Benzodiazepine receptors in the hippocampus of suicides

The characteristics of benzodiazepine binding sites (distribution, number, affinity) were defined on frozen sections of suicide's hippocampus (death by hanging) labeled with 3H flunitrazepam and compared to data previously obtained on control brains. The study was carried out qualitatively by autoradiography (distribution) and quantitatively by a biochemical technique (number and affinity). As a whole, the characteristics of BZD binding sites were not modified in relation to controls, except for a very slight decrease in affinity of subtype I.     

Preservation of fine structure in vibratome-cut sections of the central nervous system stained for light microscopy

A technique without negative effects on tissue preservation that allows precise identification and subsequent removal of central nervous system nuclei for ultrastructural analysis is described. The procedure uses 200 microns thick Vibratome-cut sections of glutaraldehyde fixed brains. These sections are stained for 25 seconds with a methylene blue solution and stored for 4 hours in 0.2 M pH 7.4 phosphate buffer in 4% sucrose for optimal visualization at the light microscopic level. The stock solution of 1 g methylene blue and 1 g sodium borate in 100 ml of distilled water, is filtered through a Millipore filter and diluted 5:95 with distilled water immediately prior to use. Regions of specific interest are then processed for electron microscopy.     

Human ocular dominance columns as revealed by high-field functional magnetic resonance imaging.

We mapped ocular dominance columns (ODCs) in normal human subjects using high-field (4 T) functional magnetic resonance imaging (fMRI) with a segmented echo planar imaging technique and an in-plane resolution of 0.47 x 0.47 mm(2). The differential responses to left or right eye stimulation could be reliably resolved in anatomically well-defined sections of V1. The orientation and width ( approximately 1 mm) of mapped ODC stripes conformed to those previously revealed in postmortem brains stained with cytochrome oxidase. In addition, we showed that mapped ODC patterns could be largely reproduced in different experiments conducted within the same experimental session or over different sessions. Our results demonstrate that high-field fMRI can be used for studying the functions of human brains at columnar spatial resolution.     

Visualization of opiate receptors and opioid peptides in sequential brain sections

Autoradiographic and immunocytochemical studies were carried out on adjacent sections from formaldehyde-perfused rat brains in order to directly correlate the distribution of opiate receptors and opioid peptides. Perfusion fixation of the brains resulted in a partial loss of specific [3H]naloxone binding with essentially no change in the pharmacological properties of the remaining sites. When the distribution of sites was compared to that of enkephalin immunoreactivity in adjacent sections, striking correlations were observed in a number of areas throughout the neuraxis. Adjacent section autoradiography-immunocytochemistry should provide a useful tool for relating the anatomical distribution of opiate receptor subtypes to different opioid peptide neuronal systems.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB- and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Variability of laminin immunoreactivity in human autopsy brain

Summary Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

A rapid, simple and sensitive method for the demonstration of central catecholamine-containing neurons and axons by glyoxylic acid-induced fluorescence. II. A detailed description of methodology.

The glyoxylic acid-induced fluorescence method for localization of brain catecholamine neurons has been modified. Fluorescence is developed rapidly in cryostat sections of brains fixed by perfusion with 0.5% depolymerized paraformaldehyde and 2.0% glyoxylic acid. Since neither freeze drying nor vibratome sectioning is required, total processing time can be less than 1 hr. Both perikarya and fine varicose axons of norepinephrine- and dopamine-containing neurons can be seen throughout the neuroaxis. The modified technique retains good cytologic integrity and may provide a useful alternative for methods combining histochemical approaches.     

Staining Senile Plaques using Bodian's Method Modified with Methenamine

A new method is presented for staining various types of senile plaques isolated from the brains of patients with Alzheimer type dementia and related diseases in paraffin embedded sections using a modified Bodian's method with methenamine. This methenamine-Bodian method made it possible to observe diffuse plaques and other amyloid deposits which are barely detected by Bodian's original method. The staining of senile plaques by the method presented here was comparable to that of immunostaining with anti-β-protein. The new method also stained neurofibrillary tangles. Therefore, the methenamine-Bodian method could be widely used for the detection of senile changes in paraffin embedded sections from autopsied human brains.     

Immunofluorescence studies on gonadotropin releasing hormone (GRH) in the fore-brain and the neurohypophysis of the green frog,Rana esculenta L.

Summary Using antibodies against mammalian LH-RH, the double antibody immunofluorescence technique has been applied to serial cross sections of the brains of adult Rana esculenta. Immunoreactive material was found in perikarya of an unpaired nucleus in front of the preoptic recess. The axons of these perikarya also contain fluorescing material. They form a single bundle which passes under the preoptic recess, than splits into two tracts, one on either side of the optic chiasm. The two tracts reunite just before entering the median eminence. The axons end near the capillaries in the outer zone of the median eminence. The possibility of two separate centres for the stimulation of gonadotropic activity in the brains of anurans is discussed.The authors wish to thank Mr. E. von der Vlist and Mr. J.J. van der Vlis for preparing the illustrations     

Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.     

Immunohistoblot analysis on whole human hemispheres from normal and Alzheimer diseased brains

We demonstrate the feasibility and usefulness of the histoblot immunostaining of cryosections of whole hemispheres of healthy and Alzheimer diseased (AD) human brains by localizing a neuron-specific marker, the anti-neuronal nuclei (NeuN) antigen. As expected, cortical NeuN-immunopositive regions were generally thinner and lighter in the AD brains than in the controls. The advantages of using whole hemisphere histoblots: (1) they provide a low-resolution overview/outline of the antigen distribution in a large surface area, (2) large, thick, and/or unfixed tissue sections from post-mortem samples (perhaps of inferior tissue quality) can be compared, and (3) subsequent immunohistochemistry can be performed on the tissue sections used for the histoblots.     

A scanning electron microscopy study of vascular casts from senile human brains.

A scanning electron microscope study was performed of vascular casts from 26 senile human brains. In 15 of these, three types of arterial deformities (glomerular loop formations, vascular wickerworks and bundles) were frequently encountered. They were compared with the appearance in microangiograms and histological sections.     

A Metachromatic Stain for Neural Tissue

The need for rapid histological feedback on neural tissue is ever present. Although there are several stains which can be readily used for staining either cell bodies or fiber tracts, adequate contrasting stains which are both rapid and easy to apply are not generally available. In 1936 Chang presented a technique for whole brains utilizing the metachromatic properties of thionin. Unfortunately this procedure was very time consuming. For the last several years we have worked with several variations of this stain and have found that thionin can be reliably used as a polychrome stain for sections of neural tissue obtained from a freezing microtome.     

采用常规电泳设备建立CLARITY透明脑技术

目的:CLARITY是一种全新的组织形态学研究技术,在脑结构与功能研究中具有重要用途。探讨能否采用常规电泳设备开展该项技术。方法:从ICR雌性小鼠取得新鲜脑组织,依次进行多聚甲醛组织固定、聚丙烯酰胺水凝胶包埋、SDS电泳洗涤,通过拍照记录结果。结果:多聚甲醛固定和水凝胶包埋后的脑组织呈乳白色,柔软富有弹性;电泳洗涤后的脑组织从周边开始逐渐透明化,至洗涤第10 d时呈现均匀的半透明状态,可用于三维神经网络原位分析。结论:研究结果为采用常规电泳设备开展CLARITY技术提供了基础数据。     

Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Combined visualization of central catecholamine-and acetylcholinesterase-containing neurons: Application of the glyoxylic acid and thiocholine histochemical methods to the same Vibratome section

Summary This paper describes a procedure for demonstration of catecholamine-and acetylcholinesterase-containing neurons in the same section of central nervous tissue. The brains are first processed according to the glyoxylic acid (GA) fluorescence method for catecholamine neurons, i.e. perfused with an ice-cold GA solution, sectioned on a Vibratome instrument, immersed in a GA solution and dried under a stream of warm air. The unmounted sections are examined and photographed in the fluorescence microscope, and then stained for acetylcholinesterase according to Holmstedt's modification of the Koelle thiocholine method (incubation for 4–6 h with acetylthiocholine as substrate and Mipafox as inhibitor of non-specific cholinesterases). The sections are then examined in the light microscope, rephotographed, and the picture compared with that following the GA reaction. The present technique makes possible, for the first time, detailed light microscopical studies of the morphological relations between central catecholamine- and acetylcholinesterase-containing neurons in the same section.     

Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains ( p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.     

Induction of NADPH cytochrome P450 reductase by the Alzheimer β-protein. Amyloid as a 'foreign body'

A large body of data suggests that the Alzheimer's amyloid peptide (Abeta) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Abeta-mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Abeta peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non-physiological substrates (such as xenobiotics like drugs or other 'foreign molecules'), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Abeta can increase the expression of CP450r, SK-N-SH human neuroblastoma cells were exposed to Abeta25-35 and Abeta1-42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Abeta peptides, neuroblastoma cells showed a clear-cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non-transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged-matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Abeta causes oxidative stress.