Neuronal alterations in hippocampus following severe hypoglycaemia: a light microscopic and ultrastructural study in the rat

Because they induce similar neuropathological changes (ischaemic cell change with microvacuolization), it has been suggested that ischaemia, status epilepticus and hypoglycaemia produce cell death by similar mechanisms, especially those resulting from intracellular calcium accumulation. We have recently demonstrated microvacuolation of neurons, mitochondrial swelling (the electron microscopic correlate of microvacuolization) and massive mitochondrial calcium sequestration (using the pyroantimonate technique) following ischaemia or status epilepticus. We therefore studied the selectively vulnerable neurons of rat hippocampus by light and electron microscopy (including the pyroantimonate technique) following 30 and 60 min of EEG isoelectricity resulting from insulin hypoglycaemia. The neuropathology at the light and EM level is unique and different from that following status epilepticus or ischaemia. The most constant finding is dark cell change of the granule cells at the tip of the dentate gyrus. In contrast to status epilepticus and ischaemia, hippocampal pyramidal neurons are far less frequently involved. Microvacuoles are rarely seen and, when present, their ultrastructural correlate is swollen Golgi apparatus, not dilated mitochondria. No intracellular calcium accumulation is demonstrable with pyroantimonate technique. Thus the cellular alterations produced by hypoglycaemia differ in character and distribution from those produced by anoxia-ischaemia. Mitochondrial calcium accumulation is not prominent in cell death from hypoglycaemia. Whether calcium toxicity plays another, subtler role in hypoglycaemic brain injury is unknown.     

Early ultrastructural changes after brief histotoxic hypoxia in cultured cortical and hippocampal CA1 neurons

Primary cortical and hippocampal neuronal cultures submitted to brief histotoxic hypoxia suffer delayed neuronal death after 24 h [Uto et al. (1995) J Neurochem 64: 2185–2192]. In this study the ultrastructural changes were monitored during the first 6 h following 5-min histotoxic hypoxia induced by exposure to 100 μM iodoacetate. In both cortical and hippocampal CA1 neurons, disaggregation of ribosomes was the earliest sign of histotoxic pathology. Vacuolizations of mitochondria, endoplasmic reticulum and Golgi apparatus, as well as fragmentation and disintegration of neurofilaments followed later. Signs of apoptotic nuclear degeneration were absent. Our observations demonstrate that, similar to that seen in ischemia, disaggregation of ribosomes after brief histotoxic hypoxia is one of the first pathological alterations heralding delayed neuronal death. Received: 14 June 1996 / Revised, accepted: 31 July 1996     

Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats

Recent data imply that mitochondrial regulation of calcium is critical in the process leading to hypoxic-ischemic brain injury. The aim was to study the subcellular distribution of calcium in correlation with ultrastructural changes after hypoxia-ischemia in neonatal rats. Seven-day-old rats were subjected to permanent unilateral carotid artery ligation and exposure to hypoxia (7.7% oxygen in nitrogen) for 90 min. Animals were perfusion-fixed after 30 min, 3 h or 24 h of reperfusion. Sections were sampled for light microscopy and electron microscopy combined with the oxalate-pyroantimonate technique. At 30 min and 3 h of reflow, a progressive accumulation of calcium was detected in the endoplasmic reticulum, cytoplasm, nucleus and, most markedly, in the mitochondrial matrix of neurons in the gray matter in the core area of injury. Some mitochondria developed a considerable degree of swelling reaching a diameter of several microm at 3 h of reflow whereas the majority of mitochondria appeared moderately affected. Chromatin condensation was observed in nuclei of many cells with severely swollen mitochondria with calcium deposits. A whole spectrum of morphological features ranging from necrosis to apoptosis was seen in degenerating cells. After 24 h, there was extensive injury in the cerebral cortex as judged by breaks of mitochondrial and plasma membranes, and a general decrease of cellular electron density. In the white matter of the core area of injury, the axonal elements exhibited varicosity-like swellings filled with calcium-pyroantimonate deposits. Furthermore, the thin myelin sheaths were loaded with calcium. Numerous oligodendroglia-like cells displayed apoptotic morphology with shrunken cytoplasm and chromatin condensation, whereas astroglial necrosis was not seen. In conclusion, markedly swollen 'giant' mitochondria with large amounts of calcium were found at 3 h of reperfusion often in neuronal cells with condensation of the nuclear chromatin. The results are discussed in relation to mitochondrial permeability transition and activation of apoptotic processes.     

Nitric oxide synthase does not mediate neurotoxicity after an i.c.v. injection of streptozotocin in the rat

Summary. In the present study we evaluated the possible role of nitric oxide (NO) in mediating neuronal damage in middle-aged rats after an i.c.v. injection of streptozotocin (STREP). An i.c.v. injection of STREP has been reported to decrease the central metabolism of glucose. This inhibition of the energy metabolism after STREP treatment might induce an excitotoxic mechanism, which may lead to the stimulation of NO synthase and, consequently to the synthesis of NO. On the other hand, STREP might induce oxidative stress directly by liberation of NO from its nitroso moiety. To investigate whether NO synthase is involved in a possible excitotoxic mechanism after STREP treatment, some of the rats treated with STREP (1.25 mg/kg in 4 μl, bilaterally 2 μl/injection site) were also treated with the NO synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME, 20 mg/kg i.p. 10 min, 6, 24 and 96 h after STREP injection). To investigate whether NO liberated from STREP may be responsible for neurotoxic effects, one additional group of control rats received an i.c.v. injection of the NO donor sodium nitroprusside (SNP, 10 μg in 4 μl). We found that STREP affected the behavioral performances in the open field and two-way active avoidance task. In addition, immunostaining for glial fibrillary acidic protein, an indicator of reactive astroglial changes to neuronal damage, showed that this was mainly located in peri- and paraventricular regions of the third and lateral ventricles, like for instance in the septum, caudate putamen and hippocampus. l-NAME treatment had no protective effect on the behavioral impairments and neuronal damage of STREP-treated rats. This suggests that the neuronal damage of STREP may still be a result of the decrease in the central energy metabolism, but without the involvement of NO synthase. This was supported by measuring, using immunostaining, the NO-mediated cyclic GMP production by the enzyme soluble guanylyl cyclase in cortical slices, i.e. l-NAME did not prevent NO production after STREP administration in vitro. In addition, it was found that SNP liberated NO in vitro, whereas in vivo SNP administration did not lead to any behavioral and neuronal deficits at all. However, the present study cannot exclude the involvement of NO liberated from STREP in neuronal damage. Received June 7, 1999; accepted September 30, 1999     

Ultrastructural consequences of radical damage before and after differentiation of neuroblastoma B-104 cells

There is abundant evidence that the pathophysiology leading to neuronal death during post-ischemic brain reperfusion involves radical-mediated damage. Although the ultrastructural alterations accompanying brain ischemia and reperfusion are well characterized, little is known about the ultrastructural alterations that are specific to radical damage. This study examines in differentiated and undifferentiated neuroblastoma B-104 cells the viability (by dye exclusion) and ultrastructural consequences of radical damage initiated by 50 μM cumene hydroperoxide (CumOOH). Differentiation was most notably associated with formation of neurites and an extensive cytoskeletal feltwork. CumOOH-induced cell death was increased after differentiation and was blocked by the iron chelator DETAPAC. The ultrastructural characteristics of radical damage here included: (1) plasmalemmal holes that appear to undergo “patching” by well-organized membrane whorls, (2) accumulation of numerous free ribosomes, (3) markedly increased vesicular trafficking about the Golgi accompanied by Golgi transformation from cisternal organization to clusters of vacuoles with numerous fusing vesicles, (4) development of large multi-layered vacuoles that include damage membranes and organelles and appear to undergo extrusion from the cell, and (5) a general loss of cytoplasmic volume. These ultrastructural alterations developed more rapidly and were consistently more advanced in differentiated cells throughout the 6-h time course. In differentiated cells radical damage also induced the disorganization and subsequent loss of the extensive feltwork of cytoskeletal elements. There was little damage to the membranes of the nuclear envelope and mitochondria. Our observations in this system are strikingly similar to ultrastructural alterations in Golgi and ribosomal organization seen in vulnerable neurons during post-ischemic brain reperfusion and suggest that these alterations during reperfusion reflect the consequence of radical-mediated damage. Received: 11 December 1995 / Revised, accepted: 26 February 1996     

GABA influences the ultrastructure composition of cerebellar granule cells during development in culture

The influence of GABA on cerebellar granule cells in culture was followed morphometrically during in vitro development by growing the cells in the absence or presence of 50 μM GABA. The presence of GABA in the culture media increased the number of neurite-extending cells by 50% after 7 days in culture. At the ultrastructural level GABA treatment led to an increased density of neurotubules, rough endoplasmic reticulum, Golgi apparatus, coated vesicles and other vesicles, whereas structures such as mitochondria and smooth endoplasmic reticulum were not affected by GABA. The density of free ribosomes showed a more pronounced tendency to decrease as a function of the culture period (1–7 days) when the cells were grown in the presence of GABA as compared to control cultures. The results strongly indicate that GABA in addition to being an important neurotransmitter serves as a trophic factor in the development of at least certain types of neurons.     

Experimental gliopathy in the adult rat CNS: Effect on the blood-spinal cord barrier

The expression of certain blood-brain barrier (BBB) properties in CNS endothelial cells appear to be dependent on astroglial interactions in vitro. However, evidence for direct astroglial support of BBB function in vivo is controversial. To determine if perivascular astroglial damage or loss would compromise BBB function in situ, localized astroglial degeneration was produced in adult rat spinal cords by systemic injections of the anti-metabolite 6-aminonicotinamide (6-AN). Between 1 and 5 days after 6-AN administration microvessels in the lumbar spinal cord (blood-spinal cord barrier) were examined for the expression of several BBB markers and for leakage of endogenous and exogenous proteins by means of immunocytochemical and histochemical procedures. Glial cells throughout the gray matter were swollen after 24 h, and by 5 days post-injection perivascular astroglia in laminae VI-VIII appeared completely degenerated. Microvessels were undamaged and continued to express BBB markers such as GLUT-I, -γ-glutamyltranspeptidase, andendothelial barrier antigen in this region in a manner comparable to control animals. These results suggest that differentiated, BBB-competent microvascular endothelia in situ may not depend on continuous astroglial support to maintain these particular BBB characteristics. However, the BBB to protein appeared to be compromised; the gray matter was immunoreactive for serum albumin and some areas were permeable to intravascularly injected horseradish peroxidase (HRP). No increase in microvascular transport vesicles was apparent, and no open, tracercontaining interendothelial junctions were detected using standard ultrastructural methods. Some venous structures were surrounded by hemorrhages and HRP reaction product. Thus, astrocytic injury may alter venous, and possibly microvascular, permeability to macromolecules. © 1994 Wiley-Liss, Inc.     

Intraendothelial accumulation of calcium in the hippocampus and thalamus of rats after systemic kainic acid administration

Summary The accumulation of calcium in the hippocampal and thalamic vascular endothelium and the perivascular space was detected histochemically by means of the pyroantimonate technique 30, 60 and 120 min after systemic kainic acid administration. An increased number of calcium pyroantimonate deposits was found in the endothelial mitochondria 60 min after kainate injection. The mitochondria were swollen at this time and vacuoles containing deposits were observed. After 120 min a pronounced perivascular glial swelling was conspicuous, besides the numerouos endothelial mitochondrial deposits. The swollen glial processes contained a large number of pyroantimonate deposits. It seems likely that the transendothelial calcium transport processes are accompanied by intraendothelial calcium accumulation and mitochondrial calcium sequestration.     

Ultrastructural study of enteric ganglia in three patients with Rett syndrome

In order to verify whether a pseudo-obstruction syndrome was associated with morphological changes in enteric ganglia, we performed an ultrastructural study on rectal biopsy specimens in three patients with Rett syndrome. Features of enteric neurons, detected to a different extent in all three biopsy specimens, included an abnormal dilatation of endoplasmic reticulum with a disorganization of cisternae of the Golgi apparatus, and masses of unidentified electron-dense granulo-filamentous material, probably of lipidic origin, observed in the perikaryon. Large electron-lucent membrane-bound vacuoles were found mostly within satellite glial cells. Sometimes, the axon terminals were swollen and showed intraxonal vacuolization. We conclude that the reported findings do not represent a specific sign of degeneration and do not constitute a significant morphological marker of disease.     

Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation

Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca²⁺]_i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca²⁺]_i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca²⁺]_i, which returned to near basal levels on restoration of O₂-containing medium. Prolonged hypoxic conditions (>60 min) caused irreversible elevation of [Ca²⁺]_i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake (“point of no return”). Pretreatment withN ^G-nitro-l-arginine methyl ester (l-NAME, 100 μM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca²⁺]_i. The hypoxia-induced elevation of [Ca²⁺]_i was also greatly attenuated ifl-NAME (100 μM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca²⁺]_i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the “point of no return”, as characterized above, greatly potentiated [Ca²⁺]_i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca²⁺]_i and subsequent cell death, was not blocked by NOS inhibitor,l-NAME.     

Ultrastructural changes in substantia nigra and striatum observed on a mouse model of Parkinson's disease induced by MPTP administration

The study was carried out on a mouse model of Parkinson's disease induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxin which damages dopaminergic neurons in substantia nigra. Occurrence of dark degenerated neurons was the most prominent ultrastructural change. They were characterized by the progressive condensation of cytoplasm and nuclear chromatin as well as by the light mitochondria and dilated cisternae of Golgi apparatus. Dark degenerated neurons were found particularly often on the 7th day after toxication, however on the last day of the observation, only a few neurons showed the features of dark degeneration. It is likely that degenerative changes led to death in the part of neurons only.     

Endocrine cells do not exist in rat brunner''s glands. An electron microscopic study

It was revealed by light microscopic experiments that in Brunner's glands and the epithelium of the human and mammalian duodenum, cells were present, with basally lying granulations. These granules could be identified at the ultrastructural level as accumulations of pleomorphe electron-dense granules ('argentaffin', 'chromaffin' and 'osmiophilic' granules). Depending on the light and electron microscopic staining techniques used, these cells were classified as 'mucous', 'serous', or 'endocrine' ('enteroendocrine', 'paracrine' and 'enterochromaffin') cells. In the present study, the following ultrastructural morphological criteria of Brunner's gland cells were correlated: size, shape and electron density of their nuclei and nucleoli with morphological changes of their cytoplasmic and intranuclear organelles, which serve the synthesis of mucopeptides. This proved that the synthesis of mucopeptides occurred in 3 phases. The first phase served the intranuclear synthesis of organelles, which latterly were used for the cytoplasmic mucopeptide synthesis. This phase was characterized by the appearance of an electron-lucent and euchromatic nucleus, which also contained an euchromatic and electron-lucent nucleolus, and a thin electron-dense nucleus membrane (euchromatic phase). Only a few freely lying ribosomes, membrane-bound ribosomes (rough endoplasmic reticulum, RER) and mitochondria were dispersed throughout the cytoplasm. Therefore, the cytoplasm appeared electron-lucent and nearly empty. The second phase was characterized by a round but heterochromatical nucleus containing also a heterochromatic nucleolus, and the appearance of receptosome- (endosome, endocytotic vesicle) and lysosome-like structures near to the cytoplasmic membrane of the basal cell portion, as well as in the basal and lateral perinuclear region (early heterochromatic phase). The receptosome-like structures were round-oval and contained a single electron-lucent inclusion vesicle, which had an empty space or an electron-opaque granule. Both inclusion structures were located eccentrically on the inner surface of the membrane of the receptosome-like structure. Here and there, it was possible to visualize great receptosome-like structures (multivesicular bodies) containing numerous membranous electron-lucent and electron-opaque inclusion vesicles. Electron-dense pleomorphe lysosome-like structures, bearing 1-4 electron-lucent inclusion vesicles with faint grains in their spaces, were also recognized in the basal cell portion, as well as in the basal and lateral perinuclear region.(ABSTRACT TRUNCATED AT 400 WORDS)     

The fragmented neuronal Golgi apparatus in amyotrophic lateral sclerosis includes the trans-Golgi-network: functional implications

The Golgi apparatus (GA) of spinal cord motor neurons is fragmented in sporadic amyotrophic lateral sclerosis (ALS), and in asymptomatic and symptomatic transgenic mice expressing the G93A mutation of the gene of the human Cu,Zn superoxide dismutase, found in certain cases of familial ALS (FALS) [Gonatas NK (1994) Am J Pathol 145 : 751–761; Mourelatos Z, et al. (1996) Proc Natl Acad Sci USA 93 : 5472–5477]. A similar fragmentation of the GA has been described in cells treated with microtubule-depolymerizing drugs, where the organelle is functional and contains both Golgi stacks and trans-Golgi network (TGN), the compartment of exit and targeting of proteins processed by the GA. To gain a better definition of the structure of the fragmented neuronal GA in ALS, four cases of sporadic ALS with numerous Bunina bodies in spinal cord motor neurons were stained with antibodies against human TGN and against the lumenal and cytoplasmic domains of MG160, a protein of the medial cisternae of the GA. The fragmented GA was stained with the three antibodies, indicating the presence of both Golgi stacks and TGN. Furthermore, the staining of the fragmented GA by the antiserum against the cytoplasmic domain of MG160 indicates that the fragmentation of the GA is not the result of a terminal and global cytoplasmic lytic event. The Bunina bodies were not stained by the anti-MG160 antibodies, suggesting that they are not derived from the GA. The perikarya of neurons with fragmented GA showed normal immunoreactivity with antibodies against the heavy neurofilament subunit and α-tubulin. However, because of the lack of appropriate antibodies the localization of proteins such as spectrin, ankyrin, centractin and others which link the microtubules with the GA were not done. The findings support the hypothesis that, in ALS, the fragmented neuronal GA is functional. Additional work with animal models of ALS may establish whether the fragmentation of the GA is a sign of early degeneration or a compensatory reaction of the injured motor neuron. Received: 7 October 1997 / Accepted: 5 November 1997     

Kynurenines in chronic neurodegenerative disorders: future therapeutic strategies

Parkinson’s, Alzheimer’s and Huntington’s diseases are chronic neurodegenerative disorders of a progressive nature which lead to a considerable deterioration of the quality of life. Their pathomechanisms display some common features, including an imbalance of the tryptophan metabolism. Alterations in the concentrations of neuroactive kynurenines can be accompanied by devastating excitotoxic injuries and metabolic disturbances. From therapeutic considerations, possibilities that come into account include increasing the neuroprotective effect of kynurenic acid, or decreasing the levels of neurotoxic 3-hydroxy-l-kynurenine and quinolinic acid. The experimental data indicate that neuroprotection can be achieved by both alternatives, suggesting opportunities for further drug development in this field.     

Ultrastructure of Opalski cells cultured in vitro

Summary The object of the present study was to analyse the ultrastructure of Opalski cells, obtained in tissue culture according to the method described by Mossakowskiet al. (1970). The electron microscopic picture of the Opalski cells was characterized by scanty endoplasmic reticulum and Golgi apparatus and a greatly reduced number of mitochondria, as compared with glial cells cultured in vitro. Their cytoplasm contained two types of spherical bodies, one of which corresponded to lysosome-like bodies; the second one, in the authors' opinion, represented an accumulation of mucopolysaccharide substances.The ultrastructural picture of Opalski cells corresponded well with their previously described histochemical properties.Work was partially supported by P. L. 480 grant, Public Health Service USA. Agreement 227704.     

Organelle changes in cat thoracic alpha- and gamma-motoneurons following axotomy

Horseradish peroxidase was used as a retrograde marker to identify the cell bodies of cat thoracic alpha- and gamma-motoneurons 8 days following axotomy. A quantitative ultrastructural comparison revealed changes in Nissl bodies, the Golgi apparatus, mitochondria and lysosomes for axotomised alpha-motoneurons, while axotomised gamma-motoneurons only showed changes in Nissl bodies and nuclear pores.     

Ultrastructural changes in the hypothalamic secretory nuclei of rats depending on the duration of clinical death.

The studies were carried out on neurosecretory nuclei of rat hypothalamus following complete circulatory arrest for 5 min (group I) and 10 min (group II). The surviving time of the animals after the experiment was 6 weeks. In group I, the ultrastructural appearance of the perikaryonic areas of the neurons indicates increased metabolic activity of these cells. Crinophagocytic bodies were noted near the Golgi apparatus. In the processes of neurons changes in the structure of the cytoskeletal elements were observed. In group II significant differences were noted, as compared with group I. They consisted in the desolation of the rough endoplasmic reticulum membranes from ribosomes, dilatation of the Golgi area cisternae, and swelling of mitochondria. In the perivascular region cells with the surface of the cytoplasmic processes membranes covered by the product of the Alcian blue reaction were noticed. In our opinion these cells may represent "cerebral macrophages". The ultrastructural changes were more pronounced in animals of group II, as compared with animals of group I.     

Extraordinary synapses of the unipolar brush cell: An electron microscopic study in the rat cerebellum

A neglected type of neuron, termed the unipolar brush cell, was recently characterized in the granular layer of the mammalian cerebellar cortex with several procedures, including light and electron microscopic immunocytochemistry utilizing antibodies to calretinin and neurofilament proteins. Although certain features of the unipolar brush cells were highlighted in these studies, the internal fine structure was partially obfuscated by immunoreaction product. In this study, rat cerebella were prepared for electron microscopy after perfusion fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where unipolar brush cells are known to be enriched, were studied by light microscopy in semithin (0.5–1 μ) sections and by electron microscopy in ultrathin sections. Unipolar brush cells were easily identified in semithin sections immunostained with antibodies to GABA and/or glycine, and cbunterstained with toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and glycine negative, while Golgi cells are darker and appear positive for GABA and, for the most part, also for glycine. Sets of identification criteria to differentiate unipolar brush cells from granule and Golgi cells in standard electron micrographs are presented. The unipolar brush cells possess many distinctive features that make them easily distinguishable from other cerebellar neurons and form unusually conspicuous and elaborate synapses with mossy rosettes. The unipolar brush cell has a deeply indented nucleus containing little condensed chromatin. The Golgi apparatus is large and the cytoplasm is rich in neurofilaments, microtubules, mitochondria, and large dense core vesicles, but contains few cisterns of granular endoplasmic reticulum. In addition, unipolar brush cells contain an unusual inclusion, which invariably lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell body usually emits a thin axon and is provided with a single, large dendritic trunk that terminates with a paintbrush-like bunch of branchlets. Numerous nonsynaptic appendages emanate from the cell body, the dendritic stem, and the branchlets. The appendages contain rare organelles and lack neurofilaments. The branchlets contain numerous mitochondria, neurofilaments, large dense core vesicles, and clusters of clear, small, and round synaptic vesicles. They form extensive asymmetric synaptic junctions with of a or two mossy fibers, which indicates minimal convergence of excitatory inputs. Under the postsynaptic densities, the branchlet cytoplasm displays a microfilamentous web. Besides their contact with mossy rosettes, the branchlets form symmetric and asymmetric synaptic junctions with presumed Golgi cell boutons that contain pleomorphic synaptic vesicles, indicating that the unipolar brush cells receive an inhibitory modulation. Some of these junctions are unusually extensive. The branchlets also form asymmetric synapses with granule cell dendrites, in which they represent the presynaptic elements, a feature never described before in the normal cerebellum. A minority of the unipolar brush cells receive mossy fiber contacts directly on the cell body, or on short dendritic branchlets emanating directly from the cell body. Such “enmarron” synapses were previously attributed to Golgi cells. Thus, the unipolar brush cells have complex synaptic features: Besides being specialized to form a powerful link with mossy rosettes, they may also have a paracrine function, and they participate with presynaptic dendrites in the cerebellar microcircuit. © 1994 Wiley-Liss, Inc.     

Cytochemical calcium localization in hypothalamo-neurohypophysial system of rats after ischemia. Preliminary observations

Using the oxalate-pyroantimonate technique ultrastructural localization of Ca2+ was determined in the nucleus supraopticus, the nucleus paraventricularis and in the neurohypophysis after complete cerebral ischemia. We observed an increased calcium accumulation in mitochondria of some neurons. Calcium precipitate was also found in vesicles, in cytoplasm of neurons and their swollen dendritic processes. Abundant Ca2+ precipitate occurred in synaptic vesicles and within synaptic cleft. It was present between disjuncted lamellae of the myelin sheath in profiles of myelinated axons present in neuropil. Diffuse precipitate was visible in the cytoplasm of pituicytes, as well as in microvesicles in the neurohypophysis. The possible role of calcium in ischemia is briefly discussed.     

The organisation of the axonal reticulum at a ligation, in in vitro incubated bovine splenic nerves

From previous studies we concluded that in noradrenergic neurons the axonal reticulum can be considered to be an extension of the Golgi apparatus, directly involved in the condensation and packaging of neurosecretion. But the precise ultrastructure of the organisation of the axonal reticulum in relation to neurosecretory granule formation remained to be elucidaded. This conversion was studied in ligated bovine splenic nerve incubated in vitro for three hours. The ultrastructure of the material accumulating proximally and distally was examined and its nature was determined by phosphotungstic acid staining and immunocytochemistry on glycolmethacrylate sections. Proximal to the ligation predominantly electron-lucent vesicles and tubules were found. Tubules of intermediate electron density appeared in between. The latter, especially in thicker sections, were seen to form complexes with tubules and granules of high electron density. All those elements were shown to be positive for dopamine-β-hydroxylase and cytochrome b561. In the distal part multivesicular bodies accumulated and they were also positive for both enzymes. From these findings it is concluded that the different types of structures accumulating proximally belong to a neurosecretory axonal reticulum. At a block the axonal reticulum is triggered to generate a reticular differentiation, in which granular densities of different size are found. This configuration compares well with that in nerve terminals and strongly suggests that granule formation is basically a local process.