Gaseous intermediates in the brain of the salmon Oncorhynchus masou

Distribution of nitroxidergic and H2S-producing neurons in the brain of the salmon Oncorhynchus masou was studied by methods of histochemical markering of NADPH-diaphorase and by immunohistochemical markering of the neuronal nitric oxide synthase and cystathionin beta-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H2S-producing systems, represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the brainstem part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensor (V, VII, and IX-X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the salmon medulla was revealed in neurons of the X nerve nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergical and H2S-producing neurons in the salmon medulla nuclei indicates that NO in salmon is the predominant neuromodulator of medulla viscerosensory systems, while H2S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NI in the descending motor systems. The results of the performed study allow suggesting that NO in the salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.     

Response of NADPH-Diaphorase-Exhibiting Neurons in the Medullar Reticular Formation to High Spinal Cord Injury

1. The effect of hemisection of the cervical spinal cord on NADPH-diaphorase staining in the reticular nuclei of the rabbit medulla was investigated using histochemical technique.2. A quantitative assessment of somal and neuropil NADPH-diaphorase staining was made by an image analyzer in a selected area of each reticular nucleus of the rabbit medulla.3. On the 7th postsurgery day, the highest up-regulation of somatic NADPH-diapho- rase staining was observed in regions regulating cardiorespiratory processes; however, the highest increase of neuropil NADPH-diaphorase staining was found in the reticular nuclei modulating the tonus of postural muscles.4. The degeneration of non-NADPH-diaphorase-stained neurons was detected throughout the reticular formation of the medulla, but the extent of neuronal death did not correlate with the up-regulation of the NADPH-diaphorase staining in the reticular nuclei of the medulla.5. The findings provide evidence that NADPH-diaphorase-exhibiting neurons are refractory to the hemisection of the cervical spinal cord and that the neuronal up-regulation of NADPH-diaphorase at the medullar level is probably not a causative factor leading to the death of the reticulospinal neurons.     

Neurochemical Organization and Connections of the Cerebral Preglomerular Complex of the Masu Salmon

Using immunohistochemical labeling of the cells containing neuronal NO synthase (nNOS), tyrosine hydroxylase (TH), GABA, and parvalbumin (PA), as well as histochemical marking of choline acetyltransferase-containing neurons, we examined the neurochemical organization of the glomerular nuclei and preglomerular complex in the brain of the masu salmon (Oncorhynchus masou). Injections of the carbocyanine dye DiI allowed us to examine projections of neurons of the preglomerular and mammillary nuclei in the salmon brain. We showed that cholinergic, GABA-, PA-, TH-, and nNOS-immunopositive neurons belonging to different morphological types are present in the glomerular and medial preglomerular nuclei. The analysis of correlations between morphometric characteristics of the cells belonging to different neurochemical types and densitometric estimates of amounts of neurochemical agents present in these cells allowed us to hypothesize that there are close morphofunctional interrelations in cell populations possessing different neurochemical and morphometric characteristics. These interrelations of the cells belonging to different chemotypes are, probably, realized as mediatory/modulatory ones. The presence of a great number of small slightly differentiated cells in the preglomerular and glomerular nuclei allows us to suppose that the growth of the greatest sensory center of the salmon brain is provided by neuroblasts that migrate from the proliferative zones in the course of postembryonal neurogenesis. It is also hypothesized that NO, TH, and GABA are involved in paracrine control of the postnatal morphogenesis of the salmon preglomerular complex. The data obtained by hodological analysis indicate that the nuclei of the preglomerular complex obtain afferent projections from the dorsomedial and ventroventral telencephalic regions, preoptic nucleus, periventricular layer of the tectum, and posterior central thalamic nucleus. Our study demonstrated the existence of reciprocal functional connections between the preglomerular complex (most important diencephalic center for transmission of sensory information) and dorsomedial and ventral regions of the telencephalon in the masu salmon.     

Distribution of cholineacetyltransferase histochemistry in isthmus and medulla of Onchorynchus masu. Tract-tracing observation on the ascending meso-pontine cholinergic system

The distribution of cholinergic neurons was studies in the brain steam, medulla and rostral spinal cord of the salmon Onchorynchus masu using histochemical choline acetyltransferase (ChAT) detection. Cholinergic neurons were observed in the isthmus, cranial nerve motor nuclei and spinal cord. In order to characterize several cholinergic nuclei observed in the isthmus of O. masu, their projections were studied by application of 1,1'-dioctadecyl-3,3,3',3,'-tetramethylindocarbocyanine perchlorate (DiI) to selected structures of the brain. The secondary gustatory nucleus projected mainly to the lateral hypothalamic lobes, whereas the nucleus isthmi projected to the optic tectum and parvocellular superficial pretectal nucleus, as it was earlier described for the other teleost group. In addition, the other isthmic cholinergic nuclei in O. masu may be homologous to the meso-pontine system of mammals. We conclude that the cholinergic systems of teleosts show many primitive features that have been presented during evolution, together with exclusive to the group characteristics.     

Localization of cystathionine β-synthase in digestive tract of the masu salmon <Emphasis Type="Italic">Onchorhynchus masou</Emphasis> (Teleostei)

By immunohistochemical method the hydrogen sulfide (H₂S)-producing system was studied in digestive tract in fry and adult masu salmon Onchorhynchus masou. The general and agerelated peculiarities have been revealed in morphology, distribution, and the amount of enteric cystathionine β-synthase (CBS)-immunopositive neurons. They have been established to be present in fry only in esophagus and stomach, while in adult fish—along the entire length of the digestive tract, the number of marked cells in esophagus, stomach, and distal intestine exceeding that in the proximal and middle intestine. In fry and in adult fish, the neurons and nerve fibers are located in branches of vagus, in intermuscular and submucosal nerve plexuses, in serosal and muscle layers. There are detected uni-, bi-, and, much seldom, multipolar neurons surrounded by numerous poorly or undifferentiated nerve cells. Besides, the CBS-immunopositive nerve fibers were present in the subepithelial area of the duct of swim bladder.     

Persistent and reparative neurogenesis in the juvenile masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> telencephalon after mechanical injury

A superficially located periventricular proliferative area with PCNA-immunopositive (PCNA+) cells, which corresponds to the pallial periventricular zone (PVZ) of other fish species, including its dorsal, lateral, and medial compartments, is discovered in the telencephalon of the juvenile masu salmon Oncorhynchus masou. The PCNA+ cells are also identified in the parenchyma of the masu salmon intact brain, and their maximum concentration is observed in the medial zone. After a mechanical injury, the zones of induced neurogenesis—neurogenic niches and sites of secondary neurogenesis surrounded by radial glial fibers—appear in the masu salmon telencephalon. The PVZ of the juvenile masu salmon pallium contains clusters of undifferentiated HuCD-immunopositive (HuCD+) neurons. A change in the HuCD+ cell topography is observed in the mechanically injured masu salmon telencephalon, namely, neurogenic niches in the lateral zone and an increase in the cell distribution density and cell migration patterns in the medial zone. A high level of persistent neurogenesis is characteristic of the juvenile masu salmon brain.     

Rapid, Corticosterone-Induced Disruption of Medullary Sensorimotor Integration Related to Suppression of Amplectic Clasping in Behaving Roughskin Newts (Taricha granulosa)

Endogenously secreted or injected corticosterone (CORT) rapidly suppresses courtship clasping in male roughskin newts (Taricha granulosa) by an action on a specific neuronal membrane receptor. Previous studies, using immobilized newts, showed that CORT administration rapidly depresses excitability of reticulospinal neurons and attenuates medullary neuronal responsiveness to clasp-triggering sensory stimuli. The present study used freely moving newts to examine clasping responses and concurrently record sensorimotor properties of 67 antidromically identified reticulospinal and other medullary reticular neurons before and after CORT injection. Before CORT, reticulospinal neurons fired in close association with onset and offset of clasps elicited by cloacal pressure. Reticulospinal neurons also showed firing correlates of nonclasping motor events, especially locomotion. Neuronal activity was typically reduced during clasping and elevated during locomotion. Medullary neurons that were not antidromically invaded (unidentified neurons) usually showed sensorimotor properties that resembled those of reticulospinal neurons. Intraperitoneal CORT (but not vehicle) reduced the probability and quality of hindlimb clasping in response to cloacal pressure, especially within 5–25 min of injection. Simultaneously, responses of reticulospinal and unidentified neurons to cloacal pressure and occurrence of clasping-related activity were attenuated or eliminated. CORT effects were relatively selective, altering clasping-related neuronal activity more strongly than activity associated with nonclasping motor events. The properties of CORT effects indicate that the hormone impairs clasping by depressing processing of clasp-triggering afferent activity and by disrupting the medullary control of clasping normally mediated by reticulospinal neurons. The rapid onset of these CORT effects implicates a neuronal membrane receptor rather than genomic action of the steroid.     

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H₂S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl-propargylglycine antagonized these l-cysteine effects. We suggest H₂ S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.     

Localization and properties of reticulospinal neurons with axons descending in the dorsolateral parts of the lateral funiculi

The localization of reticulospinal neurons responding antidromically to stimulation of fibers in the dorsolateral parts of the lateral funiculi (shown previously to be the principal collector of fibers conveying bulbar pressor influences) was determined in experiments on anesthetized and curarized cats. Most of these neurons were found to occupy the medioventral portions of the medulla, but they were concentrated in the rostral portions of the gigantocellular and ventral nuclei of the reticular formation. The velocity of conduction of excitation along axons of most reticulospinal neurons was 10–50 m/sec. Reflex responses to stimulation of the sciatic nerve with a latent period of 10–40 msec were found in 35 of 125 such cells. Stimulation of the sinus nerve did not activate them. Spontaneous activity occurred in 29 reticulospinal neurons; the mean firing rate of the various cells varied from 5 to 20/sec.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 6, No. 3, pp. 266–272, May–June, 1974.     

NO- and H<Subscript>2</Subscript>S-synthesizing neurons in the thoracic ganglion of crabs <Emphasis Type="Italic">Eriocheir japonicus</Emphasis> and <Emphasis Type="Italic">Pugettia quadridens</Emphasis> (Decapoda)

The presence and distribution of NO and H₂S-synthesizing elements in the thoracic ganglion in two crab species, Eriocheir japonicus and Pugettia quadridens, are shown using the staining methods of NADPH-diaphorase histochemistry and cystathionine-β-synthase (CBS) immunohistochemistry. NO and H₂S have been established to be synthesized by different neurons in the thoracic ganglion. The high CBS activity in efferent elements (segmental motoneurons, interneurons and nerve fibers in the limb neuropil) indicates the involvement of hydrogen sulfide in the organization of locomotor behavior in crustaceans. The presence of H₂S-synthesizing neurons in the wall of the sternal artery in E. japonicus allows suggesting the participation of H₂S in the regulation of vascular tone.     

Cystathionine β-Synthase Inhibition Is a Potential Therapeutic Approach to Treatment of Ischemic Injury

Hydrogen sulfide (H₂S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H₂S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H₂S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H₂S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H₂S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.     

Genetic differentiation between collections of hatchery and wild masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) inferred from mitochondrial and microsatellite DNA analyses

There has been very little effort to understand genetic divergence between wild and hatchery populations of masu salmon (Oncorhynchus masou). In this study, we used mitochondrial (mt) NADH dehydrogenase subunit 5 gene (ND5) and six polymorphic nuclear microsatellite DNA loci to compare the genetic variability in three hatchery broodstocks of masu salmon with the variability in eight putative wild masu populations sampled in five rivers including one known source river for the hatchery broodstocks. Both ND5 and microsatellites showed no significant genetic divergence (based on F_ST estimates) between four annual collections from the source river population, suggesting no change in genetic diversity over this time period. The F_ST estimates, an analysis of molecular variance (AMOVA), and a neighbor-joining tree using both DNA markers suggested significant differentiation between the three hatchery and all eight putative wild populations. We conclude that genetic diversity of hatchery populations are low relative to putative wild populations of masu salmon, and we discuss the implications for conservation and fisheries management in Hokkaido.     

Lanthionine Synthetase C-like Protein 1 Interacts with and Inhibits Cystathionine β-Synthase: A TARGET FOR NEURONAL ANTIOXIDANT DEFENSE*

The finding that eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is a glutathione-binding protein prompted us to investigate the potential relationship between LanCL1 and cystathionine β-synthase (CBS). CBS is a trans-sulfuration enzyme critical for the reduced glutathione (GSH) synthesis and GSH-dependent defense against oxidative stress. In this study we found that LanCL1 bound to CBS in mouse cortex and HEK293 cells. Mapping studies revealed that the binding region in LanCL1 spans amino acids 158–169, and that in CBS contains N-terminal and C-terminal regulatory domains. Recombinant His-LanCL1 directly bound endogenous CBS from mouse cortical lysates and inhibited its activity. Overexpression of LanCL1 inhibited CBS activity in HEK293 cells. CBS activity is reported to be regulated by oxidative stress. Here we found that oxidative stress induced by H₂O₂ or glutamate lowered the GSH/GSSG ratio, dissociated LanCL1 from CBS, and elevated CBS activity in primary rat cortical neurons. Decreasing the GSH/GSSG ratio by adding GSSG to cellular extracts also dissociated LanCL1 from CBS. Either lentiviral knockdown of LanCL1 or specific disruption of the LanCL1-CBS interaction using the peptide Tat-LanCL1_153–173 released CBS activity in neurons but occluded CBS activation in response to oxidative stress, indicating the major contribution of the LanCL1-CBS interaction to the regulation of CBS activity. Furthermore, LanCL1 knockdown or Tat-LanCL1_153–173 treatment reduced H₂O₂ or glutamate-induced neuronal damage. This study implies potential therapeutic value in targeting the LanCL1-CBS interaction for neuronal oxidative stress-related diseases.     

Nuclear origins of brainstem reticulocortical systems in the rat

Stereotaxic injections of 5% Fast Blue or 1% horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) were made into various cytoarchitectonic or functional regions of the cerebral cortex of anesthetized adult albino or hooded rats. Sections through the brainstems of these animals were then scrutinized for the presence of retrogradely labeled neurons. The data generated by this study indicate that at least 33 distinct nuclei or subnuclei within the brainstem reticular formation of the rat project directly to the cerebral cortex. More than half of these ascending reticulocortical systems are probably aminergic. The strongest reticulocortical projections emanate from presumed aminergic reticular-cell groups located at isthmic levels: specifically, the rostral serotonin-containing cell groups, as well as the noradrenergic locus coeruleus. However, relatively strong direct reticulocortical projections also originate from lower medullary cell groups which are probably catecholaminergic. Moderately strong reticulocortical projections emanate from cholinergic cell groups located at isthmic levels (the pars compacta of the pedunculopontine nucleus and the X area of Sakai). The most surprising finding in this study was that the classic isodendritic, nonaminergic central core of the brainstem gives rise to direct reticulocortical projections. The ventromedial areas of the medullary brainstem reticular formation give rise to the strongest nonaminergic ascending reticular projections, but all levels of the classic isodendritic reticular core give rise to direct reticulocortical projections. As a whole, cortically projecting reticular neurons are mostly small (10-25 microns in greatest diameter) or medium sized (26-35 microns in greatest diameter) neurons. Previous studies have shown that many of the cortically projecting reticular nuclei also project to the spinal cord, and within these nuclei, reticulocortical neurons often strongly resemble their reticulospinal counterparts with respect to details of neuronal morphology. This in turn suggests that some reticulocortical neurons may also project to spinal levels.     

Genetic differentiation and the problems of conservation of masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis> Brevoort, 1856 (Pisces: Salmonidae)) populations

This study focuses on the strategy for the conservation of masu salmon, Oncorhynchus masou, in the northern part of the species range (via the masu populations in Sakhalin Oblast), based on data of its population structure. It is shown that masu populations that inhabit different rivers genetically differ from each other in allele frequencies at microsatellite markers. In the Naiba River basin, at least two genetically distinct masu populations exist: in the upper reaches and in a tributary, the Bolshoy Takoy River. The masu populations on Iturup Island significantly differ from those on Sakhalin Island; within Sakhalin, the masu salmon from the Chernaya River in the southwestern part of the island is genetically distinct from the southeastern Sakhalin and Aniva Bay populations. The genetic diversity of Iturup populations is substantially lower than that on Sakhalin, probably due to their small sizes. The measures for the conservation and recovery of masu salmon populations should be based primarily on their own genetic resources, or, in the case of a lack of spawners, on the base populations of their ecological/geographical region. In the latter case, masu populations of large rivers can be considered as base ones: for southeastern Sakhalin, this is masu salmon of the Naiba River; for Aniva Bay, this is masu salmon of the Lyutoga River. Transplantation of fish, fertilized eggs, or any other genetic material from a population that is different genetically and inhabits the waters with different ecological gradients should be strongly restricted. The formosan masu salmon from Taiwan Island is studied as an example of a strict genetic isolate.     

Reproduction ecology of masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> in the Kol basin (Western Kamchatka)

In the Kol basin, one of the typical salmon rivers of Western Kamchatka, special traits of reproduction ecology of masu salmon Oncorhynchus masou are studied (characteristics and behavior of spawners during spawning, localization of spawning grounds, topography and hydrology of redds, and interaction with other species of salmonids). The masu salmon spawns in the Kol basin in tributaries of the upper reaches of the river and only in downwelling. Spawning grounds of masu samlmon are confined to stretches with overhanging banks, log jams creating shelters for spawners and favorable hydrological conditions for spawning and egg development. All over its range, masu salmon requires similar conditions for reproduction. In the north of its range, in Kamchatka, masu salmon retains the properties of the most warm-water species in the genus Oncorhynchus and selects for spawning the grounds characterized by the highest temperature in the period of spawning and development.