Inhibitory zinc-enriched terminals in mouse spinal cord.

The ultrastructural localization of zinc transporter-3, glutamate decarboxylase and zinc ions in zinc-enriched terminals in the mouse spinal cord was studied by zinc transporter-3 and glutamate decarboxylase immunohistochemistry and zinc selenium autometallography, respectively. The distribution of zinc selenium autometallographic silver grains, and zinc transporter-3 and glutamate decarboxylase immunohistochemical puncta in both ventral and dorsal horns as seen in the light microscope corresponded to their presence in the synaptic vesicles of zinc-enriched terminals at ultrastructural levels. The densest populations of zinc-enriched terminals were seen in dorsal horn laminae I, III and IV, whereas the deeper laminae V and VI contained fewer terminals. At ultrastructural levels, zinc-enriched terminals primarily formed symmetrical synapses on perikarya and dendrites. Only relatively few asymmetrical synapses were observed on zinc-enriched terminals. In general, the biggest zinc-enriched terminals contacted neuronal somata and large dendritic elements, while medium-sized and small terminals made contacts on small dendrites. The ventral horn was primarily populated by big and medium-sized zinc-enriched terminals, whereas the dorsal horn was dominated by medium-sized and small zinc-enriched terminals. The presence of boutons with flat synaptic vesicles with zinc ions and symmetric synaptic contacts suggests the presence of inhibitory zinc-enriched terminals in the mammalian spinal cord, and this was confirmed by the finding that zinc ions and glutamate decarboxylase are co-localized in these terminals. The pattern of zinc-enriched boutons in both dorsal and ventral horns is compatible with evidence suggesting that zinc may be involved in both sensory transmission and motor control.

The alteration of gamma-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure.

It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12-24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.

Zinc-enriched boutons in rat spinal cord.

The rat spinal cord reveals a complex pattern of zinc-enriched (ZEN) boutons. As a result of in vivo exposure to selenide ions, nanosized clusters of zinc selenide are created in places where zinc ions are present, including the zinc-containing synaptic vesicles of ZEN boutons. The clusters can be silver enhanced by autometallographic (AMG) development. A description of the ZEN bouton patterns is presented and discussed. The distribution of ZEN boutons could indicate that these terminal systems have a differentiated influence on sensory and motor systems.

Adrenalectomy causes loss of zinc ions in zinc-enriched (ZEN) terminals and decreases seizure-induced neuronal death.

Chelatable zinc ions from synaptic vesicles have been suggested to be involved in neuronal death caused by stroke, epilepsy and head trauma. Elevated glucocorticoid concentration exacerbates such neuron loss, while low levels protect. We have tested the notion that the neuroprotective effect of prior glucocorticoid reduction is mediated by a reduction of zinc ions contained in zinc-enriched (ZEN) synaptic vesicles. The level of vesicular zinc ions was evaluated by toluene sulfonamide quinoline (TSQ) fluorometry and zinc autometallography (ZnS(AMG)) 10 and 30 days, respectively, after adrenalectomy. The hippocampus showed significant vesicular zinc ion depletion following adrenalectomy. After the kainate injection, adrenalectomized rats showed proconvulsive seizure behavior, i.e. shortened latency to seizure onset time and increased seizure score. Additionally they showed decreased hippocampal CA3 neuronal death as compared to control animals. The present data suggest that zinc ions released from damaged ZEN terminals are involved in seizure-induced neuronal death.

Zinc-enriched (ZEN) terminals in mouse spinal cord: immunohistochemistry and autometallography.

The general distribution of zinc-enriched (ZEN) terminals in mouse spinal cord was investigated at light microscopic level by means of zinc transporter-3 immunohistochemistry (ZnT3(IHC)) and zinc selenium autometallography (ZnSe(AMG)). Staining for ZnT3(IHC) corresponded closely to the ZnSe(AMG) staining. Both appeared as dense grains of variable sizes and densities in the gray matter with a characteristic segmental laminar pattern. The white matter was unstained but contained rows of stained terminals radiating from the gray matter. In the dorsal horn, laminae I, III and IV were heavily stained, whereas lamina II appeared as the least stained area in the gray matter. Moderate staining was seen in laminae V and VI. In the ventral horn, large ZnT3(IHC) and ZnSe(AMG) grains, known from previous papers to represent ZEN terminals, were observed related in particular to motor neuronal somata and big dendrites. These ZEN terminals in the ventral horn were in general larger than those in the dorsal horn. This is the first description of the pattern of ZEN terminals in mouse spinal cord.

Zinc-enriched (ZEN) terminals in mouse olfactory bulb.

The present study was designed to localize zinc-enriched (ZEN) terminals in mouse olfactory bulb by means of ZnT3 immunocytochemistry (ICC) and zinc autometallography (AMG). The immunocytochemical staining of ZnT3 was closely correlated with the AMG pattern. ZEN terminals were defined as terminals showing both ZnT3 immunoreactivities and AMG granules. At the light microscopic level, dense staining patterns for ZnT3 immunoreactivity were seen in the granule cell layer and the olfactory glomerular layer. At the ultrastructural level, ZEN terminals were restricted to presynaptic terminals with single or multiple postsynaptic thickenings. The postsynaptic profiles contacting ZEN terminals appeared to be dendrites or somata of granule cells in the granule cell layer and periglomerular cells and mitral/tufted (M/T) cells in the olfactory glomerular layer. This suggests that two main sources of ZEN terminals are present in mouse olfactory bulb: (1) centrifugal fibres making asymmetrical synapses with granule cells and periglomerular cells, and (2) olfactory receptor terminals contacting dendritic profiles of M/T cells or periglomerular cells. The close correlation between ZEN terminals and the glutamatergic system is discussed.

The over-expression of somatostatin in the gerbil entorhinal cortex induced by seizure.

In present study, we investigated the immunohistochemical distribution of somatostatin (SRIF) in the hippocampal complex of the Mongolian gerbil and its association with different sequelae of spontaneous seizures, in an effort to identify the roles of SRIF in the self-recovery mechanisms in these animals. In the dentate gyrus and subiculum, SRIF immunoreactive (SRIF(+)) cells were similar in both the seizure resistant and the pre-seizure group of seizure sensitive gerbils. Interestingly, SRIF immunoreactivity was markedly decreased until 12 h postictal. Twenty-four hours after the on-set of seizure, the distribution of SRIF immunoreactivity in these regions had slightly increased. In contrast, in the entorhinal cortex the population of SRIF(+) cells and their density were significantly elevated compared to pre-seizure group 30 min postictal. Twelve hours after the on-set of seizure, however, the population of SRIF(+) cells and their density declined, approximately 70-80% compared to the situation at 30 min postictal. These findings suggest that the enhancement of SRIF expression in gerbil entorhinal cortex may affect tissue excitability and have a role in modulating recurrent excitation following seizures.

The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil.

Recent studies reported changes in neuropeptide Y (NPY) expression induced by seizures in the experimental epileptic models. However, there have been few reports of the alteration of NPY expression in hippocampal complexes of genetic epilepsy models. In the present study, we performed spatial and temporal analyses of NPY expression in the hippocampal complexes of the seizure-resistant (SR) and seizure-sensitive (SS) gerbils, one of the genetic models. In SR gerbils, most NPY(+) cells were located at the dentate hilus (DH) and the subiculum (SC). In the pre-seizure group of SS gerbils, neurons in the DH and SC were nearly devoid of NPY immunoreactivity. Interestingly, the acute NPY expressions were observed in these areas of the post-seizure group at 30 min, and its immunoreactivity was declined at 12 h after the onset of seizure. These findings suggest that in seizure, the deficiency of NPY in DH and SC may be one of the factors, and that the acute expression of NPY after seizure in these areas may be the compensatory response for reduction of seizure activity in this animal.

Age-related change of neuropeptide Y-immunoreactive neurons in the rat main olfactory bulb.

The change of neuropeptide Y (NPY)-immunoreactive (IR) neurons in the rat main olfactory bulb as a result of aging was investigated at several aging stages over a two-year period; postnatal 1-24 months (P 1-P 24). From P 1 to P 12, the number of NPY-IR neurons and fibers increased with highest number in P 12, and the type of NPY-IR neurons had changed from bipolar neurons with short processes to bipolar/multipolar neurons with long processes. At P 24 the population of NPY-IR neurons and fibers had significantly decreased. Furthermore, the morphology of NPY-IR neurons showed a tendency to decrease in size and processes. It is suggested that the decrease of the number and size of NPY-IR neurons and fibers may underlie the age-related changes in the olfactory processes.

Serotonergic neurons are present and innervate blood vessels in the olfactory bulb of the laboratory shrew, Suncus murinus.

The distribution and characteristics of serotonin-immunoreactivity in the olfactory bulb of the laboratory shrew (Suncus murinus, insectivore) was studied immunohistochemically. Serotonergic neurons were found only in the subependymal layer of the main olfactory bulb. These neurons were 8-12 microm in size and bipolar in shape. These serotonergic neurons had smooth nerve fibers which innervate blood vessels located mainly in the subependymal layer of the main olfactory bulb. On the other hand, other serotonergic nerve fibers with varicosities, which must be extrinsic, were detected in most olfactory layers except the olfactory nerve layer. This result suggests that intrinsic serotonergic neurons may control blood vessels and varicose serotonergic nerve fibers may act to modulate the olfactory transmission.

Distribution and characteristics of cholecystokinin-like immunoreactivity in the olfactory bulb of the cat.

The distribution and characteristics of cholecystokinin (CCK)-like immunoreactive (LIR) nerve fibers was examined in the cat olfactory bulb using immunohistochemistry. CCK-LIR cell bodies were not found; fine varicose CCK-LIR fibers were observed in the most layers. In the main olfactory bulb, no staining was seen in the olfactory nerve layer and white matter. The directions of CCK-LIR fibers in the glomerular, external plexiform and mitral cell layers were generally perpendicular to the bulbar surface; those in the deep granule cell layer were parallel; and those in the internal plexiform and superficial granule cell layers were mixed. In the accessory olfactory bulb, CCK-LIR fibers were localized only in the granule cell layer. The presence of CCK-LIR fibers of the cat olfactory bulb may be involved in the modulation of olfactory transmission.

Elevation of the gamma-aminobutyric acid transaminase expression in the gerbil CA1 area after ischemia-reperfusion damage.

gamma-Aminobutyric acid-transaminase (GABA-T) plays an important role in the metabolism of GABA, particularly in the neurons or glial cells. The present study was undertaken to determine the alteration of GABA-T expression in the gerbil hippocampus after ischemia-reperfusion. In the sham, GABA-T(+) neurons were scattered in the hippocampus proper and dentate gyrus. The intensity of the GABA-T immunoreactivity had nearly disappeared in the interneurons at 12 h after ischemia. In contrast, 24 h post-ischemia the dramatic augmentation of GABA-T immunoreactivity in the pyramidal cells was observed in the CA1 area but not in the CA2 or CA3 areas. Forty-eight hours after ischemia-reperfusion, its immunoreactivity was preserved in the CA1 neurons. These results suggest that the over-expression of GABA-T in the CA1 area may be related to delayed neuronal death after ischemia-reperfusion insult.

ReLA/P65-serine 536 nuclear factor-kappa B phosphorylation is related to vulnerability to status epilepticus in the rat hippocampus.

Although nuclear factor-kappa B (NF-κB) is essential for neuron survival and its activation may protect neuron against oxidative-stresses or ischemia-induced neurodegeneration, NF-κB activation can contribute to inflammatory reaction and apoptotic cell death after brain injury and stroke. However, there are little data concerning the specific pattern of NF-κB phosphorylations in neuronal damage/survival induced by status epilepticus (SE). In the present study, NF-κB phosphorylation showed the cellular specific pattern in responses to SE. p52-S865, p52-Ser869, p65-Ser276, p65-Ser311, p65-Ser468, and p65-Ser529 NF-κB phosphorylation was significantly decreased in the CA1 and CA3 pyramidal cells vulnerable to SE, although neuronal specific nuclear antigen immunoreactivity was strongly detected. In contrast, p65-Ser536 NF-κB phosphorylation was enhanced in these neurons accompanied by TUNEL- and Fluoro-Jade B 244signals. These findings serve as the first comprehensive description of the cellular specific distribution of NF-κB phosphorylation in response to pilocarpine-induced SE in the rat hippocampus, and suggest that enhancement in p65-Ser536 NF-κB phosphorylation may be closely relevant to neuronal vulnerability to SE, while others may be involved in neuronal survival.

Comparative studies on the distribution of glutamate transporters in the retinae of the Mongolian gerbil and the rat.

Glutamate is the major excitatory amino acid transmitter in vertebrate retinae. Glutamate transporters therefore play an important role in the precise control of glutamate concentration in the synaptic cleft by regulating extracellular glutamate concentration. In the present study, we performed an analysis of the expressions of three glutamate transporters in gerbil retina using immunohistochemistry. In the gerbil retina, excitatory amino acid carrier 1 and glutamate transporter 1 immunoreactivity was predominant in the ganglion cells but not amacrine or bipolar cells. Glutamate/aspartate transporter (GLAST) immunoreactivity was observed in the radial gliocytes of which the dense network of fine processes was localized in the inner and outer plexiform layers. GLAST immunoreactivity was also detected in astrocytes in the nerve fibre layer. These results demonstrate that three glutamate transporters show specific distributions in the gerbil retina and suggest that the glutamate re-uptake system in the gerbil retina may be different from that of the rat.

Indoleamine-accumulating cell death and endogenous glial cell reaction induced by 5,7-dihydroxytryptamine in the cat retina.

We investigated the patterns of degenerative changes of indoleamine-accumulating cells (IACs) induced by 5,7-dihydroxytryptamine (5,7-DHT, 100 microg), and the glial reaction to the neurodegenerative changes of IACs in the cat retina by using light-and electron-microscopy. The neurons accumulating 5,7-DHT in the cat retina were a few ganglion cells and displaced amacrine cells located in the ganglion cell layer (GCL), and some amacrine cells in the inner nuclear layer (INL). The cell density (per unit area, 1 mm2) of the 5,7-DHT accumulating cells in the GCL and INL was 910 and 134 cells, respectively. Most 5,7-DHT accumulating cells showed dark degeneration characterized by widening of the cellular organelles at early stage, and by darkening of the cytoplasm at a late stage. In addition, amacrine cells, showing a typical filamentous degeneration, were observed in a few cases. The degenerated neurons were phagocytosed by microglial cells and astrocytes. The immunoreactivity for glial fibrillary acidic protein (GFAP) in Muller cells was increased at early stage, but thereafter abruptly decreased. In a few cases, severe degenerative changes were observed in Miller cells. These results indicate that 5,7-DHT induces severe dark degeneration of IACs, and most degenerated cells could be eliminated by microglial cells and astrocytes in the cat retina.

Brainstem origin of the efferent components of the cervical vagus nerve in the house musk shrew, Suncus murinus.

The brainstem origin of the efferent neurons of the vagus nerve in the house musk shrew, an animal species which has been recently used in researches on emesis, was studied using the retrograde tracing method. The vagus nerve was exposed and cut at the mid-cervical level below the nodose ganglion. Horseradish peroxidase was applied to the proximal end of the cut nerve. The brainstem was sectioned and processed histochemically with the tetramethylbenzidine method. The horseradish peroxidase injection into the vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve and ambigual nuclear complex. Labelled neurons in the dorsal motor nucleus of the vagus nerve, constituting approximately 80% of the total labelled neurons, formed a longitudinal column whose length varied from 3.4 to 3.8 mm. Half of labelled neurons in this nucleus were found at the level between the area postrema and 0.6 mm rostral to it. The ambigual nuclear complex was made up of two major longitudinal divisions; the dorsal division corresponded to the ambiguus nucleus and the ventral division was identified as the external formation of the ambiguus nucleus. Our results suggest that in the Suncus murinus the neuroanatomical feature of the dorsal motor nucleus of the vagus nerve is similar to those of other mammals, but ambigual nuclear complex must be somewhat different between mammals.

Distribution of serotonin immunoreactivity in the main olfactory bulb of the Mongolian gerbil.

The distribution of serotonin immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil (Meriones unguiculatus) was examined by immunohistochemistry. Seven distinct layers of the Mongolian gerbil MOB-stained with cresyl violet were identified. Serotonin-immunoreactive (IR) cell bodies were not found in the MOB. The serotonin-IR nerve fibres had a specific laminar distribution and morphology in the gerbil MOB. Serotonin-IR nerve fibres were observed in the glomerular, external plexiform and granule cell layers of the MOB. These serotonin-IR nerve fibres showed varicosities that were larger than the thickness of the axon. The highest density of serotonin-IR nerve fibres was in glomeruli of the glomerular layer. The average fibre density in the glomerular layer was more than three to four times the density in the infraglomerular layers. Glomerular serotonin-IR fibres were much more intensively stained than infraglomerular serotonin-IR fibres. This result suggests that serotonin-IR nerve fibres of Mongolian gerbil MOB are extrinsic and may act to modulate the olfactory transmission.