Immunohistochemical staining of post-mortem adult human brain sections

One of the challenges for modern neuroscience is to understand the basis of coordinated neuronal function and networking in the human brain. Some of these questions can be addressed using low- and high-resolution imaging techniques on post-mortem human brain tissue. We have established a versatile protocol for fixation of post-mortem adult human brain tissue, storage of the tissue in a human brain bank, and immunohistochemical analysis in order to understand human brain functions in normal controls and in neuropathological conditions. The brains are fixed by perfusion through the internal carotid and basilar arteries to enhance the penetration of fixative throughout the brain, then blocked, postfixed, cryoprotected, snap-frozen and stored at -80 degrees C. Sections are processed for immunohistochemical single- or double-label staining and conventional-, electron- or confocal laser scanning-microscopy analysis. The results gained using this tissue and protocol are vital for determining the localization of neurochemicals throughout the human brain and to document the changes that occur in neurological diseases.     

Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

A probabilistic atlas and reference system for the human brain: International Consortium for Brain Mapping (ICBM).

Motivated by the vast amount of information that is rapidly accumulating about the human brain in digital form, we embarked upon a program in 1992 to develop a four-dimensional probabilistic atlas and reference system for the human brain. Through an International Consortium for Brain Mapping (ICBM) a dataset is being collected that includes 7000 subjects between the ages of eighteen and ninety years and including 342 mono- and dizygotic twins. Data on each subject includes detailed demographic, clinical, behavioural and imaging information. DNA has been collected for genotyping from 5800 subjects. A component of the programme uses post-mortem tissue to determine the probabilistic distribution of microscopic cyto- and chemoarchitectural regions in the human brain. This, combined with macroscopic information about structure and function derived from subjects in vivo, provides the first large scale opportunity to gain meaningful insights into the concordance or discordance in micro- and macroscopic structure and function. The philosophy, strategy, algorithm development, data acquisition techniques and validation methods are described in this report along with database structures. Examples of results are described for the normal adult human brain as well as examples in patients with Alzheimer's disease and multiple sclerosis. The ability to quantify the variance of the human brain as a function of age in a large population of subjects for whom data is also available about their genetic composition and behaviour will allow for the first assessment of cerebral genotype-phenotype-behavioural correlations in humans to take place in a population this large. This approach and its application should provide new insights and opportunities for investigators interested in basic neuroscience, clinical diagnostics and the evaluation of neuropsychiatric disorders in patients.     

The brain timewise: how timing shapes and supports brain function

We discuss the importance of timing in brain function: how temporal dynamics of the world has left its traces in the brain during evolution and how we can monitor the dynamics of the human brain with non-invasive measurements. Accurate timing is important for the interplay of neurons, neuronal circuitries, brain areas and human individuals. In the human brain, multiple temporal integration windows are hierarchically organized, with temporal scales ranging from microseconds to tens and hundreds of milliseconds for perceptual, motor and cognitive functions, and up to minutes, hours and even months for hormonal and mood changes. Accurate timing is impaired in several brain diseases. From the current repertoire of non-invasive brain imaging methods, only magnetoencephalography (MEG) and scalp electroencephalography (EEG) provide millisecond time-resolution; our focus in this paper is on MEG. Since the introduction of high-density whole-scalp MEG/EEG coverage in the 1990s, the instrumentation has not changed drastically; yet, novel data analyses are advancing the field rapidly by shifting the focus from the mere pinpointing of activity hotspots to seeking stimulus- or task-specific information and to characterizing functional networks. During the next decades, we can expect increased spatial resolution and accuracy of the time-resolved brain imaging and better understanding of brain function, especially its temporal constraints, with the development of novel instrumentation and finer-grained, physiologically inspired generative models of local and network activity. Merging both spatial and temporal information with increasing accuracy and carrying out recordings in naturalistic conditions, including social interaction, will bring much new information about human brain function.     

Apoptosis in human embryo development: 3. Fas-induced apoptosis in brain primary cultures

Fas (APO-1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti-Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti-Human-Fas monoclonal antibody (0.5 μg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow-cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross-linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase-8 inhibitor (IETD-fmk).These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.     

Human endorphin: comparison with procine endorphin, enkephalin and normorphine.

We have extracted a material from human brain obtained at autopsy which has morphine-like activity on the mouse vas deferens preparation. This human endorphin behaved similarly on Sephadex G-15 and cation exchange columns as did porcine material and there were no qualitative differences in pharmacological activity between this material and porcine endorphin or enkephalin. Further purification of the human material is in progress. These results add further support to the suggestion that the stereospecific binding sites in opiate responsive species are receptors for an endogenous peptide ligand with a role in brain function.     

Dopamine transport function is elevated in cocaine users

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyscho-stimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.     

Exploratory Metabolomic Analyses Reveal Compounds Correlated with Lutein Concentration in Frontal Cortex,Hippocampus, and Occipital Cortex of Human Infant Brain

Lutein is a dietary carotenoid well known for its role as an antioxidant in the macula, and recent reports implicate a role for lutein in cognitive function. Lutein is the dominant carotenoid in both pediatric and geriatric brain tissue. In addition, cognitive function in older adults correlated with macular and postmortem brain lutein concentrations. Furthermore, lutein was found to preferentially accumulate in the infant brain in comparison to other carotenoids that are predominant in diet. While lutein is consistently related to cognitive function, the mechanisms by which lutein may influence cognition are not clear. In an effort to identify potential mechanisms through which lutein might influence neurodevelopment, an exploratory study relating metabolite signatures and lutein was completed. Post-mortem metabolomic analyses were performed on human infant brain tissues in three regions important for learning and memory: the frontal cortex, hippocampus, and occipital cortex. Metabolomic profiles were compared to lutein concentration, and correlations were identified and reported here. A total of 1276 correlations were carried out across all brain regions. Of 427 metabolites analyzed, 257 were metabolites of known identity. Unidentified metabolite correlations (510) were excluded. In addition, moderate correlations with xenobiotic relationships (2) or those driven by single outliers (3) were excluded from further study. Lutein concentrations correlated with lipid pathway metabolites, energy pathway metabolites, brain osmolytes, amino acid neurotransmitters, and the antioxidant homocarnosine. These correlations were often brain region—specific. Revealing relationships between lutein and metabolic pathways may help identify potential candidates on which to complete further analyses and may shed light on important roles of lutein in the human brain during development.     

Association of childhood trauma with cognitive function in healthy adults: a pilot study

Background  

Animal and human studies suggest that stress experienced early in life has detrimental consequences on brain development, including brain regions involved in cognitive function. Cognitive changes are cardinal features of depression and posttraumatic stress disorder. Early-life trauma is a major risk factor for these disorders. Only few studies have measured the long-term consequences of childhood trauma on cognitive function in healthy adults.     

一个新的人类乙酰转移酶基因hNATL的克隆与表达谱研究

从小鼠cDNA序列入手,依据同源分析的电子克隆方法得到一个包含N末端乙酰转移酶结构域的人类新基因hNATL(Human NAT Like)。hNATL的cDNA长1803bp,编码区621bp,Genbank 登陆号AY632082。hNATL编码的蛋白长206个氨基酸残基,包含有N乙酰转移酶结构域。hNATL 基因定位于人染色体17q25．2区,包括6个外显子和5个内含子。基因表达汇编分析结果显示, hNATL在人脑和生殖腺中高表达。Northern杂交显示,在成年小鼠中hNATL在心脏,脾脏和卵巢中出现表达。整体原位杂交的结果显示,hNATL特异表达于E7．5和E8．5的小鼠胚胎脑部和HH10期鸡胚胎的头部。上述结果提示,hNATL对胚胎期脑的发育和成体中重要器官行使正常功能发挥十分重要的作用。     

Ascorbic acid, cognitive function, and Alzheimer's disease: a current review and future direction

This narrative review appraises the human and animal studies implicating ascorbic acid (AA) in normal cognitive function and Alzheimer's disease. A research framework for how nutrition affects brain aging is proposed with emphasis on AA intake, status, metabolism, and transport into brain tissue. A final synopsis highlights areas for future research regarding AA nourishment and healthy brain aging.     

Metabolic Pathways and Activity-Dependent Modulation of Glutamate Concentration in the Human Brain

Glutamate is one of the most versatile molecules present in the human brain, involved in protein synthesis, energy production, ammonia detoxification, and transport of reducing equivalents. Aside from these critical metabolic roles, glutamate plays a major part in brain function, being not only the most abundant excitatory neurotransmitter, but also the precursor for γ-aminobutyric acid, the predominant inhibitory neurotransmitter. Regulation of glutamate levels is pivotal for normal brain function, as abnormal extracellular concentration of glutamate can lead to impaired neurotransmission, neurodegeneration and even neuronal death. Understanding how the neuron-astrocyte functional and metabolic interactions modulate glutamate concentration during different activation status and under physiological and pathological conditions is a challenging task, and can only be tentatively estimated from current literature. In this paper, we focus on describing the various metabolic pathways which potentially affect glutamate concentration in the brain, and emphasize which ones are likely to produce the variations in glutamate concentration observed during enhanced neuronal activity in human studies.

     

Did brain-specific genes evolve faster in humans than in chimpanzees?

One of the most distinctive characteristics of humans among primates is the size, organization and function of the brain. A recent study has proposed that there was widespread accelerated sequence evolution of genes functioning in the nervous system during human origins. Here we test this hypothesis by a genome-wide analysis of genes that are expressed predominantly or specifically in brain tissues and genes that have important roles in the brain, identified on the basis of five different definitions of brain specificity. Although there is little overlap among the five sets of brain-specific genes, none of them supports human acceleration. On the contrary, some datasets show significantly fewer nonsynonymous substitutions in humans than in chimpanzees for brain-specific genes relative to other genes in the genome. Our results suggest that the unique features of the human brain did not arise by a large number of adaptive amino acid changes in many proteins.     

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.     

Adult human brain cell culture for neuroscience research

Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders.     

Age-related decline of serotonin transporters in living human brain of healthy males

There is growing interest in serotonin transporter (5-HTT) function in the human brain, since alteration in 5-HTT has been suggested in a variety of neurophychiatric disorders. Age-related decline in postsynaptic 5-HT receptors has been demonstrated in postmortem human studies and in vivo imaging studies, and has been assumed to be related to changes in mental function in the normal aging process. However, few studies have investigated the aging effect on 5-HTT in human brain in vivo, since the availability of suitable ligands has been limited. To investigate the aging effect on 5-HTT in living human brain, we performed positron emission tomography (PET) scans with a selective ligand for 5-HTT, [11C](+)McN5652. We examined 28 healthy male volunteers aged between 20 and 79 years. The uptake was quantified in the thalamus and midbrain by graphical analysis with the cerebellum as a reference tissue, and binding potential (BP) was used for the index of 5-HTT binding. There was a significant age-related decline in BP in the thalamus and midbrain. The decline in [11C](+)McN5652 binding was 9.6% per decade in the thalamus and 10.5% per decade in the midbrain.