An ultrastructural study of nerve profiles in the myenteric plexus of the rabbit colon

The ultrastructure of the myenteric plexus from the rabbit colon was examined in both conventionally fixed tissue and also material fixed with the chromaffin method. Montages of the ganglia were analysed semi-quantitatively. Six main types of axon profile are described and classified on a morphological consideration of the vesicle population. Most axon types formed synapses with myenteric neurons. Two kinds of chromaffin-positive nerve fibre were seen, one containing a predominance of small granular vesicles, the other containing many flattened vesicles. The difficulties in relating axon profile types to putative transmitters are discussed.     

Morphological alterations in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol

Expiants of 14-day fetal mouse spinal cord with attached dorsal root ganglia, which had become differentiated over 2–3 weeks in culture, were exposed to 1–2 μM taxol for up to 6 days. The culture medium was supplemented with nerve growth factor (300 units/ml) during exposure to the drug.By 3–6 days in taxol, unusually numerous microtubules were seen in peripheral perikaryal and proximal neuritic regions of ganglion neurons. Microtubules also engirdled massive aggregations of pleomorphic vesicular/cisternal elements in many neurons. These aggregates were visible as unusual ‘clear’ spheroidal regions in the living cells, and were often as large as the nuclei. Some of the elements comprising these striking vesicular/cisternal accumulations appeared to be portions of disrupted Golgi complexes normally polarized around the cytocentrum, as well as hypertrophied smooth endoplasmic reticulum formations. In other neuronal areas, Golgi complexes and other organelles were altered or disrupted to lesser degrees. Ordered microtubular arrays occurred along endoplasmic reticulum cisternae both in neuron somata and neurites. Over time, a plethora of microtubules assembled throughout the perikarya in various orientations apparently unrelated to microtubule organizing centers. Unlike the effects of other plant alkaloids that interact with tubulin, there was no discernible increase in filaments, although their distribution appeared altered. Concentric ordered microtubular-macromolecular lamellated complexes were seen only in neurites. Neuronal nuclei were misshapen, often displaced, and displayed fine structure reminiscent of chromatolysis. Satellite and Schwann cells contained atypically abundant microtubules, abnormal cisternae, disrupted Golgi complexes, and increased lysosomes. Some nuclei displayed abnormal chromatin, and in rare cases even microtubules.We suggest that taxol alters the distribution, integrity, and/or organization of organelle systems in dorsal root ganglion cells by engendering unusually abundant microtubules in abnormal groupings and aberrant locations in these cells.     

Development of synaptic structure and function in organotypic cultures of the rat hippocampus

The sequence of development of synapses, as well as the ultrastructure of axonal growth cones, has been investigated electron microscopically in tissue cultures of the newborn rat hippocampus. During differentiation of the tissue cultures, the formation of synapses is preceded by identifiable growth cones. A characteristic feature of axonal growth cones is the presence of numerous large clear vesicles which vary in diameter from ~100 to 150 nm. The first immature synapses were formed on the 5th, 6th or 7th day in vitro on the growth cones of differentiating neuronal processes. Axonal growth cones are occasionally found to be presynaptic to a dendrite. At first axo-dendritic synapses, most of them being en passant, arise, whereas axo-somatic and axo-spinous-dendritic synapses of different complex structures appear later.It is suggested that the earliest signs of synaptogenesis are vesicular structures (‘growth’ vesicles and few synaptic vesicles), which occur in growth cones, axons and presynaptic boutons of immature synaptic contacts even before formation of the specialized pre- and postsynaptic membranes.     

An intracellular study of myenteric neurons in the mouse colon

Intracellular recordings have been made in vitro from the myenteric neurons of the distal colon of normal littermates of the piebald-lethal mouse. Out of a total of 90 neurons, 82 were classified as S/type 1 cells and 8 as AH/type 2 cells. Seventy-eight out of 82 S cells showed spontaneous fast excitatory postsynaptic potentials (EPSPs) sensitive to d-tubocurarine (dTC, 280 microM), and 22 S cells showed spontaneous action potentials (APs). Six S cells and 1 AH cell showed spontaneous nonnicotinic slow depolarizations associated with an increase in the input resistance of the cells; during the spontaneous slow depolarization in the S cells there was an increase in the frequency of nicotinic fast EPSPs and APs. Three S cells showed spontaneously occurring regular oscillations of the membrane potential (approximately mV in amplitude and approximately 4/min). Transmural nerve stimulation produced fast EPSPs with a wide range of latencies (3 ms to 20 s) in S cells; the fast EPSPs were blocked by dTC (280 microM) or solutions containing low Ca2+ (0.25 mM) and high Mg2+ (12 mM) but not by atropine (ATR, 14 microM). Single or repetitive transmural stimulation produced slow EPSPs in 24 S cells and 3 AH cells; these were not blocked by dTC (280 microM) nor ATR (14 microM). During the slow EPSPs there was an increase in the input resistance of the cells. In those S cells that showed slow EPSPs there were many long-latency fast EPSPs; long-latency fast EPSPs were also observed in 11 other S cells that did not show a slow EPSP following repetitive transmural nerve stimulation. Long-latency fast EPSPs may be related to the firing of other neurons during their slow EPSPs. The myenteric neurons in the mouse colon have similar properties to the myenteric neurons in the guinea pig small intestine. However, the colonic myenteric neurons show more ongoing synaptic activity and more prolonged activity after nerve stimulation than myenteric neurons in the guinea pig small intestine. This activity may be due to regional differences, species differences, or preparation differences (in this study the myenteric plexus was adherent to the underlying circular muscle layer).     

Block of synaptic vesicle exocytosis without block of ca2+-influx. An ultrastructural analysis of the paralysing action of habrobracon venom on locust motor nerve terminals

The venom of the wasp Habrobracon hebetor presynaptically blocks excitatory but not inhibitory neuromuscular transmission at locust skeletal muscle. Its mode of action on excitatory motor nerve terminals has been studied at the retractor unguis muscle of Schistocerca by means of ultrastructural stereology. Paralysed and unparalysed preparations, either resting or stimulated for 7 min at 20 Hz, were compared. Paralysis does not cause structural damage to the nerve terminals but prevents the depletion of vesicles occurring upon nerve stimulation in the controls. Prolonged paralysis leads to an increase in the number and the size of vesicles resulting in an increase of total membrane per terminal cross-section by about 33% after 2 days. Stimulation causes swelling of mitochondria both in controls and in paralysed preparations, resulting from a rise of intraterminal [Ca²⁺] as is indicated by the absence of the swelling if extracellular Ca²⁺ is replaced by Mg²⁺. In addition, stimulation leads to a reduction of vesicle size, an increase in the area of axolemma and in the number of cisternae and of profiles of the smooth endoplasmic reticulum in controls but not in paralysed preparations. However, neither in controls nor in paralysed preparations is the total amount of membrane per teminal cross-section affected by stimulation. Under paralysis, vesicles tend to stick to the presynaptic membrane.It is concluded that Habrobracon venom does not block the depolarization-dependent Ca²⁺-influx into the nerve terminal and that it is unlikely to interfere with some transmitter-related process. Rather, the venom seems to block vesicle exocytosis itself. The results lend further support to the view that in insect neuromuscular synapses exocytosis is the mechanism whereby transmitter quanta are released.     

Radiation-induced damage to lacrimal glands: an ultrastructural study in Sprague Dawley rats

Injury to lacrimal glands represents a major health problem after radiation therapy of the head and neck malignancies. Accordingly, this study aimed to investigate significant ultrastructural changes of lacrimal glands and some of their underlying mechanisms following the exposure to different fractionated doses of irradiation. In this study, 28 Sprague Dawley (SD) rats were assigned to four groups (seven rats each): Group I acted as control and received no irradiation. Groups II–IV received fractionated irradiation of 5 Gy (100 cGy/fraction daily for 5 days), 9 Gy (300 cGy/fraction daily for 3 days), and 20 Gy (one fraction), respectively. One month after the experiment, examination of lacrimal glands with transmission electron microscopy (TEM) demonstrated dose-dependent ultrastructural changes in the lacrimal acinar and intralobular ductal epithelial cells. In the acinar cells, there were swollen rough endoplasmic reticulum, irregularly shaped nuclei with chromatin condensation, mitochondrial damage, and retention of secretory granules. Intaralobular ductal epithelial cells showed loss of surface microvilli and damage to mitochondria. In addition to the potential direct effects of irradiation on lacrimal acinar and intralobular ductal epithelial cells, damage to blood vessels and nerve endings seemed to mediate some of the underlying mechanisms of these irradiation-induced ultrastructural changes. In conclusion, using TEM reveals that lacrimal gland is highly sensitive to even small doses of irradiation therapy; in addition, swelling of rough endoplasmic reticulum and aberrant nuclei are the most encountered structural changes. Damage to blood vessels and nerve endings might mediate some of the underlying mechanisms of irradiation-induced secondary injury in lacrimal glands.     

Nevirapine-induced liver lipid-SER inclusions and other ultrastructural aberrations

Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae—lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.     

The origin of the autophagosomal membrane in human atherosclerotic plaque: a preliminary ultrastructural study

Autophagy is an evolutionarily conserved process that occurs ubiquitously and functions as a primary route for the degradation of damaged organelles and proteins in response to starvation, oxidative stress, and other harmful conditions. The initial event upon autophagy induction is the formation of a membranous cistern called the phagophore or isolation membrane, a cup-shaped structure that elongates, engulfs cytoplasmic “cargo”, and fuses at its rims to give rise to the autophagosome within which cytoplasmic material is enclosed. Although thoroughly studied in diverse cell culture systems, few attempts have been made to analyze the membrane dynamics during phagophore biogenesis in tissues. With respect to the cardiovascular system, no structural information is currently available regarding the sources that may contribute to the nucleation and growth of the phagophore membrane. The results presented here demonstrate that in the cells of human atherosclerotic plaque the phagophores are in contact with the endoplasmic reticulum (ER) membranes. Initially, the phagophore appears as a membrane sac that enwraps injured organelles and dysfunctional proteins and then matures into a double-membrane, closed structure often containing portions of the ER. These structural data indicate that the membrane source that elongates the phagophore might probably come from the ER. The topographical relationship between the ER tubules and the phagophore might also favor an efficient mechanism to transfer lipids from their site of synthesis to the nascent membrane, thus promoting its elongation and, ultimately, the formation of the autophagosome.     

Adrenosensitive neurons of the myenteric (Auerbach's) plexus

Experiments on the myenteric plexus of isolated strips of the small and large intestines showed the presence of adrenosensitive cells capable of responding to application of different concentrations of adrenalin (A) and noradrenalin (NA) by a distinct increase in firing rate. The greatest effect was obtained with NA; the range of action of which was wider (from 10^–9 to 10^–5 g/ml) than that of A. Addition of phentolamine to the surrounding solution in most cases prevented the appearance of these effects. Besides spontaneously discharging cells, silent neurons with no spontaneous activity also responded to the addition of A and NA. It is suggested that catecholamines may play a direct part in the modulation of processes carried out by neurons in the ganglia of the myenteric plexus.Laboratory of Physiology of the Autonomic Nervous System, I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 259–261, March, 1977.     

犬和大鼠回肠壁丛内5-羟色胺能神经元免疫组织化学观察

目的　观察犬和大鼠回肠壁丛内 5 羟色胺能神经元。方法　应用 5 羟色胺 (5 HT)抗体的免疫组织化学改良法对正常小狗 (5只 )回肠切片标本、正常大鼠 (8只 )和 5 羟色胺酸前处理大鼠 (4只 )回肠外纵肌全层铺片标本内含 5 HT免疫反应性 (5 HT IR)神经元进行了观察研究。结果　正常大鼠回壁内神经节 (丛 )内可见少数 5 HT IR核周体 ,及肠肌丛周围和节间束中含有丰富的 5 HT IR纤维。 5 羟色胺酸 (5 HTP)处理后大鼠与正常鼠相比较 ,回肠壁丛内 5 HT IR胞体和带膨体纤维的可见数稍多 ,及免疫反应性增强。正常狗远端内 5 HT IR神经元胞体和纤维非常丰富 5 HT IR基础丛内有 1～ 4个 5 HT IR神经元胞体。结论　本研究对犬和大鼠肠内 5 HT能神经元的存在提供了直接的形态学证据 ,肠 5 HT能神经元与胃肠运动的调节功能及其可能的受体机制有关     

The cytology of dopaminergic and nondopaminergic neurons in the substantia nigra and ventral tegmental area of the rat: a light- and electron-microscopic study

The results of this study support the conclusion that dopaminergic cells can be distinguished from non-dopaminergic cells, at both the light- and electron-microscopic level, by cytological features, and particularly by the pattern of Nissl substance. In both the substantia nigra and the ventral tegmental area, two main categories of cell type can be identified in Nissl preparations: (1) dark-staining, basophilic cells with large masses of Nissl substance and (2) light-staining cells with more translucent cytoplasm. The following findings provide evidence that the basophilic cells of both substantia nigra and ventral tegmental area are the dopaminergic cells. (1) There is a good correlation between the topographic distribution of basophilic cells and that of dopaminergic cells mapped by both histofluorescence and immunohistochemical methods. (2) After unilateral destruction of the dopaminergic neurons by intracerebral injection of 6-hydroxydopamine in the dopaminergic pathway, the basophilic cells in the substantia nigra and ventral tegmental area disappeared on the lesion side, while the lighter-staining cells appeared unaffected. (3) In normal rats, and in rats with unilateral 6-hydroxydopamine lesions, intraventricular injection of [3H]norepinephrine was used for specific labeling of dopaminergic neurons. In autoradiograms of semithin sections, such labeling was observed only in dark-staining and not in light-staining cells, and in cases of unilateral 6-hydroxydopamine lesion was totally absent on the lesion side. Electron-microscopy showed much of the cytoplasm of the basophilic dopaminergic cells to be densely filled with free ribosomes associated with large, well organized complexes of rough endoplasmic reticulum. The cytoplasm of the light, non-dopaminergic cells contains only sparse free ribosomes and small, widely spaced aggregates of rough endoplasmic reticulum. Both cell types occur in a similar variety of size and shape.     

The role of phospholamban and SERCA3 in regulation of smooth muscle-endothelial cell signalling mechanisms: evidence from gene-ablated mice

It is generally agreed that intracellular Ca²⁺ stores, the sarco(endo)plasmic-reticulum (SER), affect Ca²⁺ homeostasis and thus contractility of vascular smooth muscle. There is, however, no general consensus as to the magnitude of the SER contribution to Ca²⁺ handling, the basis for isoforms of the SER Ca²⁺-ATPases (SERCAs) or the role of an SER-associated regulatory protein, phospholamban (PLB). Although the biochemical and cell biological roles of the SER have been intensely studied in vitro, the development of gene-targeted and transgenic mouse models enables one to extend our information to the in vivo levels. A brief review of the role of PLB and SERCA function in vascular and endothelial cell function is presented. Studies on the PLB gene-ablated mouse indicate that vascular contractility is considerably altered. This is mirrored by changes in intracellular Ca²⁺. Moreover, differences in contractility of the gene-ablated tissues are eliminated by treatment with cyclopiazonic acid, which pharmacologically abolishes SER function by inhibiting the Ca²⁺-ATPase. Thus PLB modulation of sarcoplasmic reticulum (SR) Ca²⁺ uptake plays a major role in modulating vascular contractility. It is interesting that endothelium-dependent relaxation was decreased in the PLB-deficient aorta. This is surprising in light of the PLB distribution, thought to be limited to cardiac, slow skeletal and smooth muscle. Our data indicate the presence of PLB in endothelial cells and point to an unrecognized pathway for modulation of endothelial cell [Ca²⁺]_i and vascular contractility. Data from smooth muscle tissues of the SERCA3 gene-ablated mouse demonstrate that this isoform affects endothelium-dependent function, but not that of smooth muscle, consistent with its known distribution. This isoform appears to perform a modulatory function, rather than the more essential role of SERCA2. Gene-targeted and transgenic models provide an important avenue for understanding the role of SER in vascular signalling.     

An electrophysiological study of the projections of putative sensory neurons within the myenteric plexus of the guinea pig ileum

Conduction of action potentials in the processes of AH (afterhyperpolarizing) neurons has been examined in the myenteric plexus of the guinea-pig small intestine. AH neurons are a morphologically distinct class of myenteric neurons in which the action potentials are followed by long lasting afterhyperpolarizations and which usually lack fast synaptic inputs. These neurons have large smooth cell bodies and several long processes. We have used electrophysiological methods, combined with intracellular injection of the fluorescent dye 5(6)-carboxyfluorescein, to examine the directions of projection and lengths of axons of AH neurons. AH neurons of the myenteric plexus projected circumferentially in both directions from the cell soma for electrophysiologically determined average distances of 0.74 ± 0.05 mm. Thus, the neurons span about 1.5 mm of the circumference of the intestine. About one quarter of the AH neurons had one, or rarely two, processes that ran anally after initially projecting circumferentially. All processes conducted action potentials, with average conduction velocities of 0.23 ± 0.02 ms⁻¹.     

STCH对多巴胺能神经元保护作用的初步研究

本文通过不同手段探讨了STCH(stress and chaperone)对多巴胺能神经元的作用。采用RT-PCR检测STCH的组织表达模式;采用免疫-激光捕获显微切割技术获得单一多巴胺能神经元,采用RT-PCR检测STCH在中脑不同亚区多巴胺能神经元的表达差异;分别构建STCH过表达pEGFP-N2载体和pSuper-EGFP干扰载体,转染HEK293和SH-SY5Y细胞株,检测细胞对MPP+毒性的反应。结果显示:STCH在海马表达最低,肝脏最高;在中央灰质区的多巴胺能神经元可检测到表达,在黑质区检测不到;将STCH干扰片段转染至HEK293细胞,细胞死亡明显;转染至SH-SY5Y细胞,对细胞生长无明显影响,但细胞形态发生改变;与对照组相比过表达STCH的SH-SY5Y细胞对MPP+毒性有抵抗作用,并能抑制MPP+处理细胞中caspase-12的表达。这些结果提示:STCH对多巴胺能神经元具有潜在的保护作用,其机制可能是通过抑制内质网应激诱导的凋亡途径而实现。该结果为寻找Parkinson病的治疗靶向提供了有益的线索。     

Structural changes in nerve endings of rat median eminence superfused with media rich in potassium ions

In vitro fragments of male rat mediobasal hypothalami were superfused with Krebs--Ringer solution in the presence or absence of CaCl2. Infusions containing up to 60 mM potassium chloride were applied, at the end of which tissues were fixed in osmium tetroxide and prepared for transmission electron microscopy. Control superfusions were run in parallel. Quantitative measurements performed on electron micrographs of the outermost palisade region showed significant (20-30%) increase in caliber of axon endings after intensive potassium ion stimulation. Ultrastructurally, widespread depletion of granular vesicles and microvesicles was found. Vesicle shift to the outer zone of the terminals, formation of membrane-bound tubules of the same diameter as microvesicles, and images of attachment and collapse of vesicles into the axolemma were found, particularly after 1 min stimulation. These findings were interpreted as consistent with exocytosis. Longer stimulations were followed by the appearance of large pleomorphic vacuoles that are probably the result of post-exocytotic membrane retrieval. Axon enlargement and vesicle depletion were absent in specimens superfused with calcium-free medium containing high potassium. The functional significance of these ultrastructural changes are interpreted as supporting the hypothesis that exocytosis of calcium-loaded microvesicles can contribute to extrude this ion from median eminence nerve endings during secretion.     

The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala

Vasoactive intestinal polypeptide (VIP) neurons within the central nucleus of the rat amygdala were examined using light and electron microscopic immunocytochemical techniques. Vasoactive intestinal polypeptide-immunoreactive neurons were located in the ventral part and less frequently in the central part of the central nucleus. Vasoactive intestinal polypeptide positive terminals were distributed throughout the medial part of a cytoarchitectonically distinct central zone of the central nucleus. Three types of terminals formed synaptic contacts on VIP-immunoreactive neurons: type A containing round vesicles, type B containing many pleomorphic vesicles and type C containing fewer pleomorphic vesicles. All VIP-immunoreactive boutons observed were of type A variety, and made asymmetrical and symmetrical synaptic contacts on both VIP-immunoreactive and nonreactive neurons within the central nucleus.     

Morphological studies of electrophysiologically-identified myenteric plexus neurons of the guinea-pig ileum

The morphology of neurons in the myenteric plexus of the guinea-pig ileum has been studied by means of the intracellular application of Procion Yellow. Sixty-six electrophysiologically-unidentified cells showed a great variety of soma shapes and number of processes, the vast majority of the longer of which were circumferentially-orientated.The electrophysiological properties of an additional 47 neurons were ascertained; 29 were neurons with a slow after-hyperpolarization (AH-neurons), 14 showed fast excitatory synaptic potentials (S-neurons) and 4 were of neither category.Twenty-two of the AH-neurons had a smooth soma outline and, on average, each had 5 processes, of which the great majority of long processes were circumferentially-orientated and intraganglionic. The projection ratios of oral:circumferential:aboral processes were 6:61:9. Branching was a prominent feature of the processes.In contrast, a large soma with many broad, short processes was a feature of 8 of 14 S-neurons studied. The average number of processes was 8.6 per cell and relatively more of them were aborally-directed, giving projection ratios of 2:21:7. There was, however, such a variation and overlap in the morphology of AH- and S-neurons that it was not possible to achieve a simple, reliable classification.It is concluded that many neuronal processes may be intraganglionic and that longitudinal ones are mainly aboral. From the varied morphological characteristics of AH-neurons, it is unlikely that these neurons subserve a single function in the plexus. For the same reasons S-neurons may fulfil different physiological roles.