Cholinergic deficits and galaninergic hyperinnervation of the nucleus basalis of Meynert in Alzheimer's disease and Lewy body disorders.

AIMS: Galanin is a highly inducible neuroprotective neuropeptide and in Alzheimer's disease (AD), a network of galaninergic fibres has been reported to hypertrophy and hyperinnervate the surviving cholinergic neurons in the basal forebrain. We aimed to determine (i) the extent of galanin hyperinnervation in patients with AD and Lewy body disease and (ii) whether galanin expression relates to the neuropathological burden and cholinergic losses. METHODS: Galanin immunohistochemistry was carried out in the anterior nucleus basalis of Meynert of 27 Parkinson's disease (PD) cases without cognitive impairment (mild cognitive impairment [MCI]), 15 with PD with MCI, 42 with Parkinson's disease dementia (PDD), 12 with Dementia with Lewy bodies (DLB), 19 with AD, 12 mixed AD/DLB and 16 controls. Galaninergic innervation of cholinergic neurons was scored semiquantitatively. For a subgroup of cases (n = 60), cholinergic losses were determined from maximum densities of choline acetyltransferase positive (ChAT+ve) neurons and their projection fibres. Quantitative data for α-synuclein, amyloid beta and tau pathology were obtained from tissue microarrays covering cortical/subcortical regions. RESULTS: Significant losses of cholinergic neurons and their projection fibres were observed across all diseases. Galaninergic hyperinnervation was infrequent and particularly uncommon in established AD and DLB. We found that hyperinnervation frequencies are significantly higher in the transition between PD without MCI to PDD and that higher burdens of co-existent AD pathology impair this galaninergic response. CONCLUSIONS: Our results suggest that galanin upregulation represents an intrinsic response early in Lewy body diseases but which fails with increasing burdens of AD related pathology.

Review: Revisiting the human cholinergic nucleus of the diagonal band of Broca.

Although the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB) is the second largest cholinergic nucleus in the basal forebrain, after the nucleus basalis of Meynert, it has not generally been a focus for studies of neurodegenerative disorders. However, the nvlDBB has an important projection to the hippocampus and discrete lesions of the rostral basal forebrain have been shown to disrupt retrieval memory function, a major deficit seen in patients with Lewy body disorders. One reason for its neglect is that the anatomical boundaries of the nvlDBB are ill defined and this area of the brain is not part of routine diagnostic sampling protocols. We have reviewed the history and anatomy of the nvlDBB and now propose guidelines for distinguishing nvlDBB from other neighbouring cholinergic cell groups for standardizing future clinicopathological work. Thorough review of the literature regarding neurodegenerative conditions reveals inconsistent results in terms of cholinergic neuronal loss within the nvlDBB. This is likely to be due to the use of variable neuronal inclusion criteria and omission of cholinergic immunohistochemical markers. Extrapolating from those studies showing a significant nvlDBB neuronal loss in Lewy body dementia, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the CA2 subfield in the hippocampus which may be especially vulnerable in Lewy body disorders.

A novel method to visualise the three-dimensional organisation of the human cerebral cortical vasculature.

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aß42 were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions.

AIMS: CLARITY is a novel technique which enables three-dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinson's brain. METHODS: Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualizing under a confocal microscope. RESULTS: We found that tissue clearing speed using passive CLARITY differs according to species (human vs. rodents), brain region and degree of fixation (fresh vs. formalin-fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualized Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue. CONCLUSIONS: The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.

Phylogenetic analyses of vector mosquito basic helix-loop-helix transcription factors.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in the regulation of a wide range of developmental processes in higher organisms and have been identified in more than 20 organisms. Mosquitoes are important vectors of certain human diseases. In this study, Aedes aegypti, Anopheles gambiae str. PEST and Culex quinquefasciatus genomes were found to encode 55, 55 and 57 bHLH genes, respectively. Further phylogenetic analyses and OrthoDB and Kyoto encyclopedia of genes and genomes orthology database searches led us to define orthology for all the identified mosquito bHLHs successfully. This provides useful information with which to update annotations to 40 Ae. aegypti, 55 An. gambiae and 38 C. quinquefasciatus bHLH genes in VectorBase. The mosquito lineage has more bHLH genes in the Atonal, neurogenin (Ngn) and Hes-related with YRPW motif (Hey) families than do other insect species, suggesting that mosquitoes have evolved to be more sensitive to vibration, light and chemicals. Mosquito bHLH genes generally have higher evolutionary rates than other insect species. However, no pervasive positive selection occurred in the evolution of insect bHLH genes. Only episodic positive selection was found to affect evolution of bHLH genes in 11 families. Besides, coding regions of several Ae. aegypti bHLH motifs have unusually long introns in which multiple copies of transposable elements have been identified. These data provide a solid basis for further studies on structures and functions of bHLH proteins in the regulation of mosquito development and for prevention and control of mosquito-mediated human diseases.

Regional and progressive thinning of the cortical ribbon in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a fatal and progressive neurodegenerative disease that is accompanied by involuntary movements, cognitive dysfunction, and psychiatric symptoms. Although progressive striatal degeneration is known to occur, little is known about how the disease affects the cortex, including which cortical regions are affected, how degeneration proceeds, and the relationship of the cortical degeneration to clinical symptoms. The cortex has been difficult to study in neurodegenerative diseases primarily because of its complex folding patterns and regional variability; however, an understanding of how the cortex is affected by the disease may provide important new insights into it. METHODS: Novel automated surface reconstruction and high-resolution MR images of 11 patients with HD and 13 age-matched subjects were used to obtain cortical thickness measurements. The same analyses were performed on two postmortem brains to validate these methods. RESULTS: Regionally specific heterogeneous thinning of the cortical ribbon was found in subjects with HD. Thinning occurred early, differed among patients in different clinical stages of disease, and appeared to proceed from posterior to anterior cortical regions with disease progression. The sensorimotor region was statistically most affected. Measurements performed on MR images of autopsy brains analyzed similarly were within 0.25 mm of those obtained using traditional neuropathologic methods and were statistically indistinguishable. CONCLUSIONS: The authors propose that the cortex degenerates early in disease and that regionally selective cortical degeneration may explain the heterogeneity of clinical expression in HD. These measures might provide a sensitive prospective surrogate marker for clinical trials of neuroprotective medications.

Spatiotemporal brain imaging of visual-evoked activity using interleaved EEG and fMRI recordings.

Combined analysis of electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) has the potential to provide higher spatiotemporal resolution than either method alone. In some situations, in which the activity of interest cannot be reliably reproduced (e.g., epilepsy, learning, sleep states), accurate combined analysis requires simultaneous acquisition of EEG and fMRI. Simultaneous measurements ensure that the EEG and fMRI recordings reflect the exact same brain activity state. We took advantage of the spatial filtering properties of the bipolar montage to allow recording of very short (125--250 ms) visual-evoked potentials (VEPs) during fMRI. These EEG and fMRI measurements are of sufficient quality to allow source localization of the cortical generators. In addition, our source localization approach provides a combined EEG/fMRI analysis that does not require any manual selection of fMRI activations or placement of source dipoles. The source of the VEP was found to be located in the occipital cortex. Separate analysis of EEG and fMRI data demonstrated good spatial overlap of the observed activated sites. As expected, the combined EEG/fMRI analysis provided better spatiotemporal resolution than either approach alone. The resulting spatiotemporal movie allows for the millisecond-to-millisecond display of changes in cortical activity caused by visual stimulation. These data reveal two peaks in activity corresponding to the N75 and the P100 components. This type of simultaneous acquisition and analysis allows for the accurate characterization of the location and timing of neurophysiological activity in the human brain.

Local and global attention are mapped retinotopically in human occipital cortex.

Clinical evidence suggests that control mechanisms for local and global attention are lateralized in the temporal-parietal cortex. However, in the human occipital (visual) cortex, the evidence for lateralized local/global attention is controversial. To clarify this matter, we used functional MRI to map activity in the human occipital cortex, during local and global attention, with sustained visual fixation. Data were analyzed in a flattened cortical format, relative to maps of retinotopy and spatial frequency peak tuning. Neither local nor global attention was lateralized in the occipital cortex. Instead, local attention and global attention appear to be special cases of visual spatial attention, which are mapped consistently with the maps of retinotopy and spatial frequency tuning, in multiple visual cortical areas.

Dynamic statistical parametric mapping: combining fMRI and MEG for high-resolution imaging of cortical activity.

Functional magnetic resonance imaging (fMRI) can provide maps of brain activation with millimeter spatial resolution but is limited in its temporal resolution to the order of seconds. Here, we describe a technique that combines structural and functional MRI with magnetoencephalography (MEG) to obtain spatiotemporal maps of human brain activity with millisecond temporal resolution. This new technique was used to obtain dynamic statistical parametric maps of cortical activity during semantic processing of visually presented words. An initial wave of activity was found to spread rapidly from occipital visual cortex to temporal, parietal, and frontal areas within 185 ms, with a high degree of temporal overlap between different areas. Repetition effects were observed in many of the same areas following this initial wave of activation, providing evidence for the involvement of feedback mechanisms in repetition priming.

Spatiotemporal activity of a cortical network for processing visual motion revealed by MEG and fMRI.

A sudden change in the direction of motion is a particularly salient and relevant feature of visual information. Extensive research has identified cortical areas responsive to visual motion and characterized their sensitivity to different features of motion, such as directional specificity. However, relatively little is known about responses to sudden changes in direction. Electrophysiological data from animals and functional imaging data from humans suggest a number of brain areas responsive to motion, presumably working as a network. Temporal patterns of activity allow the same network to process information in different ways. The present study in humans sought to determine which motion-sensitive areas are involved in processing changes in the direction of motion and to characterize the temporal patterns of processing within this network of brain regions. To accomplish this, we used both magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI). The fMRI data were used as supplementary information in the localization of MEG sources. The change in the direction of visual motion was found to activate a number of areas, each displaying a different temporal behavior. The fMRI revealed motion-related activity in areas MT+ (the human homologue of monkey middle temporal area and possibly also other motion sensitive areas next to MT), a region near the posterior end of the superior temporal sulcus (pSTS), V3A, and V1/V2. The MEG data suggested additional frontal sources. An equivalent dipole model for the generators of MEG signals indicated activity in MT+, starting at 130 ms and peaking at 170 ms after the reversal of the direction of motion, and then again at approximately 260 ms. Frontal activity began 0-20 ms later than in MT+, and peaked approximately 180 ms. Both pSTS and FEF+ showed long-duration activity continuing over the latency range of 200-400 ms. MEG responses in the region of V3A and V1/V2 were relatively small, and peaked at longer latencies than the initial peak in MT+. These data revealed characteristic patterns of activity in this cortical network for processing sudden changes in the direction of visual motion.

The representation of illusory and real contours in human cortical visual areas revealed by functional magnetic resonance imaging.

Illusory contours (perceived edges that exist in the absence of local stimulus borders) demonstrate that perception is an active process, creating features not present in the light patterns striking the retina. Illusory contours are thought to be processed using mechanisms that partially overlap with those of "real" contours, but questions about the neural substrate of these percepts remain. Here, we employed functional magnetic resonance imaging to obtain physiological signals from human visual cortex while subjects viewed different types of contours, both real and illusory. We sampled these signals independently from nine visual areas, each defined by retinotopic or other independent criteria. Using both within- and across-subject analysis, we found evidence for overlapping sites of processing; most areas responded to most types of contours. However, there were distinctive differences in the strength of activity across areas and contour types. Two types of illusory contours differed in the strength of activation of the retinotopic areas, but both types activated crudely retinotopic visual areas, including V3A, V4v, V7, and V8, bilaterally. The extent of activation was largely invariant across a range of stimulus sizes that produce illusory contours perceptually, but it was related to the spatial frequency of displaced-grating stimuli. Finally, there was a striking similarity in the pattern of results for the illusory contour-defined shape and a similar shape defined by stereoscopic depth. These and other results suggest a role in surface perception for this lateral occipital region that includes V3A, V4v, V7, and V8.

Spatiotemporal imaging of human brain activity using functional MRI constrained magnetoencephalography data: Monte Carlo simulations.

The goal of our research is to develop an experimental and analytical framework for spatiotemporal imaging of human brain function. Preliminary studies suggest that noninvasive spatiotemporal maps of cerebral activity can be produced by combining the high spatial resolution (millimeters) of functional MRI (fMRI) with the high temporal resolution (milliseconds) of electroencephalography (EEG) and magnetoencephalography (MEG). Although MEG and EEG are sensitive to millisecond changes in mental activity, the ability to resolve source localization and timing is limited by the ill-posed "inverse" problem. We conducted Monte Carlo simulations to evaluate the use of MRI constraints in a linear estimation inverse procedure, where fMRI weighting, cortical location and orientation, and sensor noise statistics were realistically incorporated. An error metric was computed to quantify the effects of fMRI invisible ("missing") sources, "extra" fMRI sources, and cortical orientation errors. Our simulation results demonstrate that prior anatomical and functional information from MRI can be used to regularize the EEG/MEG inverse problem, giving an improved solution with high spatial and temporal resolution. An fMRI weighting of approximately 90% was determined to provide the best compromise between separation of activity from correctly localized sources and minimization of error caused by missing sources. The accuracy of the estimate was relatively independent of the number and extent of the sources, allowing for incorporation of physiologically realistic multiple distributed sources. This linear estimation method provides an operator-independent approach for combining information from fMRI, MEG, and EEG and represents a significant advance over traditional dipole modeling.

Functional analysis of primary visual cortex (V1) in humans.

Human area V1 offers an excellent opportunity to study, using functional MRI, a range of properties in a specific cortical visual area, whose borders are defined objectively and convergently by retinotopic criteria. The retinotopy in V1 (also known as primary visual cortex, striate cortex, or Brodmann's area 17) was defined in each subject by using both stationary and phase-encoded polar coordinate stimuli. Data from V1 and neighboring retinotopic areas were displayed on flattened cortical maps. In additional tests we revealed the paired cortical representations of the monocular "blind spot." We also activated area V1 preferentially (relative to other extrastriate areas) by presenting radial gratings alternating between 6% and 100% contrast. Finally, we showed evidence for orientation selectivity in V1 by measuring transient functional MRI increases produced at the change in response to gratings of differing orientations. By systematically varying the orientations presented, we were able to measure the bandwidth of the orientation "transients" (45 degrees).

The representation of the ipsilateral visual field in human cerebral cortex.

Previous studies of cortical retinotopy focused on influences from the contralateral visual field, because ascending inputs to cortex are known to be crossed. Here, functional magnetic resonance imaging was used to demonstrate and analyze an ipsilateral representation in human visual cortex. Moving stimuli, in a range of ipsilateral visual field locations, revealed activity: (i) along the vertical meridian in retinotopic (presumably lower-tier) areas; and (ii) in two large branches anterior to that, in presumptive higher-tier areas. One branch shares the anterior vertical meridian representation in human V3A, extending superiorly toward parietal cortex. The second branch runs antero-posteriorly along lateral visual cortex, overlying motion-selective area MT. Ipsilateral stimuli sparing the region around the vertical meridian representation also produced signal reductions (perhaps reflecting neural inhibition) in areas showing contralaterally driven retinotopy. Systematic sampling across a range of ipsilateral visual field extents revealed significant increases in ipsilateral activation in V3A and V4v, compared with immediately posterior areas V3 and VP. Finally, comparisons between ipsilateral stimuli of different types but equal retinotopic extent showed clear stimulus specificity, consistent with earlier suggestions of a functional segregation of motion vs. form processing in parietal vs. temporal cortex, respectively.

Retinotopy and color sensitivity in human visual cortical area V8.

Prior studies suggest the presence of a color-selective area in the inferior occipital-temporal region of human visual cortex. It has been proposed that this human area is homologous to macaque area V4, which is arguably color selective, but this has never been tested directly. To test this model, we compared the location of the human color-selective region to the retinotopic area boundaries in the same subjects, using functional magnetic resonance imaging (fMRI), cortical flattening and retinotopic mapping techniques. The human color-selective region did not match the location of area V4 (neither its dorsal nor ventral subdivisions), as extrapolated from macaque maps. Instead this region coincides with a new retinotopic area that we call 'V8', which includes a distinct representation of the fovea and both upper and lower visual fields. We also tested the response to stimuli that produce color afterimages and found that these stimuli, like real colors, caused preferential activation of V8 but not V4.

Functional analysis of V3A and related areas in human visual cortex.

Using functional magnetic resonance imaging (fMRI) and cortical unfolding techniques, we analyzed the retinotopy, motion sensitivity, and functional organization of human area V3A. These data were compared with data from additional human cortical visual areas, including V1, V2, V3/VP, V4v, and MT (V5). Human V3A has a retinotopy that is similar to that reported previously in macaque: (1) it has a distinctive, continuous map of the contralateral hemifield immediately anterior to area V3, including a unique retinotopic representation of the upper visual field in superior occipital cortex; (2) in some cases the V3A foveal representation is displaced from and superior to the confluent foveal representations of V1, V2, V3, and VP; and (3) inferred receptive fields are significantly larger in human V3A, compared with those in more posterior areas such as V1. However, in other aspects human V3A appears quite different from its macaque counterpart: human V3A is relatively motion-selective, whereas human V3 is less so. In macaque, the situation is qualitatively reversed: V3 is reported to be prominently motion-selective, whereas V3A is less so. As in human and macaque MT, the contrast sensitivity appears quite high in human areas V3 and V3A.

Heterotransplantation of human Burkitt's lymphoma cell lines in athymic nude mice: tumor-host relationships.

We have explored the factors which influence tumorigenicity of Burkitt's lymphoma (BL) cell lines in athymic nude mice. Four cell lines, Namalwa, CA46, JD38, and ST486 revealed tumor incidence of 63.5, 69.0, 45.5 and 10.0%, respectively, in nude mice, but there was no correlation between tumor incidence and growth rate in vivo. Thus, growth rate and tumorigenicity are dependent upon different biochemical pathways. Evidence of tumor cell heterogeneity was demonstrated in the CA46 parent cell line. Five subclones derived from CA46 revealed varying degrees of tumor incidence (but very similar growth rates) that were consistently less than the parent CA46 line. Line 5, for example, produced 5.7-fold less tumors than the parent line. None of the BL cell lines or clones produced any metastatic lesions in liver, lung, brain, bone marrow or spleen in athymic nude mice. Northern blot analysis of c-myc mRNA levels in different BL cell lines revealed a possible relationship between percent tumor takes (but not growth rates) and the level of c-myc oncogene expression. However, no correlation was observed between c-myc mRNA levels and tumor incidence or growth rates among the CA46 clones. There was no correlation between the ability of the cell lines and the subclones to either secrete growth factors or to respond to growth factors secreted by Epstein-Barr virus-induced lymphoblastoid cells or lipopolysaccharide-activated monocytes, and their growth rates or percent tumor takes in mice. Comparison of tumor incidence and growth rates in irradiated and unirradiated mice showed that host factors influenced the growth of BL in nude mice.(ABSTRACT TRUNCATED AT 250 WORDS)