The Blood-Brain Barrier in Both Humans and Rats: A Perspective From 3D Imaging.

Background: The blood-brain barrier (BBB) is part of the neurovascular unit (NVU) which plays a key role in maintaining homeostasis. However, its 3D structure is hardly known. The present study is aimed at imaging the BBB using tissue clearing and 3D imaging techniques in both human brain tissue and rat brain tissue. Methods: Both human and rat brain tissue were cleared using the CUBIC technique and imaged with either a confocal or two-photon microscope. Image stacks were reconstructed using Imaris. Results: Double staining with various antibodies targeting endothelial cells, basal membrane, pericytes of blood vessels, microglial cells, and the spatial relationship between astrocytes and blood vessels showed that endothelial cells do not evenly express CD31 and Glut1 transporter in the human brain. Astrocytes covered only a small portion of the vessels as shown by the overlap between GFAP-positive astrocytes and Collagen IV/CD31-positive endothelial cells as well as between GFAP-positive astrocytes and CD146-positive pericytes, leaving a big gap between their end feet. A similar structure was observed in the rat brain. Conclusions: The present study demonstrated the 3D structure of both the human and rat BBB, which is discrepant from the 2D one. Tissue clearing and 3D imaging are promising techniques to answer more questions about the real structure of biological specimens.

Macaque Brainnetome Atlas: A multifaceted brain map with parcellation, connection, and histology.

The rhesus macaque (Macaca mulatta) is a crucial experimental animal that shares many genetic, brain organizational, and behavioral characteristics with humans. A macaque brain atlas is fundamental to biomedical and evolutionary research. However, even though connectivity is vital for understanding brain functions, a connectivity-based whole-brain atlas of the macaque has not previously been made. In this study, we created a new whole-brain map, the Macaque Brainnetome Atlas (MacBNA), based on the anatomical connectivity profiles provided by high angular and spatial resolution ex vivo diffusion MRI data. The new atlas consists of 248 cortical and 56 subcortical regions as well as their structural and functional connections. The parcellation and the diffusion-based tractography were evaluated with invasive neuronal-tracing and Nissl-stained images. As a demonstrative application, the structural connectivity divergence between macaque and human brains was mapped using the Brainnetome atlases of those two species to uncover the genetic underpinnings of the evolutionary changes in brain structure. The resulting resource includes: (1) the thoroughly delineated Macaque Brainnetome Atlas (MacBNA), (2) regional connectivity profiles, (3) the postmortem high-resolution macaque diffusion and T2-weighted MRI dataset (Brainnetome-8), and (4) multi-contrast MRI, neuronal-tracing, and histological images collected from a single macaque. MacBNA can serve as a common reference frame for mapping multifaceted features across modalities and spatial scales and for integrative investigation and characterization of brain organization and function. Therefore, it will enrich the collaborative resource platform for nonhuman primates and facilitate translational and comparative neuroscience research.

Immp2l knockdown in male mice increases stimulus-driven instrumental behaviour but does not alter goal-directed learning or neuron density in cortico-striatal circuits in a model of Tourette syndrome and autism spectrum disorder.

Cortico-striatal neurocircuits mediate goal-directed and habitual actions which are necessary for adaptive behaviour. It has recently been proposed that some of the core symptoms of autism spectrum disorder (ASD) and Gilles de la Tourette syndrome (GTS), such as tics and other repetitive behaviours, may emerge because of imbalances in these neurocircuits. We have recently developed a model of ASD and GTS by knocking down Immp2l, a mitochondrial gene frequently associated with these disorders. The current study sought to determine whether Immp2l knockdown (KD) in male mice alters flexible, goal- or cue- driven behaviour using procedures specifically designed to examine response-outcome and stimulus-response associations, which underlie goal-directed and habitual behaviour, respectively. Whether Immp2l KD alters neuron density in cortico-striatal neurocircuits known to regulate these behaviours was also examined. Immp2l KD mice and wild type-like mice (WT) were trained on Pavlovian and instrumental learning procedures where auditory cues predicted food delivery and lever-press responses earned a food outcome. It was demonstrated that goal-directed learning was not changed for Immp2l KD mice compared to WT mice, as lever-press responses were sensitive to changes in the value of the food outcome, and to contingency reversal and degradation. There was also no difference in the capacity of KD mice to form habitual behaviours compared to WT mice following extending training of the instrumental action. However, Immp2l KD mice were more responsive to auditory stimuli paired with food as indicated by a non-specific increase in lever response rates during Pavlovian-to-instrumental transfer. Finally, there were no alterations to neuron density in striatum or any prefrontal cortex or limbic brain structures examined. Thus, the current study suggests that Immp2l is not necessary for learned maladaptive goal or stimulus driven behaviours in ASD or GTS, but that it may contribute to increased capacity for external stimuli to drive behaviour. Alterations to stimulus-driven behaviour could potentially influence the expression of tics and repetitive behaviours, suggesting that genetic alterations to Immp2l may contribute to these core symptoms in ASD and GTS. Given that this is the first application of this battery of instrumental learning procedures to a mouse model of ASD or GTS, it is an important initial step in determining the contribution of known risk-genes to goal-directed versus habitual behaviours, which should be more broadly applied to other rodent models of ASD and GTS in the future.

HumanBrainAtlas: an in vivo MRI dataset for detailed segmentations.

We introduce HumanBrainAtlas, an initiative to construct a highly detailed, open-access atlas of the living human brain that combines high-resolution in vivo MR imaging and detailed segmentations previously possible only in histological preparations. Here, we present and evaluate the first step of this initiative: a comprehensive dataset of two healthy male volunteers reconstructed to a 0.25 mm isotropic resolution for T1w, T2w, and DWI contrasts. Multiple high-resolution acquisitions were collected for each contrast and each participant, followed by averaging using symmetric group-wise normalisation (Advanced Normalisation Tools). The resulting image quality permits structural parcellations rivalling histology-based atlases, while maintaining the advantages of in vivo MRI. For example, components of the thalamus, hypothalamus, and hippocampus are often impossible to identify using standard MRI protocols-can be identified within the present data. Our data are virtually distortion free, fully 3D, and compatible with the existing in vivo Neuroimaging analysis tools. The dataset is suitable for teaching and is publicly available via our website (hba.neura.edu.au), which also provides data processing scripts. Instead of focusing on coordinates in an averaged brain space, our approach focuses on providing an example segmentation at great detail in the high-quality individual brain. This serves as an illustration on what features contrasts and relations can be used to interpret MRI datasets, in research, clinical, and education settings.

A simplified differential pacing technique for the evaluation of bidirectional cavo-tricuspid isthmus block during ablation of typical atrial flutter.

PURPOSE: Bidirectional block of the cavo-tricuspid isthmus (CTI) is an established endpoint of CTI-dependent atrial flutter (AFl) ablation. Differential pacing has been used to evaluate the CTI block. The purpose of this study is to describe a modified differential pacing technique to evaluate the CTI block. METHODS: Sixty-two patients underwent radiofrequency (RF) ablation of CTI-dependent AFl. The acute endpoints were non-inducibility of the AFl, and verification of the bidirectional CTI block by our methodology. Pacing was performed in the CS with an ablation catheter positioned immediately lateral to the CTI ablation line, and then 1-2 cm more laterally. The stimulus-to-ablation catheter atrial electrogram intervals were measured at these sites (StimCS-Abl1 and StimCS-Abl2, respectively). Pacing with the ablation catheter also was performed at these 2 sites, and the stimulus-to-CS electrogram intervals (StimABL1-CS and StimABL2-CS) were measured. The criteria for the bidirectional block were StimCS-Abl1 > StimCS-Abl2, and StimABL1-CS > StimABL2-CS. Clinical efficacy was defined as freedom from recurrent AFl during follow-up. RESULTS: Following 12.2 ± 3.7 min of RF delivery across the CTI, intervals were StimCS-Abl1 = 181.2 ± 22.7 ms and StimABL1-CS = 181.0 ± 23.6 ms, and StimCS-Abl2 = 152.2 ± 26.5 ms and StimABL2-CS = 151.2 ± 22.7 (P < 0.001). Atrial flutter was rendered not inducible in all patients, and no procedural complications were encountered. During the next 15.9 ± 0.7 months, two patients were lost to follow-up, and among the 62 other patients, one (1.7%) had flutter recurrence. CONCLUSIONS: The bidirectional CTI block can be assessed quickly and easily using only the ablation and CS catheters for differential pacing.

The Subcortical Atlas of the Rhesus Macaque (SARM) for neuroimaging.

Digitized neuroanatomical atlases that can be overlaid onto functional data are crucial for localizing brain structures and analyzing functional networks identified by neuroimaging techniques. To aid in functional and structural data analysis, we have created a comprehensive parcellation of the rhesus macaque subcortex using a high-resolution ex vivo structural imaging scan. This anatomical scan and its parcellation were warped to the updated NIMH Macaque Template (NMT v2), an in vivo population template, where the parcellation was refined to produce the Subcortical Atlas of the Rhesus Macaque (SARM) with 210 primary regions-of-interest (ROIs). The subcortical parcellation and nomenclature reflect those of the 4th edition of the Rhesus Monkey Brain in Stereotaxic Coordinates (Paxinos et al., in preparation), rather than proposing yet another novel atlas. The primary ROIs are organized across six spatial hierarchical scales from small, fine-grained ROIs to broader composites of multiple ROIs, making the SARM suitable for analysis at different resolutions and allowing broader labeling of functional signals when more accurate localization is not possible. As an example application of this atlas, we have included a functional localizer for the dorsal lateral geniculate (DLG) nucleus in three macaques using a visual flickering checkerboard stimulus, identifying and quantifying significant fMRI activation in this atlas region. The SARM has been made openly available to the neuroimaging community and can easily be used with common MRI data processing software, such as AFNI, where the atlas has been embedded into the software alongside cortical macaque atlases.

Dorsal Horn of Mouse Lumbar Spinal Cord Imaged with CLARITY.

The organization of the mouse spinal dorsal horn has been delineated in 2D for the six Rexed laminae in our publication Atlas of the Spinal Cord: Mouse, Rat, Rhesus, Marmoset, and Human. In the present study, the tissue clearing technique CLARITY was used to observe the cyto- and chemoarchitecture of the mouse spinal cord in 3D, using a variety of immunohistochemical markers. We confirm prior observations regarding the location of glycine and serotonin immunoreactivities. Novel observations include the demonstration of numerous calcitonin gene-related peptide (CGRP) perikarya, as well as CGRP fibers and terminals in all laminae of the dorsal horn. We also observed sparse choline acetyltransferase (ChAT) immunoreactivity in small perikarya and fibers and terminals in all dorsal horn laminae, while gamma aminobutyric acid (GABA) and glutamate decarboxylase-67 (GAD67) immunoreactivities were found only in small perikarya and fibers. Finally, numerous serotonergic fibers were observed in all laminae of the dorsal horn. In conclusion, CLARITY confirmed the 2D immunohistochemical properties of the spinal cord. Furthermore, we observed novel anatomical characteristics of the spinal cord and demonstrated that CLARITY can be used on spinal cord tissue to examine many proteins of interest.

Afferents of the mouse linear nucleus.

The linear nucleus (Li) was identified in 1978 from its projections to the cerebellum. However, there is no systematic study of its connections with other areas of the central nervous system possibly due to the challenge of injecting retrograde tracers into this nucleus. The present study examines its afferents from some nuclei involved in motor and cardiovascular control with anterograde tracer injections. BDA injections into the central amygdaloid nucleus result in labeled fibers to the ipsilateral Li. Bilateral projections with an ipsilateral dominance were observed after injections in a) jointly the paralemniscal nucleus, the noradrenergic group 7/ Köllike -Fuse nucleus/subcoeruleus nucleus, b) the gigantocellular reticular nucleus, c) and the solitary nucleus/the parvicellular/intermediate reticular nucleus. Retrogradely labeled neurons were observed in Li after BDA injections into all these nuclei except the central amygdaloid and the paralemniscal nuclei. Our results suggest that Li is involved in a variety of physiological functions apart from motor and balance control it may exert via its cerebellar projections.

3D imaging of PSD-95 in the mouse brain using the advanced CUBIC method.

AIMS: Postsynaptic density - 95 kDa protein (PSD95) is an important molecule on the postsynaptic membrane. It interacts with many other proteins and plays a pivotal role in learning and memory formation. Its distribution in the brain has been studied previously using in situ hybridization as well as immunohistochemistry. However, these studies are based on 2 dimensional (2D) sections and results are presented with a few sections. The present study aims to show PSD-95 distribution in 3 dimensions (3D) without slicing the brain tissue of C57BL/6 mice into sections using the advanced CUBIC technique. METHODS: Immunofluorescent staining using a PSD-95 antibody was performed on a half of the mouse brain after clarifying it using the advanced CUBIC protocol. The brain tissue was imaged using a Zeiss Z1 light sheet microscope and 3D reconstruction was completed using the Arivis Vision 4 dimensional (4D) software. RESULTS: The majority of brain nuclei have similar distribution pattern to what has been reported from in situ hybridization and immunohistochemical studies in the mouse. The signal can be easily followed in the 3D and their spatial relationship with adjacent structures clearly demarcated. In the present study, some fiber bundles also showed strong PSD-95 signal, which is different from what was shown in previous studies and need to be confirmed in future studies.

Publisher Correction: Kif1bp loss in mice leads to defects in the peripheral and central nervous system and perinatal death.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Odor Enrichment Increases Hippocampal Neuron Numbers in Mouse.

The hippocampus and olfactory bulb incorporate new neurons migrating from neurogenic regions in the brain. Hippocampal atrophy is evident in numerous neurodegenerative disorders, and altered hippocampal neurogenesis is an early pathological event in Alzheimer's disease. We hypothesized that hippocampal neurogenesis is affected by olfactory stimuli through the neural pathway of olfaction-related memory. In this study, we exposed mice to novel pleasant odors for three weeks and then assessed the number of neurons, non-neuronal cells (mainly glia) and proliferating cells in the hippocampus and olfactory bulb, using the isotropic fractionator method. We found that the odor enrichment significantly increased the neuronal cell numbers in the hippocampus, and promoted cell proliferation and neurogenesis in the olfactory bulb. In contrast, the glial cell numbers remained unchanged in both of the regions. Our results suggest that exposure to novel odor stimuli promotes hippocampal neurogenesis and support the idea that enriched environments may delay the onset or slow down the progression of neurodegenerative disorders.

Kif1bp loss in mice leads to defects in the peripheral and central nervous system and perinatal death.

Goldberg-Shprintzen syndrome is a poorly understood condition characterized by learning difficulties, facial dysmorphism, microcephaly, and Hirschsprung disease. GOSHS is due to recessive mutations in KIAA1279, which encodes kinesin family member 1 binding protein (KIF1BP, also known as KBP). We examined the effects of inactivation of Kif1bp in mice. Mice lacking Kif1bp died shortly after birth, and exhibited smaller brains, olfactory bulbs and anterior commissures, and defects in the vagal and sympathetic innervation of the gut. Kif1bp was found to interact with Ret to regulate the development of the vagal innervation of the stomach. Although newborn Kif1bp -/- mice had neurons along the entire bowel, the colonization of the gut by neural crest-derived cells was delayed. The data show an essential in vivo role for KIF1BP in axon extension from some neurons, and the reduced size of the olfactory bulb also suggests additional roles for KIF1BP. Our mouse model provides a valuable resource to understand GOSHS.

An ontologically consistent MRI-based atlas of the mouse diencephalon.

In topological terms, the diencephalon lies between the hypothalamus and the midbrain. It is made up of three segments, prosomere 1 (pretectum), prosomere 2 (thalamus), and prosomere 3 (the prethalamus). A number of MRI-based atlases of different parts of the mouse brain have already been published, but none of them displays the segments the diencephalon and their component nuclei. In this study we present a new volumetric atlas identifying 89 structures in the diencephalon of the male C57BL/6J 12 week mouse. This atlas is based on an average of MR scans of 18 mouse brains imaged with a 16.4T scanner. This atlas is available for download at www.imaging.org.au/AMBMC. Additionally, we have created an FSL package to enable nonlinear registration of novel data sets to the AMBMC model and subsequent automatic segmentation.

Melatonin receptors: distribution in mammalian brain and their respective putative functions.

Melatonin, through its different receptors, has pleiotropic functions in mammalian brain. Melatonin is secreted mainly by the pineal gland and exerts its effects via receptor-mediated and non-receptor-mediated actions. With recent advancement in neuroanatomical mapping, we may now understand better the localizations of the two G protein-coupled melatonin receptors MT1 and MT2. The abundance of these melatonin receptors in respective brain regions suggests that receptor-mediated actions of melatonin might play crucial roles in the functions of central nervous system. Hence, this review aims to summarize the distribution of melatonin receptors in the brain and to discuss the putative functions of melatonin in the retina, cerebral cortex, reticular thalamic nucleus, habenula, hypothalamus, pituitary gland, periaqueductal gray, dorsal raphe nucleus, midbrain and cerebellum. Studies on melatonin receptors in the brain are important because cumulative evidence has pointed out that melatonin receptors not only play important physiological roles in sleep, anxiety, pain and circadian rhythm, but might also be involved in the pathogenesis of a number of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and Huntington's disease.

Catheter ablation vs. antiarrhythmic drug therapy in patients with symptomatic atrioventricular nodal re-entrant tachycardia: a randomized, controlled trial.

AIMS: To conduct a randomized trial in order to guide the optimum therapy of symptomatic atrioventricular nodal re-entrant tachycardia (AVNRT). METHODS AND RESULTS: Patients with at least one symptomatic episode of tachycardia per month and an electrophysiologic diagnosis of AVNRT were randomly assigned to catheter ablation or chronic antiarrhythmic drug (AAD) therapy with bisoprolol (5 mg od) and/or diltiazem (120-300 mg od). All patients were properly educated to treat subsequent tachycardia episodes with autonomic manoeuvres or a 'pill in the pocket' approach. The primary endpoint of the study was hospital admission for persistent tachycardia cardioversion, during a follow-up period of 5 years. Sixty-one patients were included in the study. In the ablation group, 1 patient was lost to follow-up, and 29 were free of arrhythmia or conduction disturbances at a 5-year follow-up. In the AAD group, three patients were lost to follow-up. Of the remainder, 10 patients (35.7%) continued with initial therapy, 11 patients (39.2%) remained on diltiazem alone, and 7 patients (25%) interrupted their therapy within the first 3 months following randomization, and subsequently developed an episode requiring cardioversion. During a follow-up of 5 years, 21 patients in the AAD group required hospital admission for cardioversion. Survival free from the study endpoint was significantly higher in the ablation group compared with the AAD group (log-rank test, P < 0.001). CONCLUSIONS: Catheter ablation is the therapy of choice for symptomatic AVNRT. Antiarrhythmic drug therapy is ineffective and not well tolerated.

ABCA7 Mediates Phagocytic Clearance of Amyloid-ß in the Brain.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia and abnormal deposits of aggregated amyloid-ß in the brain. Recent genome-wide association studies have revealed that ABCA7 is strongly associated with AD. In vitro evidence suggests that the role of ABCA7 is related to phagocytic activity. Deletion of ABCA7 in a mouse model of AD exacerbates cerebral amyloid-ß plaque load. However, the biological role of ABCA7 in AD brain pathogenesis is unknown. We show that ABCA7 is highly expressed in microglia and when monocytes are differentiated into macrophages. We hypothesized that ABCA7 plays a protective role in the brain that is related to phagocytic clearance of amyloid-ß. We isolated microglia and macrophages from Abca7-/- and wild type mice and tested them for their capacity to phagocytose amyloid-ß oligomers. We found that the phagocytic clearance of amyloid-ß was substantially reduced in both microglia and macrophages from Abca7-/- mice compared to wild type mice. Consistent with these results, in vivo phagocytic clearance of amyloid-ß oligomers in the hippocampus was reduced in Abca7-/- mice. Furthermore, ABCA7 transcription was upregulated in AD brains and in amyloidogenic mouse brains specifically in the hippocampus as a response to the amyloid-ß pathogenic state. Together these results indicate that ABCA7 mediates phagocytic clearance of amyloid-ß in the brain, and reveal a mechanism by which loss of function of ABCA7 increases the susceptibility to AD.

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

The substantia nigra and ventral tegmental dopaminergic neurons from development to degeneration.

The pathology of Parkinson's disease (PD) is characterised by the loss of neurons in the substantia nigra parcompacta (A9), which results in the insufficient release of dopamine, and the appearance of motor symptoms. Not all neurons in the A9 subregions degenerate in PD, and the dopaminergic (DA) neurons located in the neighboring ventral tegmental area (A10) are relatively resistant to PD pathogenesis. An increasing number of quantitative studies using human tissue samples of these brain regions have revealed important biological differences. In this review, we first describe current knowledge on the multi-segmental neuromere origin of these DA neurons. We then compare the continued transcription factor and protein expression profile and morphological differences distinguishing subregions within the A9 substantia nigra, and between A9 and A10 DA neurons. We conclude that the expression of three types of factors and proteins contributes to the diversity observed in these DA neurons and potentially to their differential vulnerability to PD. In particular, the specific axonal structure of A9 neurons and the way A9 neurons maintain their DA usage makes them easily exposed to energy deficits, calcium overload and oxidative stress, all contributing to their decreased survival in PD. We highlight knowledge gaps in our understanding of the cellular biomarkers for and their different functions in DA neurons, knowledge which may assist to identify underpinning disease mechansims that could be targeted for the treatment of any subregional dysfunction and loss of these DA neurons.