Cortico-nigral projections in the cat

Using the methods of retrograde axonal transport of horseradish peroxidase and silver impregnation of degenerating axons, certain data have been obtained demonstrating that frontal, motor, orbital, insular and limbic fields of the cortex serve as sources of afferent fibers for the compact zone of the substantia nigra. The lateral zone gets projections from the same cortical areas (besides the limbic one) as the compact part and, in addition, from the parietal associative, acoustic and visual areas.     

Combined use of silver staining of the retrograde tracer WGAapoHRP-Au and pre-embedding immunocytochemistry for electron microscopy: demonstration of dopaminergic terminals in synaptic contact with striatal neurons projecting to the substantia nigra in the rat.

We investigated the applicability of the pre-embedding immunoperoxidase technique to WGAapoHRP-Au retrograde tracing. After injection of the tracer into the substantia nigra of rat, the brain was fixed and cryostat sections were immunostained for dopamine. The sections were osmicated and silver-stained to amplify the colloidal gold particles. Products of both the immunoperoxidase staining and the silver staining could be detected and distinguished by electron microscopy at low magnification. The ultrastructure was so well preserved that synaptic characteristics could be investigated. Dopaminergic terminals were demonstrated to synapse with striatal neurons projecting to the substantia nigra.     

Intranuclear ubiquitin-immunopositive structures in human substantia nigra neurons

Marinesco bodies were discovered in substantia nigra neurons of human brain in 1902. The relationships between these intranuclear inclusions and the other structures of the cellular nucleus are still obscure. The aim of this study is to elucidate the morphological and cytochemical peculiarities of intranuclear ubiquitin-immunopositive bodies in the substantia nigra neurons of human brain and to evaluate the interconnections of these peculiarities with nucleolus by means of light microscopy, immunocytochemistry, and confocal laser microscopy. It is found that up to 20% of neurons in substantia nigra of human brain contain ubiquitin-immunopositive Marinesco bodies. These rounded structures are 1–8 μm—more often 2–4 μm—in diameter. Only one-third of them are tightly adjacent to the nucleolus. By a method of silver impregnation of argentophilic proteins associated with nucleolar organizer, the absence was shown of argentophilic proteins, which are characteristic for the nucleolus, in Marinesco bodies. Special ubiquitin-positive substantially smaller structures (less than 1 μm) are revealed in the neurons’ nuclei along with Marinesco bodies. These structures are probably the initial forms in the formation of Marinesco bodies. The existence of two types of ubiquitin-immunopositive intranuclear bodies is revealed by means of confocal microscopy: one has high intensity of immunofluorescence, and the other has low intensity. Heterogeneous distribution of immunopositive product is characteristic of the former. The presence of DNA in Marinesco bodies is detected by using SYTOX Green fluorescent dye. The absence of peripheral heterochromatin zone and weak susceptibility to toluidine blue together with the presence of DNA and the absence of argentophilic proteins suggests substantial structural and chemical differences between Marinesco bodies and nucleoli, which argues against the idea that the detected bodies are modified nucleoli.     

Increased acoplasmic transport of H3-dopamine in nigro-neostriatal neurons after reserpine

Recent evidence suggests that dopamine can undergo axoplasmic transport in nigro-neostriatal neurons by binding to amine storage granules. In the present experiments it was demonstrated that reserpine pretreatment (10 mg/kg) 24 hours before stereotaxic injections of ³H-DOPA or ³H-dopamine into the substantia nigra increases the amount of ³H-dopamine transported to the neostriatum by about 300 percent. The activity recovered from the substantia nigra was significantly reduced by reserpine pretreatment however. Stereotaxic injection of ¹⁴C-leucine into the substantia nigra indicated that neither fast nor slow axoplasmic transport of protein was influenced by reserpine pretreatment in these same neurons. The increased transport of dopamine appears therefore to be due to a relatively selective action of reserpine. The results suggest that reserpine either (i) increases the binding of dopamine to newly synthesized amine storage granules, (ii) increases the number of newly synthesized amine storage granules, or (iii) accelerates the rate of transport of amine storage granules. In addition, the results support the view that reserpine can increase the membrane permeability of adrenergic neurons to the outward movement of catecholamines.     

Axonal transport in nigro-neostriatal neurons during morphine tolerance development and abstinence in rats.

Axonal transport of [³H]protein in the nigro-neostriatal pathway in rats was examined during acute and chronic morphine administration and during morphine abstinence. Two days after a microinjection of [³H]lysine into the left substantia nigra zona compacta, more than 95% of the radioactivity present in the rat forebrain was protein-bound. Examination of frozen frontal brain sections revealed that 80–90% of the labelled protein of the injected side was located in brain areas traversed by the nigro-neostriatal pathway. As a positive control, intranigrally administered colchicine reduced the amount of [³H]protein transported after 5 days to the nucleus caudatus-putamen (neostriatum) to approx 18-26% of control. In animals rendered morphine-dependent by subcutaneous implantation of tablets containing 75 mg of morphine base, 27–86% more radioactivity accumulated in the neostriatum at 3, 4 and 5 days after [³H]lysine injection. In contrast, 23–48% less radioactivity was recovered in the neostriatal areas of animals withdrawing from morphine 24 h after [³H]lysine. Gel electrophoresis of soluble and particulate [³H]protein fractions from neostriatal tissues indicated that the gel patterns of radioactivity were not altered by chronic morphine administration. Neither morphine administration nor morphine abstinence altered the rate or amount of [³H]lysine incorporation into protein of the substantia nigra. These data demonstrate that chronic morphine administration was accompanied by a generalized increase in the amount of labelled protein transported to the neostriatum but the procedure was not sufficiently sensitive to detect a minor qualitative alteration of any particular protein(s). Furthermore, these data suggest that either the capacity or the rate of nigro-neostriatal protein transport may be increased during chronic morphine administration in the rat.     

CHEMICAL SPECIFICITY OF DOPAMINE TRANSPORT IN THE NIGRO-NEOSTRIATAL PROJECTION

Axoplasmic transport of dopamine in the nigro-neostriatal system has previously been shown by the specific accumulation of labelled dopamine in the striatum following injections of labelled DOPA or dopamine into the substantia nigra. To test the specificity, 17 different labelled materials (pipecolic acid, inulin, taurine, GABA, glycine, histidine, histamine, serotonin, 5-HTP, D-amphetamine, 3-methoxytyramine, dopamine, tyramine, norepinephrine, octopamine and high and low specificity activity DOPA) were injected into the substantia nigra and the distribution of radioactivity in the brain studied after 6 and 24 h. Only the catecholamines and octopamine gave evidence of specific accumulation in the ipsilateral striatum although some of the other compounds caused diffuse labelling of the striatum along with other brain areas.     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

Electron microscopic studies of the innervation of the human spleen

Summary The innervation of four normal human spleens was investigated by electron microscopy. Unmyelinated nerve fibers accompanied the arterial vascular system up to the arterioles of the red pulp. Neither myelinated nerve fibers nor ganglion cells were seen in the splenic hilum or in the splenic tissue itself. The nerve fibers terminated against the smooth muscle cells of the blood vessels in a manner that is typical of the autonomic nervous system. The terminal axons contained small and large granular vesicles and thus were adrenergic nerve fibers. In contrast to the results of previous studies using silver impregnation methods innervation of the red or white pulp could not be demonstrated. The findings on human spleens agree with those on mammalian spleens obtained by other authors using ultrastructural and fluorescence histochemical methods.We are indebted to Prof. Dr. K. Unsicker for his help in discussing the results     

Neurochemical evidence for denervation supersensitivity: the effect of unilateral substantia nigra lesions on apomorphine-induced increases in neostriatal acetylcholine levels

Unilateral lesions of the nigro-neostriatal dopaminergic projection were induced by injections of 6-hydroxydopamine into the zona compacta of the substantia nigra. This resulted in a reduction of neostriatal dopamine to less than 6 percent of the control side. Two months later intraperitoneal injections of apomorphine (1 mg/kg) produced contralateral turning and a significant increase in neostriatal acetylcholine levels. The increase was significantly greater in the neostriatum ipsilateral to the lesion than in the intact side. Haloperidol (1 mg/kg) produced a significant decrease in neostriatal acetylcholine but this decrease did not differ between the “denervated” and intact neostriata. The nigral 6-hydroxydopamine lesions did not by themselves affect neostriatal acetylcholine levels. The fact that apomorphine produces a greater increase in neostriatal acetylcholine after lesions of the dopaminergic nigro-neostriatal projection supports earlier behavioral data suggestive of denervation supersensitivity of neostriatal dopaminergic receptors after these lesions.     

Neurochemical sequelae of kainate injections in corpus striatum and substantia nigra of the rat.

Unilateral injection of 2 μg kainic acid into the substantia nigra of the rat results in a 45% decrease in tyrosine hydroxylase activity in the injected substantia nigra and in the ipsilateral corpus striatum. In contrast, the GABAergic nerve terminals in the substantia nigra are unaffected by this treatment. Injection of kainic acid into the striatum results in a 60% decrement in the activity of glutamate decarboxylase and of endogenous GABA levels in the ipsilateral substantia nigra whereas tyrosine hydroxylase activity remains unchanged; in addition, dopamine-sensitive adenylate cyclase activity in the ipsilateral substantia nigra decreases by 74%. These findings further support the hypothesis that intracerebral injections of kainic acid cause degeneration of neurons with cell bodies near the injection site while sparing axons passing through or terminating in the region.     

Pathological changes in dendrites of substantia nigra neurons in Parkinson's disease: a Golgi study.

Neurons of the substantia nigra show severe morphological changes in Parkinson's disease. Pathological alterations of cell bodies have been described, whereas those of neuronal processes have hardly been investigated. Golgi impregnation has been the chosen method for demonstrating neuronal processes and dendritic and somatic spines. We therefore used the Golgi-Braitenberg method to qualitatively and semi-quantitatively study the substantia nigra of eight patients with Parkinson's disease compared with eight control cases. Golgi impregnation of substantia nigra neurons was good in all control cases. In full agreement with the analysis of Braak and Braak (1986) three neuronal types within the substantia nigra were found. In cases of Parkinson's disease, severe pathological changes such as decrease of dendritic length, loss of dendritic spines and several types of dendritic varicosities were found only in the melanin-containing pars compacta neurons. Pars reticulata nerve cells were intact. These findings support the predominant role played by the dopaminergic efferent pathway in the degenerative process. The afferent pathway was not affected. This suggests that the substantia nigra lesion is primary in Parkinson's disease. Loss of neurons found in H & E sections corresponded to a lesser amount of impregnated pars compacta neurons in cases with Parkinson's disease when compared to controls. Evidences exist that the duration of the disease may be related to the extent of pathologically altered Golgi-impregnated pars compacta cells. The amount of Lewy bodies in H & E sections corresponded to the quantity of round varicosities in impregnated pars compacta neurons. These round dendritic varicosities were considered to be Lewy body inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

帕金森病大鼠STN-DBS刺激8天后黑质内胶质细胞的形态学变化

目的：观察丘脑底核脑深部电刺激（STN—DBS）慢性刺激后黑质内胶质细胞和多巴胺能细胞的变化。方法：取50只健康Wistar雄性大鼠随机分为假造模组（n=10）和6-羟基多巴胺（6-OHDA）组（11=40）。立体定向单侧造模成功后将6-OHDA组随机分为假手术组（n=10）,假刺激组（n=15）,刺激组（n=15）。刺激组和假刺激组植入STN—DBS,假手术组进行手术但不植入电极。刺激组术后第8d开始每日在固定时间给予连续脉冲刺激,持续时间30mins,连续8d。假刺激组刺激方法同上但关闭电源。在STN—DBS刺激前、刺激时和刺激后观察2mins内大鼠阿扑吗啡旋转次数。刺激结束后将大鼠断头取脑固定,取左侧黑质脱水、透明、浸蜡、包埋、切片,染色。电镜下观察细胞形态并进行细胞计数。结果：帕金森大鼠植入STN—DBS刺激后症状显著改善。慢性刺激8天后刺激组黑质内的星形胶质细胞和多巴胺能细胞均较假刺激组和假手术组明显增加并有统计学意义,而刺激组的小胶质细胞较假刺激组和假手术组有所减少但无统计学意义。结论：STN-DBS慢性刺激可以促使黑质内星形胶质细胞增多,小胶质细胞减少,对黑质内爹巴胺能细胞有一定的保护作用。     

Effects of frontal motor cortex ablation on the ultrastructure of cat substantia nigra

3,4 and 5 days after the removal by suction of the left motor and premotor cortex in cats, the presence of ultrastructural changes in the substantia nigra was investigated. Whereas after 3 days dark bouton degeneration was rare, after 4- and 5-day survival it was regularly found for a distinct type of synapse containing in its bouton densely packed small round vesicles and possessing an asymmetric synaptic junction with a dendrite. Often these darkly degenerated boutons contained dense bodies which were also observed in the same type of synapse not yet exhibiting a dark axoplasm. Various inclusions, especially glycogen depots, were present in these boutons suggesting that they were in the process of degeneration. The glial reaction was comparatively severe. In addition, darkly shrunken dendrites contacted both, by intact and by altered boutons were frequently encountered as well as single degenerated neuronal perikarya. The nature of this effect, i.e. whether transneuronal or retrograde, could not be clarified. All these alterations were found bilaterally after the unilateral cortex ablation, and were confined to the substantia nigra pars compacta along its whole anterior-posterior extent. In the pars reticulata, solely traversing myelinated axons in the process of degeneration were observed. Thus, the results are in agreement with the older studies and with evidence from primates demonstrating that the substantia nigra receives a bilateral projection from the motor and premotor cortex.     

Lactacystin诱导帕金森病大鼠黑质胶质细胞的变化

目的观察蛋白酶体抑制剂Lactacystin诱导大鼠黑质胶质细胞的变化、炎性介质NF-κB的表达。方法采用立体定向术将蛋白酶体抑制剂Lactacystin 10μg注射至大鼠黑质部位,免疫组织化学法观察黑质区多巴胺(DA)能神经元、小胶质细胞、星形胶质细胞的变化,炎性介质核转录因子κB(NF-κB)的表达。结果注射Lactacystin 3周,阿朴吗啡腹腔注射后出现典型旋转行为;8周后实验组大鼠损毁侧黑质酪氨酸羟化酶(TH)阳性细胞数明显减少,黑质小胶质细胞及星形胶质细胞的数量均增加,NF-κB表达增强。结论蛋白酶体抑制剂Lactacystin能激活大鼠黑质小胶质细胞及星形胶质细胞,诱导炎性介质表达。     

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.     

Detection of the intercellular borders of the endothelium using scanning electron microscopy

Three different approaches for studying aorta endothelial cells by scanning electron microscopy (SEM) were compared to expose cell border lines and surface shape simultaneously. Staining of aorta probes with silver nitrate leads to more specific silver deposits at cell junctions, but unsatisfactory image resolution obscures any detail of cell junctions and other fine structural features. Endothelium impregnated with tannin-osmium showed enhanced cell junction detail, but this method resulted in formation of presumably artificial structures. Osmium tetraoxide was most optimal for impregnation of aorta probes, as it exposed well cell border lines, excluded artifacts of tannin-osmium staining, provided a good image resolution in SEM, and allowed to analyse the ultrastructural features of cell surface.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

6-OHDA制备的帕金森病大鼠黑质神经元的超微结构改变

目的观察6-羟多巴胺(6-OHDA)单侧注射制备的帕金森病(PD)大鼠多巴胺(DA)能神经元的超微结构改变。方法单侧微量注射6-OHDA制备PD大鼠模型,用免疫荧光组织化学方法观察正常侧与6-OHDA注射侧黑质酪氨酸羟化酶(TH)阳性神经细胞及神经纤维的变化;并利用免疫电镜技术观察大鼠正常侧与注射侧黑质致密部DA能神经元的超微结构。结果免疫荧光法显示注射侧黑质致密部TH阳性细胞数和网状部TH阳性纤维面积与正常侧的百分比平均值分别为21.83%,23.19%。免疫电镜显示:TH免疫反应阳性产物表达于PD大鼠正常侧DA能神经元的高尔基复合体质膜面及胞质内,电子密度较高,注射侧很少见或几乎未见,且注射侧线粒体嵴有不同程度的溶解,呈空泡样变或髓样变,粗面内质网脱颗粒。结论6-OHDA可引起DA能神经元发生凋亡的超微结构改变。     

A reliable method for demonstrating axonal degeneration shortly after axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.