of 11 subjects) of a data sample described in an earlier article (7).     

Volitional learning promotes theta phase coding in the human hippocampus

Electrophysiological studies in rodents show that active navigation enhances hippocampal theta oscillations (4–12 Hz), providing a temporal framework for stimulus-related neural codes. Here we show that active learning promotes a similar phase coding regime in humans, although in a lower frequency range (3–8 Hz). We analyzed intracranial electroencephalography (iEEG) from epilepsy patients who studied images under either volitional or passive learning conditions. Active learning increased memory performance and hippocampal theta oscillations and promoted a more accurate reactivation of stimulus-specific information during memory retrieval. Representational signals were clustered to opposite phases of the theta cycle during encoding and retrieval. Critically, during active but not passive learning, the temporal structure of intracycle reactivations in theta reflected the semantic similarity of stimuli, segregating conceptually similar items into more distant theta phases. Taken together, these results demonstrate a multilayered mechanism by which active learning improves memory via a phylogenetically old phase coding scheme.

Volitionally controlled—or “active”—learning has become a crucial topic in education, psychology, and neuroscience (1, 2). Behavioral studies show that memory benefits from voluntary action (3–5), putatively through a distinct modulation of attention, motivation, and cognitive control (2, 6). While these functions depend on widespread frontoparietal networks (7), a critical role of the hippocampus in coordinating volitional learning has been demonstrated in both humans (8) and rodents (9) (for a review see ref. 10). However, the mechanisms by which volition improves learning and memory are not well understood. Rodent recordings suggest that hippocampal theta oscillations (usually occurring between 4 and 12 Hz) might play a critical role, because they increase during voluntary movement (11) and active sensing (12). Consistently, human studies have shown volition-related theta power increases, although in a lower frequency range (typically between 3 and 8 Hz), during navigation in virtual (13, 14) and physical (15, 16) environments. It is believed that theta oscillations facilitate mnemonic processing by providing a temporal framework for the organization of stimulus-related neural codes (17). This is observed in the phenomenon of phase precession, where spatial locations represented by place cells in the rodent hippocampus are sequentially reactivated at distinct phases of theta oscillations (18). A similar phase coding mechanism underlies the representation of possible future scenarios in rats performing a spatial decision-making task, with early and late hippocampal theta phases representing current and prospective scenarios, respectively (19). It has been proposed (17) that these forms of neural phase coding support a range of cognitive processes, including multi-item working memory (20), episodic memory (21, 22), and mental time travel (23). In humans, this proposal has received empirical support from phase-amplitude coupling studies looking at the relationship between the amplitude of high-frequency activity and the phase of activity at a lower frequency, in particular theta (24–26). However, these analyses are agnostic to the specific content that is coupled to the theta phase and thus do not reflect “phase coding” in the narrower sense. Recent studies used multivariate analysis techniques to identify stimulus-specific representational signals at the high temporal resolution provided by human intracranial electroencephalography data (iEEG, see refs. 27, 28 for review). These analyses demonstrated the relevance of theta oscillations for hippocampal reinstatement of item-context associations (29), for the orchestration of content-specific representations of goal locations (30), and for word-object associations (31). However, it is unclear whether this mechanism is recruited when learning is volitionally controlled.Building on these empirical findings and methodological advances, we aimed to elucidate whether the improved memory performance typically observed in human active learning paradigms can be traced back to a hippocampal theta phase code. In particular, we hypothesized that during active learning, this theta phase code organizes and structures stimulus-specific memory representations. We analyzed electrophysiological activity from the hippocampus and widespread neocortical regions in epilepsy patients (n = 13, age = 33.5 ± 9.32) implanted with iEEG electrodes (total number of electrodes = 392; Fig. 1F) who performed a virtual reality (VR)-based navigation and memory task. Subjects navigated in a square virtual arena (Fig. 1A) and were asked to remember images of specific objects presented at distinct spatial locations indicated by red “boxes” located on the ground (Fig. 1B). Images were only visible when participants visited the red boxes and were hidden otherwise. Navigation occurred under two conditions: active (A) and passive (P) (Fig. 1B). In the active condition, participants could freely control their movements in visiting the stimulus sites while in the passive condition, they were exposed to the navigation path and order of image presentation generated by another participant (yoked design; Fig. 1 C and D). At the end of the experiment, the recognition memory for both the actively and passively learned items was tested (Fig. 1E). We predicted that active learning would enhance memory by promoting hippocampal theta phase coding of stimulus-specific memory representations.Open in a separate windowFig. 1.Experimental procedure, electrode implantation, and behavioral results. (A) Participants studied images presented at specific locations, indicated by red boxes located on the ground, in a square virtual environment (here shown from a bird’s eye perspective). (B) Stimulus presentation during the encoding phase of the experiment as seen by a participant. (C) Schematic timeline showing the main blocks of the experiment (A = active, P = passive, counterbalanced). (D) Detailed timeline of an example-encoding block. Participants freely determined the timings and materials of study in the active condition and were exposed to the trajectory of a different subject in the passive condition. (E) Timeline of the experiment at retrieval. (F) All electrodes included in the analyses (n = 392, MNI space), color coded by participant identity. (G) Receiver operating characteristic (ROC) curves for each subject (gray) and grand average (red). (H) Proportion of correct items for all stimuli as a function of confidence. (I) Proportion of remembered items (Left) and of high-confidence remembered items (Right) for active and passive conditions. *P < 0.05; ***P < 0.001.     

Two kinds of memory signals in neurons of the human hippocampus

Prior studies of the neural representation of episodic memory in the human hippocampus have identified generic memory signals representing the categorical status of test items (novel vs. repeated), whereas other studies have identified item specific memory signals representing individual test items. Here, we report that both kinds of memory signals can be detected in hippocampal neurons in the same experiment. We recorded single-unit activity from four brain regions (hippocampus, amygdala, anterior cingulate, and prefrontal cortex) of epilepsy patients as they completed a continuous recognition task. The generic signal was found in all four brain regions, whereas the item-specific memory signal was detected only in the hippocampus and reflected sparse coding. That is, for the item-specific signal, each hippocampal neuron responded strongly to a small fraction of repeated words, and each repeated word elicited strong responding in a small fraction of neurons. The neural code was sparse, pattern-separated, and limited to the hippocampus, consistent with longstanding computational models. We suggest that the item-specific episodic memory signal in the hippocampus is fundamental, whereas the more widespread generic memory signal is derivative and is likely used by different areas of the brain to perform memory-related functions that do not require item-specific information.

The hippocampus is essential for the formation of declarative (conscious) memory (1, 2), including both episodic memory (memory for events) and semantic memory (factual knowledge). Episodic memories represent the “what, when, and where” information about remembered events (3). Here, we focus on the neural representation of episodic memory for events, specifically words presented and later repeated in a continuous recognition memory format (4).Bilateral hippocampal lesions result in substantial anterograde amnesia for new events, whether memory is tested by recall or recognition (5). By contrast, bilateral lesions to a more anterior medial temporal lobe structure―the amygdala―have no such effect (6). One might therefore expect to find single-unit activity associated with episodic memory in the hippocampus but not in the amygdala. Yet, the earliest single-neuron studies failed to detect hippocampal neurons that fired differentially to recently presented test items vs. novel items. This was true in studies with humans (7, 8) and monkeys (9–11). One early study with monkeys identified a few such neurons in the hippocampus (12), and other studies found them in areas other than the hippocampus (e.g., inferomedial temporal cortex or inferotemporal temporal cortex) (9–11, 13, 14). Overall, this was not the pattern anticipated from lesion studies.Subsequent studies successfully detected some memory-related neural activity (15–17), observing that ∼10% of hippocampal neurons exhibited differential firing rates based on item status, with some firing more for repeated items and others firing more for novel items. Surprisingly, similar “memory-selective” neurons were also reliably detected in the amygdala at approximately the same frequency. Yet, these memory-selective neurons responded differentially to the generic, categorical status of test items (repeated vs. novel) and thus are not episodic memory signals (i.e., signals representing memory for specific events). According to neurocomputational models dating back to Marr (18), episodic memory representations in the hippocampus are supported by sparse neural codes (19–21). If memories for individual items are sparsely coded in largely nonoverlapping (pattern-separated) neural assemblies, it should be possible to find neurons that respond to particular repeated items, rather than to an item’s generic status. Two recent single-unit studies with humans detected such neurons in the hippocampus, but not in the amygdala (22, 23), apparently reflecting sparsely coded episodic memories. In the present study, we tested 1) whether the generic and the item-specific signals coexist in neural firing patterns recorded during the same memory task, and 2) whether the two kinds of signals are present exclusively in the hippocampus or are also evident in other brain regions.During a continuous recognition memory procedure, neurons were simultaneously recorded from four brain regions: hippocampus, amygdala, anterior cingulate cortex, and prefrontal cortex. Altogether, 55 continuous recognition memory sessions were completed by 34 epilepsy patients who had implanted clinical depth electrodes with microwires measuring single-unit activity (SUA) and multiunit activity bilaterally (24). We limited the present analyses to SUA. Words were presented consecutively and repeated once after varying lags; patients judged each word as either “novel” or “repeated.” Thus, repeated words differed from their earlier presentations as novel words only with respect to their combined “what, when, and where” episodic status (3).     

Three-dimensional virtual histology of the human hippocampus based on phase-contrast computed tomography

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer’s disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology.

Brain mappings of the cyto- and myeloarchitecture in larger brain areas performed postmortem are required to advance our understanding of the human brain in quantitative terms. Beyond refinements of a brain atlas, they are also essential for later integration of in vivo functional observations with high-resolution structural data (1–3). Mapping the brain, however, requires additional imaging approaches, which can visualize and quantify the three-dimensional (3D) architectonics, including data from more than a single individual (2). The potential of phase-contrast X-ray tomography also known as phase-contrast computed tomography (PC-CT) for 3D brain imaging has been recently demonstrated, both for small animals models (4–8) and the human brain (9–12). Since the entire 3D architecture on all scales is relevant for physiological functions and pathological mechanisms, multiscale implementations of PC-CT (13) are particularly suitable for brain mapping.Complementary to genomics, proteomics, and metabolics, structural data are also required to unravel mechanisms of neurodegenerative diseases. Such data must be comprehensive (large patient and control groups), quantitative, and fully digital; amenable to advanced analysis including deep learning; and intrinsically three-dimensional. Alzheimer’s disease (AD) is a case in point: Evidence for morphological changes in the hippocampus upon aging and disease can be found already in vivo with MRI. To interpret such data based on a reference model, a 3D probabilistic atlas of the hippocampus was put forward in ref. 3, combining postmortem MRI with histology. The authors concluded that, to test the hypothesis of differential involvement of hippocampal subfields in AD, a “more granular study” of the hippocampus in aging and disease would be required and hence higher-resolution and truly 3D data.To this end, we here present an advanced and multiscale implementation of PC-CT in combination with automated segmentation and statistical analysis of morphological features. In this way, a much-needed complement to conventional 2D histology is provided, sparing sample sectioning and staining. The signal is generated by the spatial variation of the real-valued part of the X-ray index of refraction n=1−δ+iβ, with δ being proportional to the electron density. Importantly, the advantage of PC-CT derives from the real-valued decrement being significantly larger than the imaginary, absorption-accounting component β; i.e., δ/β≈103 in the hard X-ray regime. Image contrast is then efficiently formed by free-space propagation, i.e., self-interference of a partially coherent beam behind the object. The fact that this does not require any additional optics between the object and the detector provides a benefit both for dose efficiency and for resolution. Several PC-CT studies have already targeted hippocampal cytoarchitecture in transgenic mouse models for AD (7, 14–17), which exhibit considerable contrast for a typical hallmark associated with this disease, namely β-amyloid plaques. In a recent study, we could also demonstrate the potential of PC-CT on paraffin-embedded hippocampal human tissue affected by AD and evaluate its capability to visualize different pathologies, including plaques, depletion of neurons, or possible recruitment of microglia to affected sites (12).In this work, we study the 3D cytoarchitecture of the human hippocampus, which serves the formation of declarative long-term memory, i.e., remote episodic or remote semantic memory, but may also affect recent memory, emotions, and vegetative functions (18). Pathologically, the hippocampus is one of the regions first affected in AD (19). As we show here, the throughput of PC-CT measurement, reconstruction, segmentation, and analysis is sufficiently high to treat data from a larger pool, here consisting of postmortem paraffin-embedded tissue blocks of several individuals, both of an AD and of a control group (CTRL), categorized by neuropathological assessment based on the National Institute of Aging – Alzheimer’s Association (NIA-AA)–recommended ABC staging (20, 21). We specifically target the dentate gyrus (DG) and its AD-caused structural alterations. As recently shown, hippocampal neurogenesis and plasticity of the entire hippocampal circuitry are linked to the DG and are found to sharply drop in AD (21). Further, we deliberately do not focus on plaques and tangles in AD, which have already been targeted by a high number of studies, but address in particular the nuclear structure of the DG neurons, since recent evidence points to a nuclear origin of AD (22) including chromatin structures (23). In addition, we also include 3D imaging examples of other parts and structures of the hippocampus and provide the corresponding statistical analysis.Fig. 1 shows a schematic of the hippocampus that is embedded in both left and right temporal lobes of the cerebral cortex, as a part of the limbic system. In Fig. 1A, the hippocampus is sketched in the sagittal plane, where it forms an elongated structure of about 4 to 5.2 cm in length (24). In Fig. 1B, the frontal plane is shown, in which the appearance is often denoted as snail shaped. Its characteristic functional units are shown in Fig. 1C, most prominently including the cornu ammonis (CA) and the DG, which is a dense zone of granule cells. In the polysynaptic signal pathway relevant in semantic memory formation, input signals from the entorhinal cortex (EC) reach first the DG, which is composed of elliptically shaped granular cells with millimeter-long dendrites. Connected through mossy fibers, information is further processed in the CA, whose neurons are characterized by their pyramidal-shaped bodies. The compartmentalization of the CA with commonly attributed subregions CA1 to -4 is not entirely standardized. The information exits the hippocampus to the inferior temporal cortex, the temporal pole, and the prefrontal cortex, constituting the gray matter (GM). There are further pathways of information processing, involving myelinated tracts in the white matter (WM) that link the hippocampus to further brain regions. The physiological relevance of the hippocampus, with respect to several important signal pathways and its pivotal role in memory function and neurodegenerative diseases, in particular in AD, underpins the necessity to study its 3D structure with cellular and subcellular resolution.Open in a separate windowFig. 1.Human hippocampus overview. (A and B) Schematics of the human hippocampus (gold) and its location, (A) in sagittal and (B) in frontal view. (C) Virtual slice through overview PC-CT data in EB configuration. The different neuronal layers are outlined: DG; CA differentiated into CA1, CA2, CA3, and CA4; WM; GM; and EC. A region with calcified blood vessels (BV) is also indicated. (D and E) High-resolution PC-CT data from an AD patient. (D) Volume rendering of calcified plaques (blue) in close proximity to the DG (gold). (E) Calcified β-amyloid-plaques (P) and calcified BVs are observed only to one side of the DG, as shown here in a maximum-intensity projection (16.2 μm thickness). Red arrows indicate the vascular connections between plaques. (Scale bars: C, 1 mm; E, 30 μm.)To cover the hippocampal cytoarchitecture in a larger patient cohort, we increase the sample throughput with respect to earlier work (12) by an optimized recording strategy enabling a large sample pool at high and comparable data quality, and we implement a multiscale PC-CT workflow for human brain tissue, based on parallel-beam (PB) recordings at high field of view (FOV) combined with zooms into region of interest (ROI) scanned at high magnification based on cone beam geometry. We then use machine learning based on the V-net architecture to segment neurons, followed by optimal transport (OT) theory to unravel pathological alterations. Note that OT enables us to identify “movement” in a patient cohort based on transport metrics in a structural feature space, which we define in the image segmentation step. OT also offers significant advantages over standard statistical tools such as t testing of a single parameter, since it can compare the entire neuron population, by metrics quantifying changes in their distribution.The implementation of PC-CT, notably regarding the multiscale configuration that comprises different zoom levels, is detailed in Materials and Methods. Beginning with overview scans with a FOV of several millimeters in expanded-beam (EB) configuration as in Fig. 1 or in PB, subregions of the hippocampus are presented at the different zoom levels in Results I: Multiscale Tomography of the Hippocampus. The structure of granule cell nuclei in the DG is then investigated in volumes of 10⁸ to 10⁹ μm³ at voxel size of px≈160 nm and in some cases even at px≈50 nm, based on geometric magnification using a divergent and highly coherent beam exiting an X-ray waveguide (WG). In the high-resolution reconstructions, neurons and in particular DG cell nuclei are segmented. Based on the segmentation masks, histograms of morphological features are obtained, containing results on the order of 10,000 neurons for each tissue sample. Histograms of five selected features in autopsies of 20 individuals (11 subjects with intermediate to high AD neuropathologic change according to ref. 20, in the following referred to as AD; 7 controls; and 2 with diffuse presentation based on ABC score) are then compared in Results II: Geometric and Statistical Analysis, using the OT tools. In a very general manner, we propose an analysis workflow to identify a pathway from healthy to pathological structure.     

Z-DNA-forming sites within the human beta-globin gene cluster.

Agarose-encapsulated, metabolically active, permeabilized nuclei from human hematopoietic cell lines were tested for Z-DNA formation in the beta-globin gene cluster. Biotinylated monoclonal antibodies against Z-DNA were diffused into the nuclei and cross-linked to DNA with a 10-ns laser exposure at 266 nm. Following digestion with restriction enzymes, fragments that had formed Z-DNA were isolated. Seventeen regions with Z-DNA sequence motifs in the 73-kb region were studied by PCR amplification, and five were found in the Z conformation.     

Growth hormone is produced within the hippocampus where it responds to age, sex, and stress

Recent studies by our group and others have demonstrated that growth hormone (GH) is produced endogenously within the hippocampal formation, a brain structure associated with learning and aspects of emotional experience. Here, we demonstrate that this endogenously produced GH is modulated by age and sex differences and the presence of estrogen. GH mRNA levels were higher in females than males, especially during proestrus, a stage of estrus when estrogen levels are elevated. Moreover, GH expression was increased in ovariectomized females that were treated with estradiol. This increase in GH mRNA in response to estrogen was followed by the appearance of GH protein and was negatively correlated with the expression levels of insulin-like growth factor-I mRNA, suggesting a feedback relationship between insulin-like growth factor-I and GH in the brain. GH mRNA levels were also elevated in primary neuronal cultures exposed to 17-beta-estradiol in vitro, further confirming the direct influence of estrogen on GH expression. Finally, exposure to an acute stressful event increased the expression and production of GH in both males and females. However, the stress-induced increase of GH in females depended on the stage of the estrous cycle in which they were exposed to the stressful event. Together, these data further demonstrate that GH is endogenously produced in the adult hippocampal formation, where it is regulated by age, estrogen, and exposure to environmental stimuli. These results suggest that GH may be involved in functions ascribed to the hippocampus, such as learning and the response to stressful experience.     

Validation of virtual colonoscopy in the detection of colorectal polyps and masses: rationale for proper study design

Summary Background. The clinicopathological and biological significance of the expression of iNOS in pancreatic cancer remains unclear. The goal of this study was to determine the possible roles and clinical significance of iNOS expression in pancreatic cancer. Methods. Seventy-two pancreatic adenocarcinoma tissue specimens were obtained by surgical resection. We investigated the immunohistochemical expression of iNOS in 72 patients with pancreatic cancer with respect to variable clinicopathological characteristics, proliferation activity (assessed by Ki-67 expression), apoptosis (assessed by TUNEL stain), and microvessel density (assessed by CD34 expression; angiogenesis). Results. Immunohistochemical investigations demonstrated immunolabeling of tumor cells with anti-iNOS antibody. Positivity for iNOS was observed in 48/72 (66.7%). The expression of iNOS protein did not correlate with age, bilirubin, tumor marker, location, size, AJCC stage, differentiation, distant metastasis, or patient survival. No significanct association was found between iNOS expression and proliferation or microvessel density in pancreatic cancer. Apoptotic index (AI) of positive iNOS expressions were significantly higher than negative expression (p<0.001). Conclusion. Inducible nitric oxide synthase (iNOS) is expressed by human pancreatic cancer, and its presence is positively correlated with apoptosis of cancer cells that could provide the basis for the development of therapeutic strategies in human pancreatic cancer.     

The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization

Glucocorticoids (GCs) exert profound effects on bone and are essential for human osteoblast differentiation. However, GCs are still interpreted as negative regulators of bone formation, mainly caused by the detrimental effects on bone after clinical use of GCs. In this paper we emphasize the importance of GCs for proper human osteoblast differentiation and matrix mineralization. We show that human osteoblast differentiation needs to be triggered by GCs in a specific time-window during the early stages of development. Exposure to GCs in the beginning of osteoblast development induces a dose dependent increase in alkaline phosphatase activity and matrix mineralization. GC-induced differentiation stimulated expression of genes involved in bone formation and suppressed genes that negatively regulate bone formation and mineralization. Furthermore we highlight the importance of local cortisol activation in osteoblasts by expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1).     

Sparse and distributed coding of episodic memory in neurons of the human hippocampus

Neurocomputational models hold that sparse distributed coding is the most efficient way for hippocampal neurons to encode episodic memories rapidly. We investigated the representation of episodic memory in hippocampal neurons of nine epilepsy patients undergoing intracranial monitoring as they discriminated between recently studied words (targets) and new words (foils) on a recognition test. On average, single units and multiunits exhibited higher spike counts in response to targets relative to foils, and the size of this effect correlated with behavioral performance. Further analyses of the spike-count distributions revealed that (i) a small percentage of recorded neurons responded to any one target and (ii) a small percentage of targets elicited a strong response in any one neuron. These findings are consistent with the idea that in the human hippocampus episodic memory is supported by a sparse distributed neural code.The hippocampus is known to play a fundamental role in declarative memory (1–4), but it is not known how mnemonic information is coded by the activity of individual hippocampal neurons. At least three different coding schemes have been considered: a localist coding scheme, a fully distributed coding scheme, and a sparse distributed coding scheme (5). In a localist coding scheme, an individual neuron (sometimes referred to as a “grandmother cell”) codes only one memory, and each memory is coded by the activity of only one neuron. In a fully distributed coding scheme, each memory is coded instead by a pattern of activity across many hippocampal neurons. Falling between these two extremes is a sparse distributed coding scheme in which each memory is coded by the activity of a small proportion of hippocampal neurons, and each neuron contributes to the representation of only a few memories. Sparse distributed coding has long been hypothesized to be the most efficient way for hippocampal neurons to encode episodic memories (remembering events) in rapid succession without overwriting previously stored memories (6–8).Most prior work concerned with the coding of declarative memory in the human hippocampus has focused on the neural representation of semantic memories (remembering facts), such as memory for famous people or landmarks (9, 10). The results of these studies suggest that long-established semantic memories may be represented by fewer than 1% of neurons in the hippocampus (11). However, neurocomputational theories are concerned with the representation of episodic memories. The purpose of our study was to test predictions of these neurocomputational theories about how episodic memories are represented by neurons of the hippocampus.The representation of episodic memory in the hippocampus typically has been investigated using recognition procedures. In recognition, the task is to discriminate between familiar items presented earlier in the experimental session (targets) and novel items not previously presented (foils). An episodic memory signal is evident when neurons exhibit different levels of activity for targets (old items) vs. foils (new items). The first recognition studies with humans (12, 13) and monkeys (14–16) failed to detect evidence of episodic memory in neurons of the hippocampus, but more recent studies have identified hippocampal neurons that differentiate targets from foils (17–21). However, these studies did not investigate how the representation of individual targets is distributed across neurons of the hippocampus. Instead, the aim was to find cells that distinguish the class of targets from the class of foils.We investigated the representation of individual targets in neurons of the human hippocampus. The participants were nine patients with pharmaco-resistant epilepsy requiring the implantation of intracranial wire electrodes for clinical evaluation and localization of seizure foci for possible surgical resection. Among them, the patients completed a total of 18 recognition memory tasks in which they first studied 32 words and then attempted to distinguish between the 32 targets that had appeared on the study list and 32 foils that had not. Each of the 64 items on the recognition test was presented only once, a format that differs from many other neurophysiology studies that present individual stimuli multiple times to identify neurons with reliable stimulus-specific firing properties. The multiple-presentation method is well-suited to the study of semantic memory (e.g., a neuron that is found to respond reliably to six presentations of the word “baby” likely is responding to its long-established semantic meaning) but is not well-suited to the study of episodic memory. When targets and foils are presented only once on a recognition test, the targets, but not the foils, are represented by an episodic memory formed earlier at the time of learning. Under these conditions, any difference in neural activity associated with targets and foils would indicate episodic memory. Note that, if the test items were presented again, the targets and foils no longer would be clearly differentiated because even the foils would be represented by a recently formed, context-specific episodic memory. Accordingly, instead of using multiple stimulus presentations during the recognition test, we examined the distribution of activity associated with once-presented targets vs. once-presented foils across all recorded neurons. The different coding schemes under consideration here make distinct predictions about the expected distributions of neural activity.     

Modelling the production of nitric oxide within the human airways.

The measurement of exhaled nitric oxide (NO) is well established for monitoring of airway inflammation in bronchial asthma. It is known that the concentration of NO determined as steady state (plateau) value at a constant expiratory flow rate depends on the flow rate chosen. This suggests that the exhaled NO is released within the conducting airways, whereas alveolar NO levels are negligible. The processes involved can be described through a lung model comprising an alveolar compartment and an airway compartment thought of as a pipe. This concept has been formulated mathematically and the models proposed in the literature are essentially equivalent. NO plateau levels obtained at different flow rates allow the estimation of 1) an effective airway wall NO concentration that represents the driving force for NO release, 2) an airway diffusing capacity for NO which depends on factors impeding or facilitating NO transport, including an increase in NO-producing surface area. Clinical studies will have to assess whether the knowledge of these or related parameters offers a significant advantage over the determination of exhaled NO at a single flow rate.     

Multiple gene deletions within the human immunoglobulin heavy-chain cluster.

Two subjects, of 11,000 healthy individuals screened, were found to be missing three and four immunoglobulin isotypes, respectively (IgA1, IgG2, and IgG4; IgA1, IgG2, IgG4, and IgE), and have been analyzed at the DNA level by means of Southern blotting and Ig heavy-chain-specific probes. A broad deletion within the heavy-chain constant region (C) gene cluster was found on chromosome 14 of both probands. Two different haplotypes are described: the first has lost the C alpha 1, C psi gamma, C gamma 2, C gamma 4, and C epsilon genes; the second lacks the C psi epsilon, C alpha 1, C psi gamma, C gamma 2, and C gamma 4 genes. These findings confirm the reciprocal order of the Ig heavy-chain genes as derived by molecular cloning. The inclusion of the C psi gamma gene within the deleted regions confirms its location between C alpha 1 and C gamma 2. From the observed frequency of the homozygous genotype, 1%-3% of healthy subjects from our population are expected to be heterozygous for multiple heavy-chain gene deletions. Cross-over between mispaired homologous regions seems to be the favored mechanism of multiple Ig gene deletions and duplications, and, generally, in the evolution of the human Ig heavy-chain gene family.     

Proinflammatory profile within the grossly normal aged human aortic wall

Studies in animal models demonstrate that angiotensin II and its downstream signaling molecules, that is, matrix metalloproteinases and monocyte chemoattractant protein-1, increase within the diffusely thickened intima of central arteries with aging. Whether such age-related changes occur within the human arterial wall is unknown. We harvested "grossly normal thoracic aortas" from 5 young (20+/-3 years) and 5 old white males (65+/-6 years) at necropsy, after death from traumatic causes. The intimae of older samples were markedly and diffusely thickened compared with younger intimae and contained increased levels of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor type 1, matrix metalloproteinases 2/9, monocyte chemoattractant protein-1, and collagen I and III proteins. In situ activities of metalloproteinases 2/9 were also significantly enhanced within old, normal aortas. The thickened intima of older aortas also contained a 5-fold increase in the embryonic form of smooth muscle myosin heavy chain-labeled cells than that of younger aortas, and these fetal-type cells were colocalized with angiotensin II protein staining. The ability of isolated smooth muscle cells to invade an artificial basement membrane in response to a monocyte chemoattractant protein-1 gradient increased with age. Furthermore, angiotensin II increased the invasive capacity of young smooth muscle cells, and this effect was reduced by a metalloproteinase inhibitor or an angiotensin II receptor blocker. Thus, in the absence of lipid infiltration, the aged human aortic wall exhibits a proinflammatory profile that renders it a fertile substrate for the development of arterial disease, for example, atherosclerosis and hypertension.     

Regional specialization within the human striatum for diverse psychological functions

Decades of animal and human neuroimaging research have identified distinct, but overlapping, striatal zones, which are interconnected with separable corticostriatal circuits, and are crucial for the organization of functional systems. Despite continuous efforts to subdivide the human striatum based on anatomical and resting-state functional connectivity, characterizing the different psychological processes related to each zone remains a work in progress. Using an unbiased, data-driven approach, we analyzed large-scale coactivation data from 5,809 human imaging studies. We (i) identified five distinct striatal zones that exhibited discrete patterns of coactivation with cortical brain regions across distinct psychological processes and (ii) identified the different psychological processes associated with each zone. We found that the reported pattern of cortical activation reliably predicted which striatal zone was most strongly activated. Critically, activation in each functional zone could be associated with distinct psychological processes directly, rather than inferred indirectly from psychological functions attributed to associated cortices. Consistent with well-established findings, we found an association of the ventral striatum (VS) with reward processing. Confirming less well-established findings, the VS and adjacent anterior caudate were associated with evaluating the value of rewards and actions, respectively. Furthermore, our results confirmed a sometimes overlooked specialization of the posterior caudate nucleus for executive functions, often considered the exclusive domain of frontoparietal cortical circuits. Our findings provide a precise functional map of regional specialization within the human striatum, both in terms of the differential cortical regions and psychological functions associated with each striatal zone.In addition to its central role in selecting, planning, and executing motor behavior (1, 2), the human striatum has been reported to be involved in diverse psychological functions, including emotion generation and regulation (3, 4), reward-related processes and decision making (5, 6), and executive functions (7, 8). These discrete functions are thought to map onto distinct functional striatal zones, which participate in separable basal ganglia-thalamocortical circuits (9–12) and are critical for the organization of behavior.Following this logic, recent attempts to parcellate the human striatum using diffusion tensor imaging (DTI) (13, 14) and resting-state functional connectivity (RSFC) (15–17) have relied on patterns of corticostriatal connectivity to identify striatal zones. Although very useful, these studies have been limited to inferring striatal function indirectly via psychological functions of connected cortical regions. In addition, it remains unclear how anatomical connections and RSFC map onto different psychological processes (18). Finally, RSFC is sometimes epiphenomenal (19), and fiber tract reconstruction with DTI is inaccurate for complex axonal projections underlying frontal corticostriatal connectivity (17).A separate body of work has attempted to differentiate striatal contributions directly to behavior empirically. However, these empirical investigations have focused on a restricted set of paradigms that may fail to capture the full range of striatal function, especially in humans. For example, converging evidence suggests a division of labor between the ventral striatum (VS) and dorsal striatum for Pavlovian and instrumental conditioning, respectively (20, 21). Within the dorsal striatum, medial regions support behavioral flexibility and lateral regions support well-learned behavior (22, 23). This work has greatly advanced our understanding of the similarities of striatal function in human and nonhuman animals. However, because of this strong reliance on classic learning paradigms, the integration of ideas about how the striatum is involved in uniquely human psychological functions, such as working memory, planning, and language, remains a work in progress.To generate a comprehensive and precise functional map of the human striatum, in terms of associations with both cortical brain regions and psychological tasks, we simultaneously analyzed corticostriatal coactivation patterns and the frequency of psychological terms in the full text of 5,809 neuroimaging studies (24). In contrast to studies of RSFC, we defined corticostriatal associations based on coactivation in task-related responses across studies. A cortical voxel and a striatal voxel were coactivated if a study reported activation in both voxels. This metric groups voxels that are associated with similar psychological processes. Similar to RSFC, this metric does not imply direct functional coupling of coactivated voxels. In contrast to existing work, we attempted to associate psychological functions with striatal areas directly, rather than inferring them indirectly based on the psychological functions of connected cortical areas. Sampling across a broad spectrum of neuroimaging studies, without regard for the psychological process under investigation, allowed a large-scale comparison of associations of striatal subregions with diverse psychological processes. This data-driven approach allowed us to (i) localize five striatal zones based on their coactivation with cortical brain areas and (ii) simultaneously characterize the association of each zone with psychological states in a relatively unbiased manner, including potential associations overlooked in both individual hypothesis-driven studies and studies of RSFC.