Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three-dimensional (3D) reconstruction. In this study, we modified the immunolabeling-enabled imaging of solvent-cleared organs (iDISCO/iDISCO+) clearing protocol to image high-resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan-neuronal marker), calcitonin gene–related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high-quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease.     

全程减压与传统减压治疗面肌痉挛的疗效对比

目的比较全程微血管减压与传统减压在治疗面肌痉挛中的不同疗效。方法研究分析298例面肌痉挛病人的临床资料,按手术方式分为两组:传统减压组186例,行面神经根出脑干区减压;全程减压组112例,行面神经根全程减压。对比分析2组病人术后2周的手术效果。结果传统减压组症状完全缓解172例(92.5%),明显减轻3例(1.6%),无效11例(5.9%);全程减压组症状完全缓解110例(98.2%),明显减轻2例(1.8%)。两组比较有效率差异具有统计学意义(P<0.05),并发症发生率差异无统计学意义(P>0.05)。结论面神经根全程减压,并隔离所有可疑责任血管是提高手术疗效的关键;术后早期症状完全缓解率是衡量手术成功的有效指标。     

岩上静脉的应用解剖及其在显微血管减压术中的意义

目的 研究岩上静脉的解剖特点，为临床施行显微血管减压术提供应用解剖学资料。方法取15例（30侧）成人尸头标本．经静脉乳胶灌注处理后，在手术显微镜下观察和测量岩上静脉的位置、形态、分支及变异等情况，以及与三叉神经、面神经、位听神经等的毗邻关系。对60例三叉神经痛或面肌痉挛病人施行显微血管减压术，术中观察岩上静脉及其属支的各项情况。结果①岩上静脉位于蛛网膜下腔间隙内，呈游离悬空状．多由2-3支属支静脉汇成．最终注入岩上窦的内、中2／3段。其属支静脉起自小脑半球的前缘部分和脑桥腹侧面，岩上静脉主干之间或两侧的岩上静脉之间存在交通连接。②根据尸头单侧岩上静脉的数量．可以将其分为单干型（9侧，30．0％）、双干型（17侧，56．7％）和三干型（4侧，13．3％）；根据55支岩上静脉注入岩上窦的位置与内听道的关系．可将其分为内侧组（17支，30．9％）、中间组（24支，43．6％）和外侧组（14支。25．5％）。临床所见与解剖结果基本一致。③岩上静脉的主干或属支与三叉神经密切相关，可与三叉神经直接接触，形成压迫。没有见到岩上静脉或其属支与面神经、位听神经相接触形成压迫的情况。结论岩上静脉是颅后窝最大的和最常遇到的静脉，其主干和属支在走行过程中与三叉神经、面神经和位听神经相毗邻．并且可以对三叉神经形成压迫。在显微血管减压术中，对作为责任血管和阻挡手术人路的岩上静脉或其属支静脉．可以完全切断。     

A quantitative and ultrastructural study of substantia nigra and nucleus centralis superior in Alzheimer's disease

Summary In four patients with presenile Alzheimer's disease (AD) and three age-matched controls a quantitative study of neurons and neurofibrillary tangles (NFT) in the substantia nigra (SN) and nucleus centralis superior (NCS) was performed. A significant neuronal loss, similar in both nuclei, was found in AD cases, while the incidence of NFT was remarkably higher in NCS. Moreover, no significant correlation between neuronal loss and number of NFT was detected. An electron-microscopic study revealed that the subcortical NFT in NCS are made up of paired helical filaments in spite of their globose round shape.Supported by CNR contract no. 83.02062.04     

Morphology and quantitative changes of transient NPY-ir neuronal populations during early postnatal development of the cat visual cortex

The early postnatal development of neuropeptide Y-containing neurons in the visual cortex of the cat was analyzed. Immunohistochemistry reveals several stages of morphological differentiation and degeneration. Completely undifferentiated neurons have very small somata with nuclei surrounded by a thin rim of cytoplasm and processes unclearly differentiated into dendrites and axons. Processes bear growth cones. Differentiating neurons show an increase in soma size and complexity of processes. Axons are recognizable. Fully differentiated neurons have well-defined axonal and dendritic patterns. Degenerating neurons are identified by thick, heavily beaded processes covered by hairy appendages and vacuolar inclusions in the somata. Cell death is expressed by shrunken somata and lysed, fragmented processes. According to their postnatal time course of differentiation and/or degeneration, NPY-immunoreactive neurons, which form several morphologically distinct cell types, are grouped into 3 neuronal populations. (1) Pseudopyramidal cells, bitufted "rectangular" cells with wide dendritic fields, unitufted cells, and small multipolar cells are located in the gray matter and have a rather primitive morphology resembling cell types found in lower vertebrate cortex and tectum. They constitute a first transient neuronal population, because all neurons are fully differentiated at birth and become largely eliminated by postnatal day (P) 12. (2) Axonal loop cells are mainly located in the white matter. Their most prominent feature is an often long hairpin loop formed by either the main axon itself or by a major collateral. The axonal branches pass through the cortex to connect the white matter and layer I. Axons do not form local plexusses and terminal elements in the gray matter. Neurons differentiate perinatally, form a first peak from P6 to P10, followed by a decrease in cell number and innervation density at P12, followed by a second peak from P15 to P20. After P20 the number of axonal loop cells steadily decreases, and they become eliminated by P48. (3) A third population consists of neurons with a higher degree of axonal ramification and a variety of axonal patterns. Early members are located mainly at the layer VI/white matter border, differentiate during the first postnatal week, and give rise to a diffuse innervation of the gray matter without forming specific terminal elements. Some of the early axonal patterns persist into adulthood, whereas others are not found in the adult brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of blood flow in the mouse dorsal spinal venous system before and after dorsal spinal vein occlusion

The availability of transgenic strains has made the laboratory mouse a popular model for the study of healthy and diseased state spinal cord (SC). Essential to identifying physiologic and pathologic events is an understanding of the microvascular network and flow patterns of the SC. Using 2-photon excited fluorescence (2PEF) microscopy we performed in vivo measurements of blood flow in the lower thoracic portion of the mouse dorsal spinal vein (dSV) and in the first upstream branches supplying it, denoted as dorsal ascending venules (dAVs). We found that the dSV had large radiculomedullary veins (RMVs) exiting the SC, and that flow in the dSV between pairs of RMVs was bidirectional. Volumetric flow increased in each direction away from the point of bifurcation. Flow in the upstream dAVs varied with diameter in a manner consistent with a constant distal pressure source. By performing ex vivo 2PEF microscopy of fluorescent-gel perfused tissue, we created a 3-D map of the dorsal spinal vasculature. From these data, we constructed a simple model that predicted changes in the flow of upstream branches after occlusion of the dSV in different locations. Using an atraumatic model of dSV occlusion, we confirmed the predictions of this model in vivo.     

慢性低氧对小鼠脑微血管内皮细胞RAGE和LRP-1表达的影响

目的研究慢性低氧状态下血脑屏障上β淀粉样蛋白（amyloidD，Aβ）转运体——晚期糖基化终产物受体（RAGE）及低密度脂蛋白受体相关蛋白-1（LRP-1）表达的变化。方法将鼠脑微血管内皮细胞（BMVECs）分别进行正常气体培养（正常对照组）以及24h、36h及48h的低氧培养，用RT—PCR法和WesternBlot法分别检测细胞RAGE和LRP-1mRNA和蛋白的表达。结果低氧24h组RAGEmRNA和蛋白表达低于正常对照组（P〈0．05），低氧48h组RAGEmRNA和蛋白表达高于正常对照组（P〈O．05），低氧24h、36h及48h组RAGEmRNA和蛋白表达呈时间依赖性增高（P〈0．05）；低氧各组LRP-1mRNA和蛋白表达较正常对照组降低，呈时间依赖性（P〈0．05）；RAGE／LRP，1mRNA和蛋白表达呈时间依赖性增高（P〈0．05）。结论在慢性低氧状态下，血脑屏障BMVECs表达RAGE上调，LRP-1下调，RAGE／LRP-1比值增高，推测可能由此增加了Ap的净入脑量。     

Qualitative and quantitative development of the visual cortex in man

Anatomical parameters derived from an analysis of 35 male brains, ranging in age from 137 days after conception (DAC) to 99 years, were studied in reference to the development of the human visual cortex (area 17). Distinct structuring of laminae V and VI of area 17 is present at 137 DAC, contrasting with the relative undifferentiation of area 18 at this time. Both the differentiation of the visual cortex into the fundamental six laminae of Brodmann and the emergence of the stripe of Gennari occur between 180 and 190 DAC. At this time, sublamina IVa emerges as a separate entity, external to a transient, trilaminated cortical complex of which the two internal components form sublamina IVc while the external component becomes a part of sublamina IVb (stripe of Gennari). Lamination in area 18 follows a different time course than area 17, appearing at about 160 DAC and achieving a definite six-layered structure at the 185th DAC. The widths of the laminae in both areas continue to fluctuate after the 190th DAC while the cellular density appears to decrease. The fresh volume of the visual cortex was determined from stained serial sections of the brain, following adjustment for artifactual shrinkage. Approximations of values utilizing the 3-, 4-, and 5-parametric logistic functions reveal that maximum growth of this area occurs at birth, with area 17 attaining 50% of its maximum volume. The maximum volume is reached at the 300th DAC and it apparently begins to decrease in the first decade. The visual cortex develops at a considerably faster rate and reaches its maximal volume much earlier than does the whole brain, cerebellum, or hippocampal formation.     

Ultrastructure of the olfactory organ in the clawed frog,Xenopus laevis,during larval development and metamorphosis

Development of the olfactory epithelia of the African clawed frog, Xenopus laevis, was studied by scanning and transmission electron microscopy. Stages examined ranged from hatching through the end of metamorphosis. The larval olfactory organ consists of two chambers, the principal cavity and the vomeronasal organ (VNO). A third sensory chamber, the middle cavity, arises during metamorphosis. In larvae, the principal cavity is exposed to water-borne odorants, but after metamorphosis it is exposed to airborne odorants. The middle cavity and the VNO are always exposed to waterborne odorants. Electron microscopy reveals that in larvae, principal cavity receptor cells are of two types, ciliated and microvillar. Principal cavity supporting cells are also of two types, ciliated and secretory (with small, electron-lucent granules). After metamorphosis, the principal cavity contains only ciliated receptor cells and secretory supporting cells, and the cilia on the receptor cells are longer than in larvae. Supporting cell secretory granules are now large and electron-dense. In contrast, the middle cavity epithelium contains the same cell types seen in the larval principal cavity. The VNO has microvillar receptor cells and ciliated supporting cells throughout life. The cellular process by which the principal cavity epithelium changes during metamorphosis is not entirely clear. Morphological evidence from this study suggests that both microvillar and ciliated receptor cells die, to be replaced by newly generated cells. In addition, ciliated supporting cells also appear to die, whereas there is evidence that secretory supporting cells transdifferentiate into the adult type. In summary, significant developmental additions and neural plasticity are involved in remodeling the olfactory epithelium in Xenopus at metamorphosis. J. Comp. Neurol. 398:273–288, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical evaluation of the microvascular density through the expression of TGF‐β (CD 105/endoglin) and CD 34 receptors and expression of the vascular endothelial growth factor (VEGF) in oligodendrogliomas

Angiogenesis has been proposed as essential for the growth of solid tumors. The determinants of this process, the growth factors and the vascular endothelial receptors have brought a potential in the tumor prognostic determination as well as perspectives of “targets” for antiangiogenic therapy. In oligodendrogliomas (OL), angiogenesis is little known and/or has generated conflicting results. In order to clarify angiogenesis in OL, we have evaluated the immunohistochemical expression of vascular endothelial growth factor (VEGF) and the microvascular density (MVD) through the expression of TGF‐β (CD105/endoglin) (MVD‐CD105) and CD34 (MVD‐CD34) receptors using the Chalkley point method in 30 OL. No significant immune reaction was found for the VEGF. There was expression in <10% of tumor cells and/or staining of weak intensity in 15 (50.0%), >10% of cells and moderate intensity staining in 1 (3.33%), and negative expression in 14 (46.67%). If present, the expression was restricted to tumor and endothelial cells. Our findings suggest that VEGF has little influence on OL angiogenesis. All specimens showed CD105 and CD34 expression in the intratumor vascular endothelium, suggesting involvement of CD105 in OL angiogenesis. The mean ± SD MVD‐CD105 and MVD‐CD34 were 10.83 ± 2.24 and 11.00 ± 2.76 in OL (P = 0.086; r = 0.319); 10.00 ± 2.00 and 10.40 ± 3.02 in OL grade II (n = 15) (P = 0.547; r = 0.105), and 11.67 ± 2.22 and 11.53 ± 2.45 in OL grade III (n = 15) (P = 0.817; r = 0.551), respectively. The absence of correlation between DMV‐CD105, DMV‐CD34 and tumor grades suggests that anti‐CD105 and anti‐CD34 antibodies have different vascular specificities. MVD‐CD105 was greater in OL grade III than in OL grade II (P = 0.0032), indicating an increase in the vascular neoformation, something which must be evaluated as a possible prognostic factor in OL. Both TGF‐β and CD105 bring perspectives as “targets” for antiangiogenic treatments in OL.     

Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after spinal cord injury

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are γ-aminobutyric acid (GABA) ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 ± 0.03) than in the control group (1.34 ± 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 ± 0.013 μm) as compared to the controls (0.24 ± 0.008 μm). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 ± 0.022 μm) as compared to controls (0.28 ± 0.014 μm). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 ± 0.08) as compared to controls (1.41 ± 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection. © 1996 Wiley-Liss, Inc.     

兔大脑中动脉阻断所致局灶性脑缺血脑组织内皮抑素蛋白及mRNA表达增加

目的内皮抑素（endostatin）是强烈的抗血管再生因子。本文探讨大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）致局灶性脑缺血后脑组织内皮抑素蛋白及mRNA基因表达的变化,同时检测缺血脑组织血管内皮生长因子（vascular endothelial growth factor,VEGV0的含量。方法24只新西兰白兔随机分为正常对照组（n=5）、假手术组（n=4）、缺血2小时组（n=5）、缺血24小时组（n=5）及缺血48小时组（n=5）共5组。酶联免疫吸附试验测定VEGF含量,免疫组化分析内皮抑素蛋白变化,原位杂交检测内皮抑素mRNA表达。结果与对照组相比,MCAO局灶性脑缺血后内皮抑素蛋白和mRNA表达均明显增加,至少分别增加了50％（P〈0．01）和70％（P〈0．05）,同时缺血脑组织VEGF含量也明显增加,至少增加了270％。结论缺血导致脑组织内皮抑素表达增加,且内皮抑素的增加与缺血后脑组织VEGF变化无相关性,但可能抑制脑缺血后的血管再生,从而加重脑缺血损伤。     

Inhibition of mammalian target of rapamycin complex 1 in the brain microvascular endothelium ameliorates diabetic Aβ brain deposition and cognitive impairment via the sterol-regulatory element-binding protein 1/lipoprotein receptor-associated protein 1 signaling pathway

Aims

Mammalian target of rapamycin complex 1 (mTORC1) is highly activated in diabetes, and the decrease of low-density lipoprotein receptor-associated protein 1 (LRP1) in brain microvascular endothelial cells (BMECs) is a key factor leading to amyloid-β (Aβ) deposition in the brain and diabetic cognitive impairment, but the relationship between them is still unknown.

Methods

In vitro, BMECs were cultured with high glucose, and the activation of mTORC1 and sterol-regulatory element-binding protein 1 (SREBP1) was observed. mTORC1 was inhibited by rapamycin and small interfering RNA (siRNA) in BMECs. Betulin and siRNA inhibited SREBP1, observed the mechanism of mTORC1-mediated effects on Aβ efflux in BMECs through LRP1 under high-glucose conditions. Constructed cerebrovascular endothelial cell-specific Raptor-knockout (Raptor^fl/+) mice to investigate the role of mTORC1 in regulating LRP1-mediated Aβ efflux and diabetic cognitive impairment at the tissue level.

Results

mTORC1 activation was observed in HBMECs cultured in high glucose, and this change was confirmed in diabetic mice. Inhibiting mTORC1 corrected the reduction in Aβ efflux under high-glucose stimulation. In addition, high glucose activated the expression of SREBP1, and inhibiting of mTORC1 reduced the activation and expression of SREBP1. After inhibiting the activity of SREBP1, the presentation of LRP1 was improved, and the decrease of Aβ efflux mediated by high glucose was corrected. Raptor^fl/+ diabetic mice had significantly inhibited activation of mTORC1 and SREBP1, increased LRP1 expression, increased Aβ efflux, and improved cognitive impairment.

Conclusion

Inhibiting mTORC1 in the brain microvascular endothelium ameliorates diabetic Aβ brain deposition and cognitive impairment via the SREBP1/LRP1 signaling pathway, suggesting that mTORC1 may be a potential target for the treatment of diabetic cognitive impairment.     

兔大脑中动脉阻断所致局灶性脑缺血脑组织内皮抑素蛋白及mRNA表达增加(英文)

目的内皮抑素(endostatin)是强烈的抗血管再生因子。本文探讨大脑中动脉闭塞 (middle cerebral artery occlusion, MCAO)致局灶性脑缺血后脑组织内皮抑素蛋白及mRNA基因表达的变化,同时检测缺血脑组织血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。方法24只新西兰白兔随机分为正常对照组(n=5)、假手术组(n=4)、缺血2小时组(n=5)、缺血24小时组 (n=5)及缺血48小时组(n=5)共5组。酶联免疫吸附试验测定VEGF含量,免疫组化分析内皮抑素蛋白变化,原位杂交检测内皮抑素mRNA表达。结果与对照组相比,MCAO局灶性脑缺血后内皮抑素蛋白和mRNA表达均明显增加,至少分别增加了50%(P<0.01)和70%(P<0.05),同时缺血脑组织VEGF含量也明显增加,至少增加了270%。结论缺血导致脑组织内皮抑素表达增加,且内皮抑素的增加与缺血后脑组织VEGF变化无相关性,但可能抑制脑缺血后的血管再生,从而加重脑缺血损伤。     

Appearance of tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie preceeds the onset of clinical disease

Summary We have consistently observed tubulovesicular structures in brain tissues during the terminal stages of naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species and virus strain. In NIH Swiss mice inoculated intracerebrally or intraocularly with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, tubulovesicular structures, measuring 20–50 nm in diameter, were particularly prominent in dilated, pre-and postsynaptic neuronal processes, occasionally being mixed with synaptic vesicles. These structures appeared 13 weeks following intracerebral inoculation, 5 weeks before the onset of clinical signs, when spongiform changes were also detected. The number and density of tubulovesicular structures increased steadily during the course of clinical disease, and were particularly abundant in mice 47 to 51 weeks after intraocular inoculation. In hamsters infected with the 263 K strain of scrapie virus, these structures were initially detected 3 weeks following intracerebral inoculation and increased dramatically at 10 weeks postinoculation. The appearance of tubulovesicular structures before the onset of overt disease in mice inoculated with CJD virus by either the intracerebral or intraocular route, and before the appearance of other neuropathological changes in hamsters infected with scrapie virus, indicate that they represent either a part or aggregate of the infectious virus or a pathological product of the infection.Presented in part at the 64th annual meeting of the American Association of Neuropathologists, held in Charleston, South Carolina, June 9–12, 1988 and at the 7th annual meeting of the American Society for Virology, Austin, Texas, June 12–16, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Ministry of Health and Social Welfare, Poland     

Ultrastructural characterization of pharyngeal and esophageal motoneurons in the nucleus ambiguus of the rat

The viscerotopic organization of the upper alimentary tract has been established in the nucleus ambiguus, but there is little information about the morphology of the individual neurons innervating the pharynx and esophagus. We studied the ultrastructure of pharyngeal (PH), cervical esophageal (CE), and subdiaphragmatic esophageal (SDE) motoneurons labeled by retrogradely transported wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) in the compact formation of the nucleus ambiguus. WGA-HRP was injected into the lower pharynx, or the cervical and subdiaphragmatic esophagus of male rats. The retrogradely labeled PH neurons in the rostral portion of the compact formation were large (26.1 × 50.1 μm, 906.7 μm²), polygonal, and contained well-developed cell organelles with a round nucleus. Subsurface cisterns connected with rough endoplastic reticulum were often present near the postsynaptic membrane. Both CE and SDE neurons in the compact formation were medium-sized, round or oval, and contained well-developed cell organelles, although the SDE neuron was significantly larger than the CE neuron (24.9 × 33.6 μm, 593.0 μm² in the SDE neuron, and 19.5 × 30.2 μm, 440.3 μm² in the CE neuron). The average number of axosomatic terminals in a sectional plane was largest in PH neurons (29.0), smaller in CE neurons (7.9), and smallest in SDE neurons (4.2). The number of axosomatic terminals containing round vesicles (Gray's type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray's type II) in PH and CE neurons, but there were few Gray's type II axosomatic terminals in SDE neurons. Desmosome-like junctions at somato-somatic or somato-dendritic apposition were often present in the area surrounding SDE neurons. There were also small unlabeled neurons (9.5 × 18.1 μm, 131.8 μm²) in the compact formation of the nucleus ambiguus. The small neurons contained poorly developed cell organelles and an irregular shaped nucleus with invaginated nuclear membrane, and had no Nissl bodies. These results indicate that PH neurons have the characteristics of somatic motoneurons, and that CE and SDE neurons are similar to visceral motoneurons. © 1996 Wiley-Liss, Inc.     

Immunohistochemical localization of beta-amyloid precursor protein sequences in Alzheimer and normal brain tissue by light and electron microscopy.

Immunohistochemical staining with antibodies directed against four segments of the amyloid precursor protein (APP) was studied by light and electron microscopy in normal and Alzheimer (AD) brain tissue. The segments according to the Kang et al. sequence were: 18-38 (T97); 527-540 (R36); 597-620 (1-24 of beta-amyloid protein [BAP], R17); and 681-695 (R37) (Kang et al. [1987]: Nature 325:733-736). The antibodies recognized full length APP in Western blots of extracts of APP transfected cells. They stained cytoplasmic granules in some pyramidal neurons in normal appearing tissue from control and AD cases. In AD affected tissue, the antibodies to amino terminal sections of APP stained tangled neurons and neuropil threads, and intensely stained dystrophic neurites in senile plaques. By electron microscopy, this staining was localized to abnormal filaments. The antibody to the carboxy terminal segment failed to stain neurofibrillary tangles or neuropil threads; it did stain some neurites with globular swellings. It also stained globular and elongated deposits in senile plaque areas. The antibody against the BAP intensely stained extracellular material in senile plaques and diffuse deposits. By electron microscopy, the antibodies all stained intramicroglial deposits. Some of the extracellular and intracellular BAP-positive deposits were fibrillary. Communication between intramicroglial and extracellular fibrils was detected in plaque areas. These data suggest the following sequence of events. APP is normally concentrated in intraneuronal granules. In AD, it accumulates in damaged neuronal fibers. The amino terminal portion binds to abnormal neurofilaments. Major fragments of APP are phagocytosed and processed by microglia with the BAP portion being preserved. The preserved BAP is then extruded and accumulates in extracellular tissue.     

Distribution of benzodiazepine receptors in the rat superior colliculus: a light and electron microscope quantitative autoradiographic study

The distribution of benzodiazepine (Bdz) receptors of the central type was analysed in the superficial grey layer of the rat superior colliculus from light and electron microscope autoradiographs, using the highly specific partial reverse agonist [3H]Ro 15-4513, a radioligand which can be crosslinked to its binding sites by ultraviolet rays. Biochemical characteristics of the binding were first defined by liquid scintillation count on unfixed cryostat mesencephalic brain slices. Saturation curves (1.6-20 nM) and Scatchard plot indicated that the radioligand bound with a high affinity (Kd = 11 nM) to a single population of sites (Bmax = 650 fmol/mg dry tissue). A slight primary chemical fixation of the brain did not significantly modify the binding characteristics. The consolidation of the specific binding by ultraviolet light on prefixed brain slices was found to be optimal after a 45-min illumination period. The distribution of Bdz sites on light and electron microscope autoradiographs was then analysed by applying these binding conditions. Prefixed brain slices (50 micron thick, Vibratome) were incubated in the 15 nM radioligand in the absence (total binding) or in the presence (non-specific binding) of the non-radioactive antagonist Ro 15-1788 (10(-5) M). Quantitative light microscopic study of Epon-embedded semithin sections showed that 95% of the silver grains of the specific label were located on the neuropil to the detriment of the neuronal and glial cell compartments. In the electron microscopic study, the distribution of the specific binding sites was statistically analysed over a total of more than 10 identified single or junctional tissue compartments, using the 50% probability circle method (Williams, 1969). Apart from a slight labeling of varicose profiles, the specific labeling was found to be concentrated on two particular tissue compartments: the percentage of grains associated with contacts between varicosities and dendrites was 32%, and that associated with axodendritic synapses was 16% of the total specific labeling measured over all compartments combined. A low proportion (33%) of the labeled axodendritic interfaces was characterized by a synaptic differentiation. These results suggest that both synaptic and non-synaptic Bdz receptors are present in the rat superior colliculus, and may each modulate neuronal cell activity in a different way.     

The postnatal development of the presynaptic grid in the visual cortex of rabbits and the effect of dark-rearing

We have quantitatively investigated the postnatal development of synaptic contacts in the visual cortex of rabbits and the effects of long-term dark-rearing on this development. The synaptic contacts are visualized in full en face extension using 0.5 μm sections of E-PTA treated tissue²¹.In early postnatal life the mean size and the size distribution of the synaptic grids are much the same as in adult rabbits. Only minor changes are observed. The variations in size distribution at different levels below the pial surface, characteristic for the adult animal²¹, are also present in early postnatal life. Prior to eye opening many synaptic grids lack dense projections or are only partly covered. After eye opening the dense projections mature but are still different from the adult grids at postnatal days 21–27. The number of complex grids, annulate or horseshoe-shaped, gradually increases and is above adult level at days 21–27.Dark-rearing has no effect on size and size distribution of the synaptic grids and does not affect the frequency of complex grids. Careful observation reveals, however, that the dense projections do not develop normally. They remain small and fuzzy as in the visual cortex of young rabbits, and many grids are only partly covered.These observations demonstrate that the synaptic contacts are to a great extent genetically predetermined. They are also formed with a specific size which does not change during development and is not affected by dark-rearing. The gradual maturation of the dense projections and the retardation of their development during dark-rearing indicates that visual experience interferes with the development of these synaptic substructures. Because of the complementarity of dense projections and vesicle attachment sites the maturation of dense projections and the effect of dark-rearing can be interpreted as change in the efficacy of synaptic transmission.