Rosa Álvarez-Otero

Calbindin and calretinin immunoreactivities identify different types of neurons in the adult lamprey spinal cord

The central pattern generator for locomotion in vertebrates is composed of different spinal neuronal populations that generate locomotor movement. In the lamprey spinal cord, several classes of interneurons have been identified based on morphologic and physiological criteria and integrated in the spinal cord circuits implicated in the generation of locomotion. However, the lack of histochemical markers for most of the interneurons makes it difficult to study whole populations along the spinal cord. We have investigated the immunoreactivity with antibodies raised against calbindin and calretinin. Several types of neurons could be classified: (1) strongly immunoreactive neurons located dorsomedially, (2) moderately immunoreactive neurons located laterally, (3) small weakly immunoreactive neurons, d) ventromedial neurons, (4) liquor contacting cells, and (5) motoneurons. The ventromedial group of calbindin‐immunoreactive neurons also is immunoreactive for serotonin and, therefore, represents the ventromedial group of dopamine/serotonin spinal neurons. Some of the lateral calbindin‐immunoreactive neurons may be CC‐type cells (cells with caudal‐crossed axons), because they are retrogradely labeled by tracer injections into the contralateral spinal cord. Other well‐characterized cell types, such as sensory dorsal cells, lateral interneurons, descending propriospinal edge cells, and spinobulbar giant interneurons are negative for both calbindin and calretinin. Therefore, calbindin and calretinin are useful markers for the study of cell populations that may be integrated in locomotor circuits. J. Comp. Neurol. 455:72–85, 2003.

Development and organization of the lamprey telencephalon with special reference to the GABAergic system.

Lampreys, together with hagfishes, represent the sister group of gnathostome vertebrates. There is an increasing interest for comparing the forebrain organization observed in lampreys and gnathostomes to shed light on vertebrate brain evolution. Within the prosencephalon, there is now a general agreement on the major subdivisions of the lamprey diencephalon; however, the organization of the telencephalon, and particularly its pallial subdivisions, is still a matter of controversy. In this study, recent progress on the development and organization of the lamprey telencephalon is reviewed, with particular emphasis on the GABA immunoreactive cell populations trying to understand their putative origin. First, we describe some early general cytoarchitectonic events by searching the classical literature as well as our collection of embryonic and prolarval series of hematoxylin-stained sections. Then, we comment on the cell proliferation activity throughout the larval period, followed by a detailed description of the early events on the development of the telencephalic GABAergic system. In this context, lampreys apparently do not possess the same molecularly distinct subdivisions of the gnathostome basal telencephalon because of the absence of a Nkx2.1-expressing domain in the developing subpallium; a fact that has been related to the absence of a medial ganglionic eminence as well as of its derived nucleus in gnathostomes, the pallidum. Therefore, these data raise interesting questions such as whether or not a different mechanism to specify telencephalic GABAergic neurons exists in lampreys or what are their migration pathways. Finally, we summarize the organization of the adult lamprey telencephalon by analyzing the main proposed conceptions, including the available data on the expression pattern of some developmental regulatory genes which are of importance for building its adult shape.

GABAergic neuronal circuits in the cerebellum of the dogfish Scyliorhinus canicula (Elasmobranchs) : an immunocytochemical study

The presence of GABAergic cells and fibres in the corpus cerebelli and auricles of a dogfish, Scyliorhinus canicula, was studied with immunocytochemistry. In both locations, two types of GABA-immunoreactive (GABA-ir) neurons were observed: stellate cells in the molecular layer and Golgi cells in the granular layer. Stellate cell axons gave rise to numerous GABA-ir boutons distributed throughout the molecular layer and a smaller number of boutons which contacted Purkinje cell perikarya. No GABA-ir baskets around Purkinje cells were observed. Golgi cells of the granular layer gave rise to numerous GABA-ir boutons which were located around cerebellar glomeruli. Purkinje cell perikarya and their axon terminals in the cerebellar nucleus were not GABA-immunoreactive. These findings are discussed in terms of the phylogeny of cerebellar circuits.

Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter

Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35Sor with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.

Organisation of the cerebellar nucleus of the dogfish, Scyliorhinus canicula L.: A light microscopic, immunocytochemical, and ultrastructural study

Elasmobranchs possess a well‐developed cerebellum with an associated cerebellar nucleus. To determine whether the organization of this nucleus is comparable with that of the deep cerebellar nuclei of mammals, we studied the dogfish cerebellar nucleus with light microscopic methods (Nissl stain, Golgi method, reduced silver stain, NADPH‐diaphorase histochemistry and immunocytochemistry) and with electron microscopy. We found the dogfish cerebellar nucleus to consist of about 1,050 large neurons, the ratio of Purkinje cells to cerebellar nucleus neurons being about 17:1. Immunocytochemistry showed large glutamatergic neurons in the main portions of the nucleus and small glutamate‐ and/or α‐aminobutyric acid (GABA)‐immunoreactive cells in the subventricular region of the nucleus. Large glutamatergic neurons corresponded to bipolar or triangular cells revealed by Golgi methods. Application of horseradish peroxidase to the cerebellar cortex produced the labelling of beaded fibres of Purkinje cells in the cerebellar nucleus. Unlike in mammals, GABAergic innervation of the cerebellar nucleus was scarce: Purkinje cell axon terminals in the cerebellar nucleus did not appear to be GABA‐immunoreactive, most GABAergic fibres being found in the subventricular neuropile. Some fibres immunoreactive to serotonin and somatostatin were also observed in the subventricular neuropile of the cerebellar nucleus. Three neuron types were distinguished with electron microscopy (types A to C). Type A cells were abundant and smooth‐surfaced, and appeared to correspond to Golgi‐impregnated neurons and large glutamate‐immunoreactive cells. Type B neurons were scarce and possessed dendrites covered by sessile or stalked spines. Type C neurons were small cells located mainly in the medialmost region of the nucleus and corresponded to subventricular glutamate‐ and GABA‐immunoreactive cells. Six types of synaptic bouton were observed (types I to VI). The most abundant (type I boutons) made symmetrical contacts and appeared to correspond to Purkinje cell axons. Type I boutons were the only type observed on perikarya and initial axon segments of type A cells. Type IV and type V boutons made complex glomerular‐like asymmetrical contacts with spines of type B cells. Type VI boutons appeared to correspond to peptidergic and/or monoaminergic axons. The functional significance of these results is discussed.

Characterization of melatonin synthesis in the gastrointestinal tract of rainbow trout (Oncorhynchus mykiss): distribution, relation with serotonin, daily rhythms and photoperiod regulation.

Melatonin is synthesized in peripheral locations of vertebrates, including the gastrointestinal tract (GIT). In teleost, information regarding this topic is scarce. Here we studied the presence and synthesis of melatonin at the rainbow trout GIT. Different sections of trout GIT (from esophagus to hindgut) were dissected out and assayed for contents of melatonin, serotonin (5-HT) and its metabolite, 5-hydroxyindole acetic acid, as well as for aanat1, aanat2 and hiomt mRNA abundance. A trout group was pinealectomized to evaluate changes in plasma and gut melatonin content. Finally, the daily profile of melatonin and 5-HT content, and aanat1, aanat2 and hiomt mRNA abundance were analyzed in gut of trout kept under normal lighting, and then under constant darkness. Melatonin was detected in all GIT regions with higher concentrations in the muscular wall than in the mucosa, a similar trend to that of 5-HT. In contrast, transcripts of melatonin synthesis enzymes were more abundant in the mucosa. Pinealectomy did not affect melatonin levels in midgut and hindgut either at day or at night. Additionally, no daily rhythms could be defined for melatonin content in gut tissues but increases during late light phase and at midnight occurred. However, aanat1, aanat2 and hiomt mRNA abundance showed clear daily rhythms with peaks at night. These rhythms remained with a 3-h phase advanced peak in fish exposed to constant darkness. Our results provide clear evidence for a local synthesis of melatonin in trout GIT that might be influenced by the content of 5-HT in the tissue. The process is affected by environmental light cycle and is likely to be under circadian regulation.

Changes in the levels and phosphorylation status of Akt, AMPK, CREB, and FoxO1 in hypothalamus of rainbow trout under conditions of enhanced glucosensing activity

ABSTRACT There is no available information about mechanisms linking glucosensing activation in fish and changes in the expression of brain neuropeptides controlling food intake. Therefore, we assessed in rainbow trout hypothalamus the effects of raised levels of glucose on the levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking these processes. We also aimed to assess the changes in the levels and phosphorylation status of two proteins possibly involved in the modulation of these transcription factors: Akt and AMPK. Therefore, in pooled preparations of hypothalamus incubated for 3 and 6 h in the presence of 2, 4 or 8 mmol l−1 d-glucose, we evaluated the response of parameters related to glucosensing mechanisms, neuropeptide expression and levels and phosphorylation status of the proteins of interest. The activation of hypothalamic glucosensing systems and the concomitant enhanced anorectic potential occurred in parallel with activation of Akt and inhibition of AMPK. The changes in these proteins relate to neuropeptide expression through changes in the level and phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses to increased glucose. Summary: The activation of glucosensing systems in rainbow trout hypothalamus leading to changes in the regulation of food intake occurs in parallel with changes in the abundance and phosphorylation status of Akt, AMPK, CREB and FoxO1.

A tract-tracing study of the central projections of the mesencephalic nucleus of the trigeminus in the guppy (Lebistes reticulatus, teleostei), with some observations on the descending trigeminal tract

We studied the central projections of the mesencephalic nucleus of the trigeminal nerve (MesV) in the guppy (Lebistes reticulatus), after application of horseradish peroxidase or fluorescein dextran amine into the eye orbit. A small number (1 to 13) of large mesencephalic trigeminal neurons were solid labeled in the ipsilateral rostral mesencephalon. At the level of the trigeminal nerve entrance, the united process of each mesencephalic trigeminal cell bifurcates, giving rise to a peripheral branch that exits in the trigeminal nerve and a descending branch that runs caudally in a medial bundle separated from the descending trigeminal tract. This bundle passes close to the visceromotor nuclei of the medulla oblongata. Descending processes give rise to short collaterals to the descending nucleus of the trigeminus and the ventrolateral reticular area. Most MesV descending fibres terminate in this ventrolateral field at the transition of the medulla to the spinal cord, but one or two fibres could be followed to the C6 level, where they give rise to collaterals to the dorsal funicular nucleus. No collaterals directed to the trigeminal motor nucleus, the cerebellum, or the mesencephalic tegmentum were observed. These projections were also compared with those of the descending trigeminal tract.

Glucosensing in liver and Brockmann bodies of rainbow trout through glucokinase-independent mechanisms☆

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) are present in the liver and Brockmann bodies (BB) of rainbow trout, and are affected by stress. We evaluated in these tissues changes in parameters related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and SGLT-1 6h after intraperitoneal injection of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment), or d-glucose (hyperglycaemic treatment). Half of tanks were kept at normal stocking density (NSD; 10kgfishmass·m(-3)) whereas the remaining tanks were kept at high stocking density (HSD; 70kgfishmass·m(-3)). The results provide for the first time in fish evidence for the presence of putative glucosensor systems based on mitochondrial activity and sweet taste receptor in liver whereas in BB systems based on LXR, mitochondrial activity, sweet taste receptor, and SGLT-1 could be operative. We also obtained for the first time in fish evidence for the functioning of integrative metabolic sensors in response to changes in nutrient levels since changes in the mRNA abundance of sirtuin 1 (SIRT-1) were observed in response to increased glucose levels. The stress conditions elicited by HSD altered the response of the glucosensor systems based on mitochondrial activity, sweet taste receptor, and SGLT-1 in the liver, and LXR and SGLT-1 in the BB.

An immunocytochemical and ultrastructural study of a specialized glial region of the medulla oblongata of the adult and juvenile grey mullet

Electron microscopy together with glial fibrillary acidic protein (GFAP) and vimentin immunocytochemistry reveals the presence of a specialized glial region in the octavolateral area of the medulla oblongata of adult and juvenile grey mullets, Chelon labrosus. Glial cells, which can he characterized as ependymal and subependymal cells, originate in the walls of the lateral recess of the fourth ventricle. They contain numerous gliofilaments and are connected through frequent gap junctions. Electron microscopy also reveals the lack of nerve cell parikarya and processes in this region. Immunocytochemistry reveals that the glial cells are strongly GFAP-positive in both adults and juveniles, whereas vimentin is only detected in this region in juveniles. The meninges associated with this glial region contains connective fibres, formed of very thick collagen fibres, that arc metachromatic and PAS-positive under light microscopy. These findings strongly support a structural role for this medullary specialization. Differences between adults and juveniles in the distribution of GFAP- and vimentin-immunoreactive structures in other regions of the medulla oblongata are also reported.