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Morphometric characteristics and relative numbers of neurons belonging to different neurochemical types in the nuclei of the Preglomerular complex and also in the Glomerular nucleus of the Oncorhynchus masou brain.
1. Introduction
The revealed peculiarities of structural and neurochemical organization and description of basic histogenetic processes (proliferation, migration and neuronal cell differentiation) during the brain forming in fish, which have signs of fetal organization, widen the existing knowledge about histogenesis of these structures in postembryonic development. It seems conceivable, that during postembryonic development in teleost fishes some neurotransmitters and gaseous mediators (NO and H2S) act as factors, which initiate and regulate the cellular and the tissues processes of genetic program during the brain development. We suppose the presence of epigenetic control of adult neurogenesis in salmon brain via highly coordinated nonsynaptic cell–cell signaling. This communication engages the neurotransmitters GABA and dopamine whose extracellular concentrations depend on neuroblasts number and high affinity uptake systems in neural stem cells. Neuroblasts release GABA providing a negative feedback control of stem cell proliferation and instructing them on the size of the neuroblast pool. We suggest that in salmon brain exist strong control mechanisms of neuroblast production. The data provided by our study add to our general understanding, that peculiarities of distribution of classical neuromediators (GABA, catecholamines) and gasotransmitters (NO and H2S) are directly connected with ability of the fishes brain to grow during the animal entire life. We suggest, that some classical neuromediators (GABA, catecholamines) and gasotransmitters (NO and H2S) not only regulate functional activity of neurons and modulate synaptic transmission in mature neural networks, but also are regarded as inductors of the fishes brain development (morphogenetic factors) in postembryonic ontogenesis. We propose that dopamine and GABA act as homeostatic signals to regulate neuroblast production. This confirmation is proved by finding of the phenotypically immature elements, expressing the above mentioned molecules in proliferating brain areas, in the three-year-old salmon brain, and of elements, which owe morphology of radial glia. The presence of enzymes, synthesizing gasotransmitters in the brain areas, which are expressing proliferative cell nuclear antigen PCNA, have proved their participation in regulation of postembryonic neurogenesis.
In the fishes, which preserve fetal state during long time (salmon and carp), such markers as NO and H2S in periventricular proliferative areas may present in different ratios. This is consistent with the hypothesis that in functionally similar complexes in animals the different signal transduction systems may be involved. In contrast to widespread neurogenetic model Danio rerio, the development of the salmon and sturgeon nervous system occurs during long time. As it follows from our data, the development of different CNS structures in the Oncorhynchus masou brain is characterized by evident heterochrony, so the cells of caudal brain regions gain features of phenotypical specialization earlier than in the forebrain structures. We suggest that the brain of these animals during a long time preserves the signs of fetal organization and low differentiated cells presence confirms this hypothesis.
Last years, certain attention of neuroscientists of different profile was turned to participation in the work of the brain «gaseous intermediaries»: nitric oxide (NO) and hydrogen sulphide (H2S). Their presence is found in the brains of representatives of different groups of vertebrates: from the Agnatha to human. The few data points to a high degree of variability in the distribution of NO-ergic neurons in the fish brain [1-3], and information about the involvement of nitric oxide and hydrogen sulphide in the functional activity of nervous system of fish is unordered and contradictory. This draws attention to the fact that the relative number of NO-synthesizing neurons and glial cells in the sensory, motor and integrative centers of the brain fish significantly exceeds that of terrestrial vertebrates, in particular, mammals [1, 4, 5]. This implies a wide and varied participation of NO in the metabolism of neurons and glial cells in the central nervous system of fish compared with mammals. However, information about the relationship of the NO-producing neurons of the brain of fish with the systems of classical neurotransmitters such as acetylcholine, catecholamines and GABA, are practically absent. Virtually nothing is known about the distribution of H2S-producing systems in the CNS of bony fishes. These investigations are of particular importance in connection with the emerging data on morphogenetic the role of classical and gas intermediary in the formation of the central nervous system of vertebrates [6].
The brain of fish has a unique vertebrates feature - it grows with the organism during all life. In connection with this fish is a model object for the study of embryonic and postembryonic development of the CNS, to influence these processes of various factors. It is shown that in the brain of adult vertebrate a system of cambial elements remains, the activity of which allows to replenish the population of neurons and glial cells in the course of a long period after birth [7]. Currently the mechanisms of pre-and postnatal morphogenesis of the brain in the fish, which for a long time secures the larval state, virtually have not been studied [8-10].
Especially it concerns the role of the so-called «radial glial cells» in the processes of morphogenesis of the brain, the availability and distribution of proliferative areas in the brain of adult fish. The results of the research on Danio rerio showed that the newly formed cells moving from periventricular areas deep inside the brain, where they differentiate into neurons [11]. It was found that the centers of proliferation are localized along the rostro-caudal axis of the brain [7]. The interest to the study of these processes in fish is caused by the fact that the «radial glia» may be connected with the processes of migration and differentiation of neurons and glial cells in the prenatal period, large quantities present in the brain of a fish and in the adult state (unlike other vertebrates). However, in spite of the available literature information, participation of the radial glia (RG) is in the process of neurogenesis adult animals and little studied. One of the reasons for the lack of such information is a small number of examined in the terms of species and groups of fish, the absence of reliable markers of the RG in lower vertebrates.
Sturgeon and salmon fish, which have become the main objects of our research, represent the most ancient group of vertebrates, which are the most primitive branches ray-finned fish [12-13]. The information about the development of the brain sturgeon and salmon, the relations of embryonic and a definitive parts in the structure of the pre-and postnatal neurogenesis, organization and formation of the neuromediating and modulating brain systems in the literature are extremely limited. This concerns especially the sturgeon fishes, the evolution of which was carried on the pedomorphosis way, which is characterized by the slowing of organs or of their systems and the preservation of the adult embryonic status of relevant features.
The purpose of this chapter is to explore the organization, projection features and relationships of signal-transduction systems, producing a classic neurotransmitters (catecholamines, acetylcholine, gamma-aminobutyric acid-GABA) and gazotransmitters (nitric oxide and hydrogen sulphide), in the brain of fish and evaluate their participation in the processes of the post-embryonic morphogenesis the CNS.
2. Methods
Molecular-biological approaches associated with identifying of histochemical and immunohistochemical activity of mediators or enzymes of their synthesis were used for characteristics of neurotransmitter systems structures of the brain and spinal cord fish. Specific antibodies are also used by us in identifying of proliferative cell nuclear antigen (PCNA), transcription factor Pax6 and calcium binding protein parvalbumin. To investigate the relationship of brain applied marking nerve fibers using carbocyanin dye DiI. To track ascending mediatorically specific projections of catecholaminergic cells was used the immunofluorescence method of marking tyrosine hydroxylase in combination with the marking of the DiI.The histochemical reaction on NADPH-diaphorase (NADPH-d, NF 126.96.36.199). Experimental procedures were conducted in accordance with European Community guidelines on animal care and experimentation. The animals were deeply anesthetized with 0.03% tricain methanesulfonate (MS-222, Sandoz) and perfused transcardially with 50 ml of 0,63% saline followed by 200 ml of a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were then removed from the skull, postfixed in the same fixative for 5 hours, washed in PB at 4°C overnight and then placed in a 30% sucrose solution for cryoprotection.
Fifty-micron-thick transverse sections were cut on a cryostat and collected in cold PB and, after several washes in PB, processed for NADPH-diaphorase histochemistry. Free-floating sections were incubated in a medium made up of 1mM β-NADPH, 0.8 mM nitro blue tetrazolium, and 0.06% Triton X-100 in 0.1 M phosphate buffer (pH 7.6), at 37˚C for 2 hours [14]. All chemicals were purchased from Sigma. After incubation, the sections were rinsed in PB, mounted on gelatin-coated glass slides, and air-dried overnight. The following day they were dehydrated cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany).
In order to determine the specificity of the histochemical reaction, the following controls were carried out: incubation without the substrate β-NADPH, and incubation without the chromogen nitro blue tetrazolium in order to rule out possible nonspecific formation of reaction product. In all cases, no residual reaction was observed.
For histochemical staining of cholinergic neurons in the brain of fish we used marking of choline acetyltransferase (ChAT; NF 126.96.36.199.). Method was performed on fishes whose brains were fixed at 4°С for 2 h in 1% solution of paraformaldehyde based on cacodylate buffer (0.1 M) with sucrose (0.32 М; рН 5.0). The material was washed out in cacodylate buffer (рН 5.2) with sucrose for 18 h with sevenfold change of this solution. Frontal and sagittal 50-μm-thick slices were prepared with a freezing microtome. To exclude nonspecific transferase activity, 20 mM diisopropyl fluorophosphate (DFP), 10 % sucrose, and 25 mM cacodylate buffer were added to the incubation medium (рН 6.0) cooled to 4°С ; this medium was placed on an ice bath (0-4°С) for 1 h. After preincubation, the slices were placed in the incubation medium (рН 6.0) with the following final concentrations (mM): cacodylate buffer, 25; DFP, 1.0; choline chloride, 4.0; lead nitrate, 1.0; acetyl-CoA, 0.3, and 5% sucrose. The sliced were thermostated at 37°С for 2 h, washed out in distilled water, and treated in 5% solution of ammonium sulfide. Then, the slices were post-fixed for 5 min in 5% solution of formaldehyde based on cacodylate buffer (0.1 М; рН 5.2) with sucrose (0.32 М), dehydrated, and embedded in balsam. To estimate the specificity of reactions to ChAT, we carried out a few control experiments. In the first control series, we excluded DFP from the incubation medium. In the second control series, cetyl-CoA or choline chloride were absent in the incubation medium. In the third control series, we added chloracetylcholine-perchlorate (10 mM) to the DFP-containing pre-incubation medium; the incubation period was increased to 1.5-2 h. In all control experiments, a positive reaction was absent.
Immunohistochemical methods. Fishes were kept in aquaria with aerated seawater at 15-17°С. Before experiments, fishes were anesthetized in the cuvette with 0.1% solution of tricaine methanesulfonate (MS-222; Sigma, USA) in seawater for 10-15 min. The brains of fishes were fixed for 2 h at 4°С in 4% solution of paraformaldehyde dissolved in phosphate buffer (0.1 M, рН 7.2). For morphological analysis, the obtained material was embedded in paraffin according to a standard technique and stained by Nissl. In the course of immunohistochemical studies, we identified the elements containing GABA, tyrosine hydroxylase (TH), parvalbumin (PA), neuronal nitric oxide synthase (nNOS), proliferative cells nuclear antigen (PCNA), transcription factor Pax6 and cystathionine β-synthase (CBS). For this purpose, we used indirect avidin-biotin-peroxidase (ABC technique) or streptavidin-biotin staining. The material was washed out for 24 h in 30% sucrose solution. Transverse 50-μm-thick slices of the fish brain were prepared using a freezing microtome. Free-floating slices were incubated at 4°С for 48 h in the presence of monoclonal mouse antibodies against GABA (ICN Biomedicals, USA; dilution 1:4000) and tyrosine hydroxylase, TH (Vector Laboratories, USA; dilution 1:5000), PCNA (Dako, Denmark; 1:4000), monoclonal antibodies against human transcription factor Pax6 (Chemicon, USA; 1:3000), monoclonal antibodies frog against PA (ICN, Biomedicals, USA; 1:4000), rabbit polyclonal antibodies against nNOS (ICN, Biomedicals, USA; 1:5000), monoclonal antibodies mouse against CBS (Abcam ab54883, England 1:5000). Then, the slices were incubated with secondary biotin-conjugated horse antibodies against mouse immunoglobulins (Vector Laboratories, USA) for 2 h at room temperature and washed out three times in 0.1 М phosphate buffer. To reveal localization of NO-ergic neurons and fibers, we used a technique of indirect streptavidinbiotin immunohistochemical labeling of NOS. The slices were incubated with primary polyclonal rabbit antibodies against nNOS (IСN Biomedicals, USA; dilution 1:5000) at 4°С for 24 h. After three washings out in phosphate buffer, the slices were incubated with secondary biotin-conjugated goat antibodies against rabbit immunoglobulins (Biomedicals, Germany) at room temperature for 2 h. The material was washed out three times in phosphate buffer. Then, the slices were incubated in the presence of the streptavidin-peroxidase complex (Biomedicals, Germany) at room temperature for 2 h and again washed out three times in phosphate buffer. Immunohistochemical reactions were visualized using a standard avidinbiotin system (ABC; Vectastain Elite АВС Kit; Vector Laboratories, USA). To identify the reaction products, the slices were incubated in a substrate for detection of peroxidase (VIP Substrate Kit; Vector Laboratories, USA); the process of staining was controlled under a microscope. Then, the slices were washed out in three changes of phosphate buffer, mounted on slides, dehydrated using a standard technique, and embedded in balsam. To estimate the specificity of the immunohistochemical reaction, we used a technique of negative control. The masu brain slices were incubated in a medium containing 1% nonimmune horse serum (instead of primary antibodies) for 48 h, and then all procedures were performed as was described above. In all control experiments, the immunopositivity in the studied cells was absent.
To study projections of the preglomerular complex and glomerular nucleus, we used the carbocyanine dye 1,1΄-dioctadecyl-3,3,3΄,3΄-tetramethylindocarbocyanine perchlorate, DiI (Aldrich, Sigma, USA). The brains of fishes were fixed for 24 h in 4% solution of paraformaldehyde; then, crystals of the above dye were introduced in the region of the anterior and medial preglomerular and mammillary bodies. The obtained preparations were incubated in 4% solution of paraformaldehyde with the addition of 0.01% ethylenediamine tetraacetic acid (EDTA) at room temperature. Frontal, sagittal, and horizontal slices (50 μm thick) were prepared using a vibratome (VIBRATOME 3000; Sectioning system, Germany) and embedded in glycerine. To visualize the marker, we used an optical system, AXIOPLAN-2, Imaging (Gerinang, Germany). Preparations with DiI-marked structures were photographed using a optical system AXIOPLAN-2, Imaging (Gerinang).
Immunofluorescent labeling of tyrosine hydroxylase (TH) combined with retrograde labeling of neurons with the carbocyanine stain DiI was used to study the brains of Amur bitterlings Rhodeus sericeus. Specimens were fixed in 4% paraformaldehyde for one day, after which crystals of stain were placed in the ventral part of the telencephalon. Specimens were incubated in 4% paraformaldehyde supplemented with 0.01% ethylenediaminetetraacetate (EDTA) at room temperature for one day. Frontal, sagittal, and horizontal vibratome sections of thickness 50 μm were cut and incubated with primary mouse monoclonal antibodies against TH (Vector Laboratories, Burlingame, USA) diluted 1:1000 at 4°C for two days. Sections were then incubated with secondary fluorescent antibodies conjugated with Alexa 546 (Invitrogen Molecular Probes, USA) diluted 1:300 overnight. TH localization was studied using a Leica DM 4500 fluorescent microscope (Germany). Labeled TH and the carbocyanine label were visualized using a Leica TSC SPE confocal laser system (Germany).
Immunoperoxidase labeling of fragmented DNA chains, (TUNEL-labeling). To reveal apoptotic cells, we used a technique for immunoperoxidase labeling of fragmented DNA chains. After 2-h-long fixation in 4% solution of paraformaldehyde based on 0.1 M phosphate buffer (рН 7.2), dissected parts of the brain were washed out for 24 h in 0.1. M phosphate buffer. Then, these samples were put in 30% solution of sucrose based on phosphate buffer (0.1 М) for cryoprotection and kept in this solution up to full immersion. Frontal and horizontal slices (20 μm thick) were prepared using a freezing microtome. To identify TUNEL-positive structures, we used a immuno-peroxidase identification system, ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., USA). For blocking endogenous peroxidase, the slices were incubated in 1% solution of hydrogen peroxide for 3 min and then washed out two times for 5 min in phosphate buffer. The slices were covered with a smoothing buffer (75 μl) and kept for 10 sec at room temperature. Then, the slices were slightly dried, subjected to the action of TdT enzyme (55 μl/5 cm2), incubated in a humid chamber for 1 h at 37°С, and immersed in a stop buffer for 10 min. The slices were washed out in phosphate buffer at room temperature (three times for 1 min with changing of the solution), again dried, covered with antidioxygenin conjugate (65 μl/5 cm2), and incubated in a humid chamber for 30 min. To detect the reaction products, cerebral slices were incubated in the substrate for identification of peroxidase (VIP Substrate Kit; Vector Labs, USA) with control of the development of color under a microscope, washed out in three changes of phosphate buffer, and mounted on glass slides. The cell nuclei were subjected to final staining with methyl green according to the technique of Brasher [15]. The preparations obtained were dewatered using a conventional technique and embedded in balsam. Morphometric processing was performed using an inverted-stage microscope, Axiovert 200M, equipped with a module, ApoTome, and digital cameras, Axio Cam MRM and Axio Cam HRC (Carl Zeiss, Germany).
The measurements were performed at ×400 magnification in five randomly chosen fields of vision for each studied region. The proliferation index (PI) and apoptosis index (AI) were calculated per 1 mm2 of the section using the following formulas:
PI = (n of the PCNA-positive nuclei × 100%) ÷ total n of the nuclei and
AI = (n of TUNEL-positive fragments × 100%) ÷ total n of nuclei
Parametric comparison (Student's t-test) was used for estimation of the intergroup differences. The data obtained were processed using Statistica and Excel software. Numerical data are presented below as Means ± s.e.m.
3. Participation of classic neurotransmitters in the postembryonic neurogenesis in the fish brain
Studies suggest that in the salmon′s brain at different stages of postembryonic development two forms of intercellular communications exist. The first form occurs in the early stages of postembryonic development and represents cells cooperation, carried out paracrinic in the period when cells have not developed processes and synaptic structure yet. However, such little differentiated cells are already able to express the specific synthetic machinery for some neurotransmitters and their synthesizing enzymes, gaseous intermediates, transcription factors and other substances (Fig. 1A-D). We suppose that most of the synthesized signals in this period are involved in the regulation of neuronal targets, differentiation and expression of their specific phenotype, acting as a morphogenetic factors. This is consistent with the Ugryumov concept [6] regarding the development of the mammalian brain in the embryonic period of ontogenesis. Already in the early stages of post embryonic morphogenesis of masou salmon, simultaneously two systems of neurochemical signaling exist, the dopaminergic and GABA-ergic systems, providing paracrinic and perhaps autocrinic influence on target cells until the formation of synaptic contacts and the beginning of neurotransmission with specific interneuronal connections. Study on the eal Anguilla anguialla showed that the maximum concentration of dopamine D1 receptors is found in periventricular zones [16] which represent a matrix areas of the brain, where neurogenesis continues throughout the life of the animal. Consequently, the cells located in proliferating areas are targeted for the regulatory impact of dopamine. These cells are localized on the territory of the largest vascular plexus (forebrain and caudal medullar), and synthesize in these regions some substances, like a dopamine and GABA, which then may be excreted in the portal system blood flow and further into the general circulation system, providing regulatory endocrine effects on the peripheral organs [17]. Thus, there is considerable justification to suggest that in the hypophysotrophic areas of the diencephalon and medulla oblongata of the brain of juvenile salmons O. masou, dopamine and GABA in undifferentiated cells of periventricular and subventricular areas are inducers of development (morphogenetic factors).
Along with the specified form of intercellular signaling in salmon brain, in ontogenesis there is the development of specific system of forebrain activation and development of the system of remote intercellular signaling. The source of these directed connections are the nuclei of preglomerular complex [18]. Development of projective systems of salmon take place simultaneously with the formation of the structure of preglomerular complex [19]. In the brain of non mammalian vertebrates the volume of sensory projection zones increases during all their life and is provided due to the proliferation of neural stem cells located in the areas of special neurogenetic niches [10]. It is connected with the necessity of adaptation of the CNS of such animals to increase the size of the body and increased inflow of primary sensory information. We believe that a dopamine, GABA-and NO-ergic systems in the brain of salmon participate in regulation of some basis histogenetic processes, such as a cell migration and differentiation of neuro- and gliospecific lines, because the nuclei of preglomerular complexes contain morphologically and neurochemically heterogeneous cell populations (table. 1) represented by the different stages of ontogenesis of major cell types. Cells formed in the proliferative (PCNA-containing) diencephalic areas migrate to the region of preglomerular complex, where their subsequent differentiation and growth take place. These processes are regulated by dopamine and GABA, that indicates the presence of D1 and D2 dopamine receptors [16, 20] and GABAB benzodiazepine receptors [21] in these nucleus of fish. A critical step prevalence of paracrinic relations in the salmons brain can be considered the period before the formation of the blood brain barrier (BBB), which in salmon brain is formed during the first year of life (according to [22]). In the next period of ontogenesis, the formation of the specific connections and the development of cellular processes of neurons and synaptogenesis take place. Today much data exist about the participation of radial glia in the processes of postembryonal neurogenesis by asymmetric mitoses in which one daughter cell remains in the periventricular area and has a rounded shape, while the other has a long process, which may later be eliminated through somal translocation [23]. It was shown that during embryogenesis of human, the predecessors of dopaminergic neurons in the basal part of the midbrain have the morphology of radial glia [24]. Immunolabeling of radial glia cells in salmon′s brain in different ages (Fig. 2 (A-D), as well as evidence that the TH-and GABA-ip cells were located on the territory of PCNA-ip proliferative zones and together with PCNA marked the neuromeric structure of diencephalon and medullar part of the brain, certainly shows that dopamine and GABA-ergic signaling participates in the processes of postembryonic neurogenesis of the salmon′s brain, as inductors of development. Our data are consistent with the labeling of some rhombomeres in the brain in an embryo of sharks Scyliorhinus canicula [25].
|Neuronal nitric oxide synthase (nNOS)
|Choline acetyltransferase(ChAT)
|Tyrosine hydroxylase(ТН)
|Size of
number (%)
|Size of
number (%)
|Size of
number (%)
|Size of
|Size of
Differentiation of cells in various parts of the salmon′s brain presents a heterochronical process. In caudal part of brain some reticulospinal cells, cells of nucleus raphi, nuclei of V, VII, IX and X pairs of cranial nerves, much earlier acquire the features of phenotypic specialization than in the structures of forebrain. Measurements of fractal dimension and some morphometric parameters (total length of branches, number of terminal branches, number of branching points, and cell area) were used for the quantification of morphological patterns of two spinal neuron groups in young Oncorhynchus masou at two ontogenetic stages [26]. During the 1st and 2nd years of life, the neurons of brainstem and spinal cord have enough developed dendrites and axons, which, however, have growth cones, indicating the continued postembryonic period of growth and development of these structures and their further differentiation. During the second year of life, the values of morphometric parameters and fractal dimension of neurons increased in both groups. Basic morphometric values correlated with fractal dimensions and conformed to morphological changes in the dendritic tree of the investigated neurons in ontogenesis. During the third year of life, in the nuclei of the brain and spinal cord large-differentiated cells expressing TH, GABA and parvalbumine in the motoneurons of ventral spinal column, nuclei of craniocerebral nerves, reticulospinal cells and some diencephalic nuclei were revealed [27].
Along with systems synthesis of classical neurotransmitters, immunolocalisation of transcription factor Pax6 was investigated, the marking of which adequately reflects neuromeric structure of the salmon′s brain in different ages (Fig 3 A, B). The early juveniles (3 and 6 months old) are the Pax6-ip cells do not have any processes and formed a small clusters corresponding to forebrain prosomeres (P1-P3), and in the medulla, such accumulations marked the rostral (R1-R2) rhombomeres (Fig. 3B). On the boundary of neuromeres labeling of PCNA and Pax6 were absent (Fig.3 A, D). In three-year old salmons the marking Pax6 was found in the cells and radial fibers, located in the periventricular and subventricular areas of diencephalon that corresponds to the data of labeling of Pax6 radial glia in the areas of postnatal neurogenesis of mammals [10]. On borders of the forebrain neuromeres the immunolabeling Pax6 in 3-years old individuals was absent. Expression of Pax6 was also found in glomerular nucleus and nuclei preglomerular complex that suggests about morphogenetic processes on the territory of the largest sensory center during postembryonal period. Immunolocalisation of Pax6 in specific cell clusters of glomerular nucleus, appropriated to some neuroanatomical zones, in which the differentiation of neurons, conducting various types of sensory signalization was revealed (Fig 3C. D). Studies suggest that factor Pax6 participates in the regionalization of the structure of the brain in postembryonal period, and its expression in different ages of salmon brain shows that the processes of neurodetermination and migration of cells, formed in proliferative areas of the brain in these age periods are regulated by means of this transcriptional factor. In the literature there are discrepancies regarding the organization and topography of dopamine, GABA-and NO-ergic complexes in the brain of different teleost fishes. Significant differences in the organization of the mediator systems in different fish species become more explainable, given the above mentioned scheme. We believe that neurotransmitter systems in the brain fish should be considered not only from the standard point of view of their definitive neuroanatomical structure, but must also take into account data on heterogeneous molecular phenotype of dopaminergic, GABA and, apparently, NO-ergic systems. Thus, for the establishment of homology, along with the systematic position, it is advisable to take into account the age, stage of development, physiological status and sex of the animal. In adult masou salmon and chum salmon the cells of Dc area of the telencephalon reach a high level of specialization and corresponding to the Ramon-Molener classification can be attributed to allodendric type.
Such cells have been found only in the most mature individuals (of 4-5 years old) going to spawn. One of the forms of specialization of these cells is that they have a network of basal spiny dendrites. This corresponds to the estimated specialization of such cells as associative spiny interneurons participated in communications with other parts of the dorsal area in telencephalon. Widespread TH and GABA in the telencephalon of adult chum salmon indicates that species to this period of development, along with paracrinic (volume) neurotransmission, there is a distant form of neurotransmission, which is becoming the predominant further ontogenetic development and ageing of the animal. We suggest that the acquisition of spiny apparatus by the neurons in the dorsal (Vd) and internal (Vi) areas ventral zone can be considered as one of the stages of ontogenetic development of neurons in the brain, indicating the age-related changes in the organization of the salmon′s dopaminergic system. Formation of the system of neurochemical communication in the CNS of masou salmon in postembryonal period consists of two main stages. At the first stage the undifferentiated cells are located in matrix areas of the brain and expressed of specific syntheses (catecholamenes, GABA, NO, some transcription factors). These substances are acting in paracrinic interaction and involved in regulating basis histogenetic processes: cell proliferation, cell migration, differentiation of target cells and expression of a specific phenotype. Under the influence of these factors on the second step is the formation of specific relations, development of processes of neurons and sinaptogenesis.
As a model to test an alternative hypothesis, we studied the CNS of amur bitterling Rhodeus sericeus (Cyprinidae), coming to sexual maturity in the first year of life. The literature of the late twentieth century actively discussed some issues relating to the organization and topography catecholaminergic system of vertebrate's brain detectable by methods formaldehyde-induced fluorescence (FIF) and IHC labeling of tyrosine hydroxylase. In this period, a hypothesis was formulated about the existence of dopamine deposited system in the brain of fish [28]. Data about neuromeric organization and molecular markers that define dopaminergic phenotype of neurons in Danio rerio, had recently published [29]. In the bitterling brain 3 main types of cells were verified. The first type consists of small round cells in the periventricular nucleus of the diencephalon and second one are formed by large pear-shaped or fusiform cells [30]. The cerebrospinalliquor-contacting cells (CSL) are the most common third type of catecholaminergic cells. According to the Meek classification [28], large cells and CSL-contacting cells at Amur bitterling can be attributed to the elements of dopamine deposited system. In the hypothalamus of bitterling were discovered a few CSL-contacting cells with a low level of activity TH, but cells were marked by gliocsalic acid. Some fish have similar features morphology of CSL-contacting cells (in particular, the presence of apical dendrite, turned into the lumen of the cerebral ventricle), and these cells are a FIF-positive, but do not contain enzymes synthesis of catecholamines (TH-negative). It was the reason for the assumption that such cells are not synthesizes catecholamines by themselves, but receives CE from external sources, in particular, liquor or from large dopaminergic neurons [31]. Data labeling catecholaminergic systems on other groups of vertebrates show that dopamine and norepinephrine dissolved in the cerebrospinal fluid are of greater importance for non mammalian vertebrates; but in mammals, the CSL-contacting cells at all have not been identified [32]. These confirm the observations obtained by us on the masou salmon.
Lack of Cyprinidae fish glomerular nucleus largely hinders establishing of homology between ascending sensory projections in the telencephalon with those of other fishes [33]. To identify sources of CA-ergic innervation of the ventral part of the telencephalon of bitterling investigated the projection of this area of the brain. Tracing part of dopaminergic fibers in the ventral telencephalon bitterling showed that along with intratelencephalic cell groups exists the extratelencephalic sources of innervation of the dorsal and ventral nuclei [30]. Sources of dopaminergic projections in the ventral part of the telencephalon are two populations of cells in posterior tuberculum of bitterling, namely large cells and small rounded cells. Such cells are projected on the dorsal and ventral areas of ventral telencephalic part respectively and are considered by us as the morpho-functional equivalents of meso-striatal and meso-limbic systems of mammals. Identification dopaminergic fibers in the dorsal region of telencephalon of D. rerio [31] suggests that teleostea have also equivalents of meso-pallial system.
The peculiarities of localization of medullary neurons, morphology of the dendrites, and trajectories of the axon projections in the medulla of the Amur bitterling allow us to differentiate three groups of ТН-positive neurons, namely interfascicular cells, units related to the lobus vagus, and cells localized within the area postrema (Fig. 4 A-D). The 3-year-old masou salmon in all the above mentioned areas of the brain stem were also identified large TH-ip cells with clear features of phenotypic differentiation. However, along with differentiated ТН-positive elements of masou salmon we revealed previously not described alternative TH-positive elements, namely small undifferentiated cells, located on the territory of proliferative periventricular and subventricular zones [27]; numerous radial fibers, having different localizations in medullar part of CNS (Fig. 2E). We believe that the presence of such elements with clear features of fetal organization, as well as radial fibers in the brain 3-year-old masou salmon connected with the processes of postembryonic (adult) morphogenesis of the brain. The differentiated TH-ip neurons in salmon brain are functionally active to this period of ontogenesis elements of CA-ergic system. Study of the CA-ergic system in the medullary part of bitterling found pronounced features of specialization associated with the organization of medullary CA-ergic complexes. Analysis of these characteristics showed that of bitterling CA-ergic cells in neuronal networks of the medulla can fulfill the functions of local interneurons, projection long axon neurons, neurosecretory units, or sensory units. The morphology of interfascicular ТН-positive cells in the Amur bitterling brain allows one to regard their functional specialization as local interneurons, since they form intensely branched dendritic networks (Fig. 4A, B). All three groups of medullary TH-ip neurons of bitterling project their terminals to the longitudinal catecholaminergic tract. Therefore, it is appropriate to hypothesize that all these cells are relatively long-axon neurons projecting to the rostral part of reticular formation, isthmus, and secondary gustatory nucleus which are relay centres, between the primary sensory nuclei of medulla oblongata and sensory centers of the ventral thalamus. The ТН-positive cells of the vagus region and area postrema (supposedly dopaminergic) have access to the fourth ventricle; likely, these neurons are chemosensory units responsible for the relations between the cerebrospinal fluid and neuronal medullary systems (Fig. 4С, D). On the other hand, these two neuronal groups in the Amur bitterling differ from others in an extremely high level of TH activity; it cannot be ruled out that they can serve as a source of dopamine coming to the cerebrospinal fluid. The morphology of these neurons allows one to hypothesize that each of the three groups of medullary CA-ergic neurons in the Amur bitterling is involved in realization of at least two functions of the above-listed ones, while the cells associated with the lobus vagus can combine all three functions. In the masou salmon brain phenotypically mature types of TH-ip cells localized in similar areas of the medulla oblongata, can have a similar functional specialization (Fig. 2E). However, part of the identified by us TH-ip elements is located in the proliferative (PCNA-marked) areas of medulla oblongata [27] at the earlier stages of ontogenesis mark neuromeric structure of medulla oblongata. At later stages localization TH found in the fibres of radial glia in interfascicular region, on the territory of fossa romboidea, as well as in populations of small cells in periventricular and subventricular areas (Fig. 2E).
In the Amur bitterling the density of the distribution of such phenotypically not mature cell forms in the periventricular area of the brain is significantly lower than the masou salmon. We believe that the features allocation in medullar part of masou salmon and Amur bitterling confirm the assumption about the participation of dopamine as a morphogenic factor regulating brain development of fish in postembryonal period.
4. Participation gaseous intermediators in the modulation of classical neurotransmitters in fishes brain
Study of the modulating influence gaseous intermediators to the classical system of neurotransmitters in the brain of fish previously had not been carried out. In our studies showed that the total nitroxidergic products in the nuclei of the brain stem in different fish species significantly exceeded the measure set for other groups of vertebrates and, particularly mammals. So, it is normal for different fish species NO-producing neurons were verified somato- and viscerosensor and visceromotor nuclei of medulla oblongata (V, VII, IX, X nuclei of craniocerebral nerves, Fig. 5), efferent octavo-lateral neurons, the nuclei of the isthmus, secondary gustatory nuclei, the nuclei of oculomotor complex (III, IV and VI nuclei of cranial nerves). Most of these nuclei in fish brain are cholinergic centers of brain stem involved in the innervation of brachiomotor muscles and some sensory inputs from the somatosensory, gustatory extra- and intraoral system, mechanosensory, octavolateral receptors. In fish due to low level of cephalization brain the most of the sensory inputs from the somatosensory (nucleus V), octavolateral, gustatory extraoral (nucleus VII), intraoral (nucleus IX) are concentrated on the territory of medullary part; therefore this sector is perceived by a large volume of incoming sensory information (see the diagram on Fig. 5). Despite significant interspecific morpho-adaptative differences, in Perciformes and Cyprinoid fish were identified similarities in the organization of medullar and spinal NO-producing centres. Participating NO in modulation of sensor systems in forebrain of mammals it was proved today [34]. We assume that in the medulla fish NO performs modulation of primary sensory centers, located in the nuclei of craniocerebral nerves. In the masou salmon brain all of the above mentioned nuclei, located in the stem and isthmus region are cholinergic and express nNOS (see the diagram on Fig. 5). Primary sensory nuclei (V, VII, VIII, IX and X), and secondary relay nuclei (secondary gustatory nucleus, the nucleus of the isthmus) in tha masou salmon brain, processing heteromodal sensory the information in the nuclei of preglomerular complex modulated by NO (Fig. 5). We assume that in the masou salmon brain NO is modulator of sensory and motor cholinergic centers.
The most important sensory center, conducting nociceptive information in fish's brain is a nucleus raphi. We found that in different species of teleost fish the most of the neurons of the nucleus raphi superior and nucleus raphi inferior are expressed NADPH-d. This confirms the data installed on mammalian and human brain, that NO is a mediator of nociception [35]. The presence of nNOS-producing neurons and high level of activity NADPH-d in the nuclei of raphi, dorsal spinal cord fibers and sensory part of the nucleus of trigeminal nerve indicates participation of nitric oxide in the modulation signals of nociceptive and somatosensor centers of the medulla oblongata in fishes brain. Study of the localization of nNOS in some periventricular hypophysotropic nuclei in diencephalon of adult specimens of Amur bitterling showed that TH-ip and NO-producing system in periventricular and subventricular nuclei in general have similar localization and area of colocalisation these transmitters is periventricular nucleus of posterior tuberculum, where nNOS and TH were localized in small cells, forming ascending projections on the ventral telencephalon. In these cells NO can modulate synaptic plasticity of dopaminergic neurons and regulate the excretion of dopamine.
Study of physiological activity of hydrogen sulfide in the nervous system of mammals began recently [36], and identifying its role in the central nervous system of fish previously had not been carried out. The results of researches conducted on fish suggest that hydrogen sulfide acts as an intermediary, regulating a number of enzymatic reactions cells. Distribution of the enzyme synthesis of H2S in the CNS of fish is expressed species-specific features, perhaps reflecting their adaptation character and functional status of the animal. Cystathionine β-synthase in the brain of masu salmon Oncorhynchus masou and carp Cyprinus carpio was found in neurons of the ventral spinal column and medulla oblongata, fibers and cells of the cerebellum, optic tectum, and telencephalon. In all brain areas, the intensity of CBS labeling in neurons varied between moderate and high. We found interspecies differences in the immunolocalizatoin and optical density of CBS in different brain structures of masu salmon and carp. In carp, the medulla oblongata and spinal cord contained intensely marked vessels that were absent in masu salmon. In the brain of carp, H2S presumably functions as a predominant vasoregulator. H2S-producing systems in the brain of bony fishes have specific characteristics of organization and strong species-related differences that correlate with the specificities of neuromediators, for example, NO-producing, systems.
Comparative studies of localization CBS and densitometric data in various structures of the masu salmon and carp showed interspecific differences, having obviously adaptive value (Fig. 10). In different areas of the masu salmon brain revealed varicose or smooth microcytosculpture of afferents and their endings, which may indicate to synaptic or paracrinic (volume) methods of H2S release in different areas of the fishes brain.Currently shows the involvement of GABA in the regulation of the endocrine activity of hormones preoptico-pituitary complex at the early juveniles Salmo trutta fario [37]. On larval and early juvenile of this salmon species showed the participation of GABA-ergic innervation in the regulation of synthesis of peptide hormones of the pituitary, namely metencephalin and galanin [37]. In our research on different age groups of masu salmon, it was found that GABA-ip neurons are present in various parts of the brain: in the medulla oblongata, periventricular nuclei of diencephalon, mesencephalic tegmentum, the brain stem, the cerebellum and spinal cord (Fig. 11). In addition to the neural localization of GABA, it was identified small undifferentiated cells and radial fibers, localized in areas where the proliferative activity of cells persists in adults animals (Fig. 11A, D). These zones have been identified in the diencephalon on the territory of preoptical area, posterior tuberal, thalamic and hypothalamic areas; in the region of the central gray matter of mesencephalic tegmentum; in the interfascicular area of brain stem and in the periventricular zones in nuclei IX-X pairs of cranial nerves and the spinal cord. Patterns of distribution GABA-ergic elements in the masu salmon brain is similar with the distribution of TH-ip structures. This similarity manifests itself in the presence of both phenotypically mature cell forms and undifferentiated elements with periventricular and subventricular localization and marking of neuromeric structure of the brain. This immunomorphology of GABA-ergic structures, discovered in the different age groups of masu salmon, may indicate that, like dopamine, GABA should be also considered as morphogenetic factor affecting of postembryonic brain development.GABA-ergic neurotransmission characterized by a high variability of synaptic responses. In mammals, hydrogen sulfide regulates the condition of GABA-receptor of different subtypes, localized pre-and postsinapticaly [38]. In adult masu salmon in different areas of the brain and spinal cord, containing large projection cells, namely the dorsal tegmental nuclei, medial reticular formation, reticulospinal cells, neurons in the ventral spinal column were installed joint localization of GABA, PA and CBS (Fig. 7). These large-cells structures in the fish brain participate in the organization of fast motor responses [39]. In medullary regions of the medial RF and VSC of the masu salmon, the level of colocalization of CBS, GABA, and PA is rather high. It is believed that the presence of PA promotes the formation of buffer calcium systems that provide generation of repeated action potentials in neurons with high-frequency discharges (Fig. 7). The high level of colocalization of PA, cytochrome oxidase, and 2-deoxyglucose also indicates that the PA content is typical of neuronal systems characterized by a high level of oxidative metabolism [40]. It was demonstrated that the concentration of intracellular calcium in neurons and glial cells upon the action of H2S reversibly increases (due to the release of calcium from intracellular stores and its entry into the cell through the plasma membrane) [41, 42]. Such adenylate cyclase-dependent mechanisms of activation can also be realized in the magnocellular CBS-and PA-ip populations of myelencephalic cells of the masu salmon brain identified in our experiments. As was found, inhibition of H2S synthesis results in a significant decrease in the level of intracellular calcium. This confirms the conclusion on the appreciable effect of H2S-dependent pathways on the time characteristics of processes related to calcium homeostasis in the neurons [41].
Significant heterogeneity of CBS-ip, GABA-ip,and PA-containing subpopulations of neurons in all regions of the masu brain is indicative of the fact that such units belong to different neurochemical and electrophysiological systems. The density of CBS-, PA-, and GABA-ip cells in the masu salmon is maximum and constant in the magnocellular caudal cerebral regions, namely in the regions of localization of the reticulo-spinal neurons, "high-frequency" Mauthner cells, and ventral spinal cord (VSC) neurons. Cells of these types in fishes are involved in the organization and control of rapid motor reactions [43]. Н2S-dependent regulation of calcium release with participation of PA can influence the parameters of impulse activity due to shortening of the refractory period in the corresponding neurons after generation of action potentials and, therefore, can provide the animal with certain behavioral evolutionary preference. Thus, in the population of large inhibitory neurons containing enhanced concentration of intracellular Ca2+, the excretion of GABA in our opinion can be arranged with the help of hydrogen sulphide.
Study of the relationship between NO and H2S-producing systems in the masu salmon brain revealed that they were separate, non-overlapping system of intra-and intercellular signaling. The study of the distribution of NADPH-d positive, nNOS-and CBS-ip elements in different areas of the masu salmon brain, and some features immunolabeling of cells and fibers indicate that NO and H2S-producing systems are independent neural complexes that perform specialized functions in the work of local neural networks.
In the dorsal region of the telencephalon in masu salmon NO is predominant gasotransmitter, the effects of which release by paracrinical way. In the ventral region of the telencephalon prevails system of hydrogen sulfide synthesis. In the ventral region of the telencephalon high activity CBS was revealed. Perhaps this system has synaptic localization, significant morphological heterogeneity of cells in the dorsal nucleus (Vd) and varicose cytosculpture of the afferents. Apparently, in the telencephalon of masu salmon way to release the gasotransmitters affect the nature of their neuromodulatory effects.
In the periventricular area of diencephalon and optic tectum masu salmon were populated by both CBS and nNOS and NADPH-d-producing cells. The presence of NO and H2S-producing elements in these areas indicates possible participation of hydrogen sulfide and nitric oxide in morphogenesis these compartments of a brain. In masu salmon brain has been identified CBS-ip fibers of varicose type that penetrate the layer of Purkinje cells. The presence of such fibers and CBS-ip endings in interganglionic plexus of corpus cerebelli, possibly reflecting the sinaptical method of release of H2S in this area of the masu salmon brain. The presence of NO-ergic cells and fibers was shown in the cerebellum on different species of fish by histochemical marking of NADPH-d [4, 44, 45]. Detection of nNOS in eurydendroid cells of masu salmon cerebellum confirms received our earlier data on histological labelling of NADPH-d in the neurons of this type of fish [46]. According to Ikenaga with co-authors [47], most of the eurydendroid cells in fish are aspartat-ergic and receive GABA-ergic impulses from the Purkinje cells. According to our data, the population of eurydendroid cells of masu salmon in cerebellum contains GABA-ergic and PA-ergic cells. Identified in the of masu salmon cerebellum thin nNOS-ip fibers, in our opinion, are the axons of eurydendroid neurons. Thus, nitric oxide, and being located in the projection eurydendroid cells, can acts as a modulator of aspartat-ergic signals in structure of efferent fibres to various parts of the masu salmon brain. Localization of nNOS, NADPH-d and CBS in interfascicular cells of masu salmon, by the classification [48], identified for the first time. We believe that interfascicular CBS-and nNOS-ip neurons of masu salmon are separate subpopulations of cells of the reticular formation, which modulating GABA-and cholinergic system in the medulla oblongata, respectively.
Secondary gustatory nucleus is seen as a visceral integrative centre in medulla oblongata in fishes brain [46]. In Carp in this nucleus was found the CBS immunolocalization, and in the masu salmon the secondary gustatory nucleus is CBS-immunonegative, but contains NADPH-d and nNOS. We believe that with the participation of H2S and NO-producing systems in the brain fish is carried out sensory modulation functions related to the evaluation of food in space and coordination of mechanosensor, visual and gustatory features. In Carp brain the main neurotransmitter of the gustatory system is hydrogen sulfide, and in the masu salmon brain is nitric oxide, which confirms the assumption about the use of fish of various signal transductor systems to transfer the neurochemical information in functionally similar complexes.
We have revealed the existence of NO and H2S-producing neurons in brainstem and isthmus regions of masu salmon brain. nNOS-ip and NADPH-d positive neurons were discovered in the composition of somato- and viscerosensoric (V, VII, IX-X) and visceromotoric (III, IV and VI) of craniocerebral nerves, octavo-lateral efferent complex, medial reticular formation. CBS in the medulla of masu salmon was detected in neurons of the nucleus X nerve, reticulospinal cells and ventro-lateral reticular formation. Distribution of NO and H2S-producing neurons in the nuclei of medulla oblongata of masu salmon indicates that NO is the predominant neuromodulator of somato-and viscerosensoric and visceromotoric systems of medulla oblongata, and H2S probably modulates viscerosensoric systems associated with the nucleus X nerve, and descending motor systems. NO and H2S-producing systems in the fishes brain: 1) are independent neural complexes which are carrying out specialized functions in the work of local neural networks; 2) represent separate, non-overlapping systems of intra-and intercellular signaling, modulating the activity of choline-, GABA-and catecholaminergic systems, respectively; 3) regulate the processes of adult neurogenesis in the matrix areas of the brain.
5. Gaseous mediators as a regulators of adult neurogenesis
Unlike mammals, the fish brain has a high neuronal plasticity and can produce new cells throughout life [49]. The results of our investigations indicate the existence of nNOS and NADPH-d in neurons and glial cells in the masu salmon brain. It is shown that NO plays the role of signaling agent, regulating the processes directed growth of axons and dendrites and migration of differentiating neurons [50]. It is established that in the subventricular zone of mammalian forebrain is surrounded by NO-producing neurons [51, 52]. Cells expressing nNOS were identified among progenitor cells of dentate girus in the hippocampus of Guinea pig [53]. These areas of the brain are considered zones adult neurogenesis in which the proliferation of the cells is maintained throughout the life of animals and human. The results of our investigation (Fig. 8A, B) suggest that in the periventricular area of the medulla oblongata in masu salmon containing PCNA-ip proliferating cells in different age periods, NO can act as a regulator of adult neurogenesis, which confirms the data obtained on mammals.
In the periventricular area of the medulla oblongata, ventral and lateral areas of the cerebellum of carp are considered matrix areas of the brain of this species [54], identified highly CBS-immunogenic cells, without any processes (Fig. 8C, D). The sizes of cells, their location in the brain and correlations with H2S-producing neurons indicate the presence of H2S-producing of glia in the matrix zone of carp brain. In similar areas of the masu salmon brain such cells were not found. As currently participation of gaseous mediators in the regulation of post-embryonal neurogenesis of mammals was shown [55], we believe that in carp brain H2S can act as such an agent, as the presence of CBS in proliferative areas of brain we consider as one of the proofs of this. One of the mechanisms regulating the in fish producing the large number of cells, educated including postembryonal period is apoptosis [7]. Study of a 60-day old sturgeon fry showed the presence of intensively proliferating zones containing PCNA-ip cells in forebrain. The active proliferation of cells in this period of the sturgeon′s development is complemented by the formation of secondary neurogenetic zones.
The 3-year olds sturgeons' zone proliferation and apoptosis in various parts of the intact CNS saved (Fig. 9A, B). The highest proliferative activity was detected in periventricular zone of medulla oblongata, that allows considering this area as a major area of adult neurogenesis (Fig. 14A, 15B). In the medial reticular formation, dorsal nuclei of the thalamus, the inner fibrous layer of tectum opticum and lateral hypothalamus were discovered maximum number of apoptotic elements. This circumstance allows us to suppose that these regions in the sturgeon brain correspond with the areas of postmitotic neuroblasts localization. In the sensory centers (tectum opticum and nuclei V, VII and X nerves were revealed variable ratio processes of proliferation and apoptosis (Fig. 10A), which indicates different rates of growth and differentiation of visual and chemosensory centers of the sturgeon brain. In contrast to mammals in which central divisions of sensory systems are completely formed and correspond strictly to the number of sensory receptors at the moment of birth and/or immediately after this event, sensory projections in the fish brain continue their growth and development during the entire life. Such a peculiarity of the fishes is related to the fact that the CNS organization must adapt to a significant permanent increase in the size of the body and, correspondingly, to a rise in the volume of incoming sensory information. Our studies of projections of the somato- and viscerosensory nuclei of the myelencephalon and tectum opticum of the sturgeon confirmed in general the hypothesis that adult neurogenesis and apoptosis exert significant effects on the peculiarities of postnatal development of the sensory systems. Our findings agree with the published data on intensification of differential growth in primary sensory regions in the lobe of the nucl. vagus of the carp, as well as in the Danio retina and tectum, compared with other cerebral regions [56].
Up to now, it remains unknown whether all types of neurons develop and are integrated into the corresponding networks of the growing brain of fishes. It seems probable that some initial level of organization of neuronal networks in fishes is already preformed at the moment of their hatching, and only some types of neurons continue their formation and integration into existing networks during the later period of life. It is believed that the weak ability for substitution or development of new neurons in the mammalian brain is related to the limited ability of such cells in animals of this class to be integrated into mature neuronal networks [58]. It is hypothesized that neurons formed de novo in adult animals are distinguished by a higher plasticity compared with that of preexisting cells [59, 60]. This viewpoint agrees well with our findings on the sturgeon and allows us to suppose that postembryonic (adult) neurogenesis correlates with coordinated growth of the sensory systems and sensory structures of the brain. Therefore, this phenomenon can open possibilities for the processing of new ontogenetic experience. Incorporation of new cells into the neuronal networks existing earlier in the sensory regions is directly related, first of all, to an increase in the size of the brain in the course of growth of the fish. However, it should be taken into account that fishes, immediately after hatching, possess relatively well preformed sensory and motor systems making possible rather rapid training for complex behavioral habits, e.g., active catching of food and avoidance of predators. This indicates that some parts of the CNS of fishes, which are responsible for information processing and realization of functional needs of the organism necessary within a certain life period, begin to function before hatching. The later postembryonic growth can be considered a process of delayed development related to the maintenance of the functions necessary in future, e.g., for the formation of zoosocial communication or sexual behavior. Therefore, our conclusion that some parts of the sturgeon brain remain, in fact, in the neotenic state over a rather long postembryonic period seems to be quite logical. This hypothesis explains the high indices of proliferative activity in some brain regions in cartilaginous ganoid fishes.
The particular relevance of the results obtained acquire the communications regulatory functions of nitric oxide and hydrogen sulfide, regarded as regulators of adult neurogenesis in the fish brain. We have highlighted nNOS-ip fiber varicose type in subventricular area of the spinal cord, as well as the presence of PCNA-and nNOS-ip cells in the composition of the periventricular area of diencephalon and medulla oblongata in sturgeon and salmon indicates the presence of NO-producing elements in zones containing proliferating cells. On the other hand, detection of NO-ergic activity in TUNEL positive areas of the brain sturgeon indicates the involvement of nitric oxide in the regulation of apoptosis. Thus, it is possible that in the brain sturgeon NO is as proapoptogenic and regulatory proliferation factor exercising to maintain a balance between the two processes. Cytotoxic and neuroprotective effects NO can be viewed as interrelated elements of a single action: if the excess output of NO potentiates the mechanisms of apoptosis in the zones of localization of postmitotic neuroblasts, the factors reducing NO production can be considered as compensatory. In the basis of post embryonic morphogenesis of sturgeon′s A. schrenckii brain, and particular, development of sensor systems are founded on certain ratio of the processes of proliferation and apoptosis, having NO-dependent mechanism of regulation.
6. Conclusion
Thus, we believe that the peculiarities of the distribution of the investigated systems synthesis of classic neurotransmitters (GABA, catecholamines), as well as gaseous mediators (NO and H2S) is directly linked to the ability of the brain fish grow throughout life. We interpret the obtained results in this context. This led us to the conclusion that some of the classic neurotransmitters (dopamine, GABA), as well as gaseous intermediaries (NO and H2S) are not only regulators of the functional activity of neurons and modulators of synaptic transmission in mature neural networks, but also are considered as inductors of development (morphogenetic factors) in the brain during postembryonic fish ontogenesis. Proof of this is a detection of the phenotypic immature elements in the masou salmon brain adult age, expressing the above mentioned molecules in proliferative areas of the brain, as well as elements that have the morphology of the radial glia. Presence of markers of gaseous intermediaries in the areas of expressing proliferative nuclear antigen (PCNA), proves the involvement of gaseous intermediaries in the regulation of post-embryonal neurogenesis. The fish with the prolonged cycle of development (salmon, and carp) such markers (NO and H2S) in periventricular proliferative areas of the brain may differ, which is consistent with the notion that in functionally similar complexes in animals can be used different signal transduction systems. Development of the nervous system salmon and sturgeon, in contrast to the widespread neurogenetic model D. rerio occurs over a long period of time. According to our data, the different structures of the CNS of masou salmon characterized by severe heterochrony, i.e. the cells of caudal parts of the brain in much earlier than neurons of forebrain departments, acquire the features of phenotypic specialization. We are convinced that the brains of these animals for a long time keeps features of fetal organization, and the presence of first and second years of life low differentiated phenotypically immature cell forms, confirms this hypothesis. The data presented in this study open a new trend in investigation of cellular mechanisms of shaping in structural organization in the postembryonic fishes brain and in examination of morpho-functional manifestations concerning histogenetic processes in different periods of postembrionic ontogenesis. The new priority data received are connected with development of nervous tissue in the pacific salmon brain and with dynamic of the brain shaping and distribution of classical neurotransmitters and gaseous mediators in a context of incessant postembryonic neurogenesis.
This work was supported by the Grant of Far Eastern Branch of Russian Academy of Sciences № 12-III-A-06-095.


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Chevrolet-branded cars have been sold in the majority of automotive markets worldwide, " In Oceania, Chevrolet is represented with its own Australian subsidiary, Holden, with returned into the region at 2018 after a 50-year absence with the launching of their Camaro and Silverado pickup truck. Back in 2005, Chevrolet has been relaunched in Europe, chiefly selling vehicles built by GM Daewoo of South Korea together with the tagline"Daewoo was grown enough to eventually become Chevrolet", " a move rooted in General Motors' attempt to construct a worldwide new around Chevrolet. With the reintroduction of all Chevrolet to Europe, GM planned Chevrolet to be always a mainstream value manufacturer, whereas GM's traditional European standard-bearers," Opel of Germany, and Vauxhall of uk would be transferred upmarket. But, GM reversed the move around in overdue 2013, announcing that the brand could be withdrawn from Europe, with the exception of their Camaro and Corvette at 2016. Chevrolet cars will continue to be marketed from the CIS states, for example Russia. After General Motors completely obtained GM Daewoo in 2011 to create GM Korea, the previous using the Daewoo automotive brand had been stopped at its own indigenous South Korea and succeeded by Chevrolet.
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In this day and age it's difficult to imagine how or why handicapped discrimination can occur with regard to using a travel scooter and traveling by air. However, last night my little family of four were DENIED ACCESS by Air France at LAX, simply because they didn't know what to do with our little travel scooter, or didn't like it. They did say if we would like to come back tomorrow to try again…
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Considering a home purchase in Fairfax Station? can help.
Tantalyzing Home For Sale in Northern Virginia
Welcome home to this 4 bedroom, 3 full, and 1 half bath Dutch Colonial on a full acre! Being nestled behind trees provides privacy, yet lot of natural light to shine through hardwood clad family room on to the breakfast area. kitchen and … [Read more]
4 beds, 3 full, 1 part baths
Home size: 2320
Added: 08/30/19, Last Updated: 09/01/2019
Property Type: Detached for Sale
MLS Number: VAFX1084916
Subdivision: Colchester Hills
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Nesbitt Realty is a mom-and-pop real estate shop! For all things real estate, call Julie at (703)765-0300.


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2019 ASEAN-Republic of Korea Commemorative Summit
The 2019 ASEAN-Republic of Korea Commemorative Summit will take place in Busan from November 25 – 26 to commemorate the 30th anniversary of the dialogue relationships between Korea and ASEAN countries. In conjunction with this meeting, the 1st Mekong-Republic of Korea Summit will also be held on Wednesday, November 27. This year's event is the first international summit gathering that Korea is hosting under the Moon Jae-in administration.
ASEAN stands for the Association of Southeast Asian Nations, the political and economic regional grouping founded in 1967 to jointly address regional issues. It consists of ten member countries: Malaysia, Singapore, Brunei Darussalam, Cambodia, Indonesia, Laos, Myanmar, Philippines, Thailand & Vietnam. The integration is underpinned by respect for diversity and a young population, which is expected to serve as the main driver of the region's further growth. Visit the official website to learn more about this international summit.
Korea hosted two special summits with ASEAN in Jeju and Busan in 2009 and 2014, respectively, and the third special summit will be held in Busan this year. This makes Korea the first nation to host all of the three special summits in its own country with ASEAN out of all the countries that have a dialogue relationship with the regional body, which is a testament to ASEAN's trust in and support of Korea's commitment to cooperation with the region.
The 1st Mekong-Republic of KOREA Summit will be the first summit-level meeting between Korea and the Mekong region, a result of continued cooperation which began in 2011 when a ministerial meeting took place between the two. The summit will serve as a foundation to further cooperation toward mutual growth with Mekong countries, which are some of the fastest growing regions of ASEAN.
KTO Media Fam Trip in Early November
In KTO recent media fam trip, we brought 3 pairs of Malaysian YouTubers (The Grim Film, 碰碰PongPong & Fu Love) and a Blogger (Placesandfoods.com) to visit BEXCO in Centum City, Haeundae-gu, Busan, which is the venue of ASEAN-ROK Commemorative Summit. They were very excited to be there where Malaysia leaders will be discussing and developing plans for youth empowerment, education, and employment with other ASEAN leaders. For more funny and heart-warming moments of the fam trip, stay tuned to their YouTube channels, Instagram and website.


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RUTH Duckworth will be hitting the streets to raise money for charity as she takes part in the London Marathon.
Ruth, aged 27, from Redditch, put her name down for the gruelling 26 mile race after her mother was diagnosed with breast cancer.
She will raise money for cancer reasearch and also guide dogs for the blind.
Ruth said: "I wasnt a runner at all, although I did actually swin for Bham, but three years ago my mom had cancer, so now I am devoted to raising money for breast cancer and guide dogs.
"I chose guide dogs purely for my best mate, he's my totally insane two-year-old yellow labrador. His brother and sister were picked for guide dogs as well as his aunties too. He was just a bit too enthusiastic!
"I got him while my mom was having treatment and he got me through. One of my team mates, Blind Dave, is running seven marathons in seven continents, and he's totally blind, and there's me going to struggle doing one. So to be on his team feels amazing.
"However my fiance's Grandad is blind also, and while he hasn't a guide dog, he copes fairly well, it just makes you realise how much you take little things for granted and are very lucky."
Ruth can be sponsored via her sponsor page at www.justgiving.com/ruthduckworth81 or by going to www.guidedogs.org


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My name is Ling
Contact me
Born Taiwanese by default and bred in South Africa.
Life has been a whirlwind of moving around in South Africa, this beautiful country is where I call home. I moved to Cape Town to pursue my studies in architecture at UCT, thus the start of my gastronomic adventures began. The perfect setting to explore creativity and passion for the simple things in life.
The aim of this blog is to share my experiences with other food lovers who may have a similar interest in food and travels.
I hope to share my adventures and hear about yours too.
Eatery Adventures is my diary snippets of my travel, events and home cooking adventures. The ultimate goal is to share my eatery palate of an Asian South African and explore global cultures through food.
Disclaimer for Eatery Adventures
If you require any more information or have any questions about our site's disclaimer, please feel free to contact us by email at email@example.com.
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All the information on this website is published in good faith and for general information purpose only. does not make any warranties about the completeness, reliability and accuracy of this information. Any action you take upon the information you find on this website (www.eateryadventures.co.za), is strictly at your own risk. will not be liable for any losses and/or damages in connection with the use of our website.
From our website, you can visit other websites by following hyperlinks to such external sites. While we strive to provide only quality links to useful and ethical websites, we have no control over the content and nature of these sites. These links to other websites do not imply a recommendation for all the content found on these sites. Site owners and content may change without notice and may occur before we have the opportunity to remove a link which may have gone 'bad'.
Please be also aware that when you leave our website, other sites may have different privacy policies and terms which are beyond our control. Please be sure to check the Privacy Policies of these sites as well as their "Terms of Service" before engaging in any business or uploading any information.
By using our website, you hereby consent to our disclaimer and agree to its terms.
Should we update, amend or make any changes to this document, those changes will be prominently posted here.


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Rita from Fairfield Age: 33. Need an adequate man, which will be fun and good in bed. Write, await the ))
It all started when I was years old, I had only the B. My little dog came at my feet. I have a little boty schitzu and hes sucks cock I put peanut butter on my dick and balls he loved but how can I get him to fuck me in the ass. I was moving to where I am today in Colorado, before we left, I was spending some nights att my Grandmothers house, and I took the dog outside with me, hid behind a shed, and I opened his mouth and let him suck on my dick. Anyone gay zoophyte know, that tongue of animals is much longer and they love to lick all. Hot teens playing with I have 2 dogs, a boston terrier, and a great perrinie a sheep dog, kinda.
Donna from Fairfield Age: 28. Bright, luxurious and always positive girl will brighten up your vacation. My skills will surprise you.
Dog Sucks Man Penis Videos
I am also into fucking k9 females. He was working my cock and balls nice and steadily until my cock was beginning to hurt a little bit. A young girl sucks the I have seen a vid where a golden sucking a guys penis. A Young Boy sucks the I visited my aunt in the country one summer when I was like 10 or 11, and her dog, a terrier, was always sniffing my crotch. I tried it a second time, but he forcefully bites my cock, and tries to bite my hands and feet.
Gina from Fairfield Age: 29. looking for a guy for regular and pleasant meetings.
Katie from Fairfield Age: 28. I would like to meet a reliable, business and not married man of my years. I'm charming and funny.
Yeeeeeaah, we got rid of em, we moved. Incest is hot on the right conditions billy Says. I had a pitbull lick my cock, my dad walked in and he took off his clotthes and shoved his huge dick into his mouth than he took it out and sucked my dick, i love to fuck my dad and my dog. I was a repressed teen in a really conservative family, so I thought this was a safe way to explore.
Marion from Fairfield Age: 35. For regular sex will meet with a guy.


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We reckon RTE must hold some sort of record for news and weather bloopers at this point.
Blooper King Aengus Mac Grianna may have left the news desk for good last November, but there's no shortage of broadcasters to keep his legacy alive.
This week's "I didn't know I was on air" gaffe comes courtesy of weather woman Siobhan Ryan, whose pre-broadcast prep was aired to the nation last night.
Ah shtop! ???? pic.twitter.com/ti6fsvOISf
— UnaN (@PlectraElectra) July 27, 2018
In the clip, the broadcaster can be heard telling someone off camera to "shtop" before she mutters to herself to "keep sentences short".
Reddit user Lady_Fifington shared the entire clip, and it's actually difficult to watch.
They may be unable, but we wouldn't change them for the world.
READ MORE: Una Healy Splits From Husband Ben Foden Amid 'Cheating Claims'


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The synod nominating committee will soon be meeting to assemble a slate of candidates for election at the May 1-3, 2020, synod assembly. There will be elections for synod council, disciple committee, consultation committee, and nominating committee.
The current synod nominating committee is requesting the names of people who the committee may consider for these positions. It is especially helpful to have names of laypersons and also persons of color or whose primary language is other than English. Please send suggestions with contact information along with a brief reason this person is being recommended to the nominating committee chair, Pastor Nate Liedtke at firstname.lastname@example.com.


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Professional Qualifications
BA (Hons) Degree Counselling- 2019
FDA Degree in Counselling-2018
Introduction to counselling - 2016
Transactional Analysis 101 - 2017
I believe YOU know what is best for YOU! Together we can discuss your goals during our sessions, what it is YOU want to use our time together for. You may need a space to talk and be heard without judgement. We can explore your feelings, thoughts and behaviour. We can look at patterns of your behaviour, explore how they assist you, are they still assisting you and if and how YOU would like to change them.
At all times I believe YOU are in charge of your life. I can support you and together achieve your goals for therapy.
Personal Approach
Transactional Analysis


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Riverside is an idyllic rural retreat tucked away in a sleepy hamlet on the Somerset Levels; built in Victorian times, two workers cottages have been sympathetically transformed into one big country cottage sleeping 12 guests in 6 bedrooms. There's a games room and a secure garden with a hot tub beneath a leafy arbour; Riverside is very well equipped and interiors are in keeping with the style and age of the property without being old-fashioned - it's all very stylish and the entire ambience is one of calm and space. With the riverside setting and far reaching views across the countryside, this is the perfect place to come and unwind for a family holiday, hen weekend, celebration, or corporate break.
The Somerset Levels is a unique landscape of wetland habitats rich in wildlife. Right on the doorstep are walks along the riverbank to welcoming country pubs; climb to the top of Burrow Mump, stroll to the Willows & Wetlands Visitor Centre just up the road, discover the delights of cider farms, charming villages and old churches out round the lanes. Have a mooch around one of the market towns nearby - Langport or Somerton, or for serious retail therapy go to Clarks' Village or to Taunton where there's something for everyone – a golf course, a bowling alley, multiplex cinema, and of course plenty of shops and places to eat.
Spend the day exploring the Levels and on into the Mendip Hills; visit Wells to see the Cathedral and the Bishop's Palace, take the kids to Wookey Hole or Cheddar Gorge; head over to the Jurassic Coast and have a spuddle about in the rockpools at Charmouth, or catch a fishing trip at Lyme Regis or Beer. Book now for a relaxing stay at Riverside.
- Sleeps 12 in 6 bedrooms
- Hot Tub and Games Room
- Play equipment
- Enclosed garden
- BBQ (charcoal 1 bag provided)
- SKY TV (movies package)
- Consideration required for neighbouring property
- 2 x Dogs by prior arrangement £50 Each
- Pool table and table football
- 6 shower/bathrooms (3 en suite)
- WiFi
- 55 minutes to South Coast
- Short breaks available
- Non smoking
- No Stag parties/Hen parties
- No fireworks, sky lanterns or drones
Ratings & Awards Accessibility
- Car Required
- Child Friendly
- Dogs Welcome
- Non Smoking
- Not Suitable for Wheelchairs
- Suitable for Elderly / Infirm
Child / Baby Facilities
- Cot Available
- High Chair
- Stair Gate
- Toddler Beds
- Toys & Games
Eco Features
- Eco Waste Management
- High Level of Insulation
- Low Energy Lighting
- Recycling Available
Entertainment Facilities
- Board Games
- Books
- DVD Player
- Internet Access
- Mobile Phone Service
- Pool Table
- Radio
- Satellite / Cable
- Snooker / Billiards
- Table Football
- Table Tennis
- WiFi
General Facilities
- Catering Available
- Central Heating
- Coffee Maker
- Cooking Utensils
- Dishwasher
- Dog-bed and Bowl
- Fireplace
- Freezer
- Fridge
- Grill
- Hair Dryer
- Iron & Board
- Linen Provided
- Local Maps
- Log Fire
- Microwave
- Oven
- Shower
- Toaster
- Washer/Dryer
- Washing Machine
- Woodburner
Leisure Facilities
- Hot Tub
- Pampering Available
Local Activities
- Climbing
- Cycling
- Festivals
- Fishing
- Golf
- Horse Riding
- Rafting
- Shopping
- Sightseeing
- Walking
Local Amenities
- Bike Hire
- Cafe
- Farm Shop
- Firewood
- Gallery
- Pub
- Restaurant
- Shops
- Theatre
Outdoor Facilities
- Balcony / Terrace
- Barbecue
- Climbing Frame
- Garden
- Outdoor Lighting
- Parking for several vehicles
- Parking Off Street
- Patio
- Patio Furniture
Type of Location
- Moors
- Peaceful
- Rural
- Scenic
- Village
- Click to view full details
Location: Riverside, Curload, Stoke St Gregory Taunton, Somerset, TA3 6JE
"We have stayed in many holiday cottages in the past and Riverside Cottage is the best by far. Everyone has enjoyed it so much. The children have had a fantastic time enjoying the games room and we have all enjoyed using the hot tub. The cottage has everything you could wish for to make it a home-from-home. An amazing holiday. Thank you."
Book a stay at Riverside from £1,200.00
More Houses in Somerset
Hurstone Sleeps 16
Walnut Arbour Sleeps 14
Pound Farm Sleeps 21


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Here at the King-Logan household, it's time for new phones, so my husband and I decided to shop online to see if we could get a better deal. Two smartphones at the same time adds up, you know what I mean? Naturally, we started our search at everyone's favorite online store, Amazon.
One thing we noticed as we clicked through dozens of options was the differences in the product descriptions. Some gave us a thorough description of the product, others gave us just the technical stats, and still others gave us almost nothing to go on.
Which options do you think we were most attracted to and considered most seriously? You guessed it! Of course, we were more interested in the products with better descriptions.
The experience made me appreciate more than ever how important it is for us artists to include meaningful descriptions of our artwork on our e-commerce websites, too. Yes, it's true, a picture is still worth a thousand words. But in the world of online shopping, buyers want to learn as much as they can about an item before they buy it. So, let's give people everything they need to feel confident in choosing what we have to offer.
Whether your art is hyper-realistic or completely nonrepresentational, you can learn to write powerful, attention-getting descriptions for your work. Here are several ideas to get you started:
1. Tap into emotion. I think artists sometimes shy away from talking about emotion in descriptions because they want viewers to have their own emotional responses to the work. The thing is, viewers will look at the work first and have that personal reaction no matter what. Then when they go to read about what kind of emotion you had in mind, they'll either feel affirmed because their response mirrored your intention or be intrigued by having a different response. Either way, it will add to the emotional impact for them, which is generally what motivates someone to buy a work of art.
2. Think about the senses. Buyers can already see the colors and shapes within your work, but is there something more you could provide in terms of the senses? This tip works best for representational artists who might include descriptions of the sounds, textures, temperatures, and other sensory aspects of the subject matter.
3. Discuss your inspiration. Art lovers never tire of learning more about the artistic thought process, so let them in on what you had in mind when you were creating the work. Why is the subject, style, or technique important to you? What inspired you? Why is this work meaningful to you? Was the act of creating transformative in some way? Viewers really do want to know.
4. Sprinkle in some high-powered adjectives. If you feel like your descriptions need a little more finesse, or if you feel like you're using the same words over and over again, think about injecting one or two interesting adjectives that will make your writing more lively. Or maybe I should say animated. Or maybe I could use scintillating. Anyway, if you're stumped for ideas, hop onto thesaurus.com and ask it for great suggestions.
5. Keep it short and basic. Having said all that, there isn't really any need for you to write long descriptions. Most people will only stay with you for about 100 to 200 words, so give them the relatively short sound bite they're looking for. While you're at it, be sure to include the most basic of all descriptions, including what type of art it is (painting, photograph, sculpture, etc.), what medium you used, and whether it's an original or a reproduction. In fact, the entire description can be written in very simple, plain language. Don't feel like you have to get artsy or poetic with your writing.
Still wondering if it's really worth all the effort? All I can say is, think about your own online shopping experiences. A great product description just may be the thing that clinches the deal.
What's worked for you in terms of writing artwork descriptions? Please share your ideas in the Comment box below. Let's connect!


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I am using a fresh install of Bitcoin and have no wallet with coins at this time, and want to use the Bitcoind service in short time (loading blockchain using bitcoind will take days because of slow pc, can not wait for that also because PC can't run all day). So I decided to download the blockchain from http://eu1.bitcoincharts.com/blockchain/.
But how do you use it? I am using Ubuntu 10.4 and Bitcoin 0.7.0. I already placed the downloaded files in .bitcoin directory and then run bitcoind but then it just started downloading the blockchain from the network, ignoring my downloaded files.
Thank you in advance.


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OU180140 - GS27 Accessoires
Warning: Last items in stock!
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Extreme sweetness
Machine washable
Clean your car like a professional with this Scratch Free Triple Action Wash Mitt GS27®. Its black part allows to rub gently, and carries the dirt without spreading it. Its honeycombed part makes it possible to demuse effectively without any risk of scratches and its gray micro-fiber surface makes it possible to overcome stubborn stains.
Use: Rinse your vehicle with clean water. Then dip your glove in a water seal diluted with a GS27® shampoo, and rub your bodywork. Start with the high parts of your vehicle to finish at the bottom. Do not forget to rinse the Scratch Free Triple Action Wash Mitt GS27® regularly with clean water.


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A young, skilled, 6.2 million strong workforce in Bogota ensures the scalability of any operation. The city's 133 institutions of higher education enroll over 872 thousand students and graduate over 184 thousand professionals and technicians per year. In addition, Colombia has one of the most flexible labor regimes in Latin America.
Source: National Ministry of Education, Labor Observatory for Education.*Invest in Bogota estimation for 2018 based on Ministry of Education, Labor Observatory for Education. The average growth of the last 5 years is used.
Among university students, Business Administration, Engineering, Social Sciences and Humans, are some of the most popular fields of study.
A Flexible Work Regime
According to the Global Competitiveness Report (2018) of the World Economic Forum, Colombia's Labor Market is one of the most efficient in Latin America.
Labor Market Efficiency* (2018)
Updated (mm/dd/yyyy): 12.19.2019


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Home Economics Disaggregated impacts of CAP reforms : proceedings of an OECD workshop.
Goals of the European Union sugar reform
The goals of the reform were as follows.
Major features of the reform
On 24 November 2005, EU Ministers of Agriculture reached agreement on the reform
of the sugar regime. The major features of the reform were as follows.
Table 8.1. Evolution of EU reference prices for sugar
The key objectives of the reform were to reduce sugar output by about 6 million tonnes. It was decided not to introduce compulsory quota cuts for EU manufacturers, but to implement a voluntary restructuring scheme. Under the system of voluntary quota renunciation, which was funded by a restructuring levy on production, the least efficient producers were encouraged to exit the industry early on, with the highest level of compensation (EUR 730/tonne) offered in the 2006/07 and 2007/08 seasons. The scheme came into operation on 1 January 2006; however, by the end of January 2007, the deadline for renunciations for 2007/08, less than 2.2 million tonnes had been renounced and the programme appeared to be in trouble. In September 2007, new elements to the restructuring scheme were agreed to by the EU Council of Ministers in order to improve its attractiveness. These included additional payments of EUR 237.50/tonne to farmers who gave up quotas, and fixing the share of the growers and machinery contractors to 10%. A waiving of the restructuring levy was introduced on withdrawal of sugar production during the 2007/08 season if renounced for the 2008/09 season, and a two- stage mechanism was announced for renunciation with producers allowed to submit volumes on 31 January and 31 March 2008.
This revised package substantially improved incentives for producers to renounce production quotas. After the modifications in the rules for quota renunciation were approved in 2009, 3.3 million tonnes of quota were given back for 2008/09 and 0.1 million tonnes for 2009/10. Aggregating the figures shows that there was a decrease of almost 5.217 million tonnes, white value, in the EU's supported sugar production since the sugar market reform came into effect in 2006. The Commission has practically reached its initial goal of reducing sugar production by 6 million tonnes.
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They are a person of curiosity. A random rearrangement of the letters in your name anagram will give Hbaanzej. Your profession was philosopher and thinker. They will make profit at the business fields. It may be true that they are a person of old fashioned.
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People with this letter are the person of logic. They like the stable things in their life.
J is for jahahzeb, that you always bring. They want to live the present to the fullest. They are loved by their cousins and siblings and in turn they also give abundance of love to them. Year Girl Name by Alphabet.
We'll save it, review it, and post it to help others. However, they will never allow themselves to walk on an unfair path, even if it is the only way that helps them to accomplish their plan. A pleasant outward appearance is mostly seen for these natives.
Jahanzeb – meaning | Baby Name Jahanzeb meaning and Astrology
They have to deal with their ego very often. These people are mostly interested in elevated aspects of their life. Record your pronunciation Recording. That is why they receive appreciation from other people. Boy Name by Alphabet. Recordings from children under 18 are not allowed.
Jahanzeb – Names Encyclopedia
Origin and Meaning of Jahanzeb. Ahmed Jahanzeb is a pop music and classical music singer-songwriter and musician. Those people whose name starts with the letter J are loved by many people.
What they lack is the power to remain stable at hazardous situations. He has had major accomplishments since An inherent leadership quality is found in these people. They are serious minded person. Sometimes in some cases it can be seen that their few wish never have any bound. Name by Birth Date. We noticed you have a microphone. A ajhanzeb for aware, of your surroundings N is for neighborly, friendly to all acquaintances Z is for zeal, your zest for life. A is for acts, the daily acts of kindness shown to others.
Try to become less adhered to material property and learn to have only as many, as you may give back. They need to be more determined at their goal and job.
Twitter Facebook Pinterest YouTube. They are a person of emotion. How do you pronounce that?
Statistics and meaning of name Jahanzeb
Ahmed is also known as The Wonder Boy. The natives with name Jahanzeb overtly conscious about their goals. And when time progresses a jqhanzeb attitude is seen in them.
Dashing in nature, these men and women love to have quality time with their loved ones, friends.


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Price: $14.99
Product ID : LBTP001
Weight: 1.00 lbs
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This elegant, all-over floral panel made from high-gauge lace is embellished with a delightful printed array of scrolling garden flowers and then gently crushed to create a classic, yet updated look. Brewster Lace tailored panels dress your windows with soft, colorful, light-filtering texture. Panels feature 1.5" rod pockets for trouble free installation, measure 50" wide, and are sold seperately. Machine wash, warm water, gentle cycle. Do not bleach. Line dry. Warm iron if necessary. 100& polyester.
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Profusion Publishers
Profusion is an independent British publishing house, based in London. The Profusion Crime Series publishes outstanding and popular crime writing and true crime stories from Eastern Europe.
London, UK