Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

Proteomic and Bioinformatic Tools to Identify Potential Hub Proteins in the Audiogenic Seizure-Prone Hamster GASH/Sal.

The GASH/Sal (Genetic Audiogenic Seizure Hamster, Salamanca) is a model of audiogenic seizures with the epileptogenic focus localized in the inferior colliculus (IC). The sound-induced seizures exhibit a short latency (7-9 s), which implies innate protein disturbances in the IC as a basis for seizure susceptibility and generation. Here, we aim to study the protein profile in the GASH/Sal IC in comparison to controls. Protein samples from the IC were processed for enzymatic digestion and then analyzed by mass spectrometry in Data-Independent Acquisition mode. After identifying the proteins using the UniProt database, we selected those with differential expression and performed ontological analyses, as well as gene-protein interaction studies using bioinformatics tools. We identified 5254 proteins; among them, 184 were differentially expressed proteins (DEPs), with 126 upregulated and 58 downregulated proteins, and 10 of the DEPs directly related to epilepsy. Moreover, 12 and 7 proteins were uniquely found in the GASH/Sal or the control. The results indicated a protein profile alteration in the epileptogenic nucleus that might underlie the inborn occurring audiogenic seizures in the GASH/Sal model. In summary, this study supports the use of bioinformatics methods in proteomics to delve into the relationship between molecular-level protein mechanisms and the pathobiology of rodent models of audiogenic seizures.

Delving into the significance of the His289Tyr single-nucleotide polymorphism in the glutamate ionotropic receptor kainate-1 (Grik1) gene of a genetically audiogenic seizure model.

Genetic abnormalities affecting glutamate receptors are central to excitatory overload-driven neuronal mechanisms that culminate in seizures, making them pivotal targets in epilepsy research. Increasingly used to advance this field, the genetically audiogenic seizure hamster from Salamanca (GASH/Sal) exhibits generalized seizures triggered by high-intensity acoustic stimulation and harbors significant genetic variants recently identified through whole-exome sequencing. Here, we addressed the influence of the missense single-nucleotide polymorphism (C9586732T, p.His289Tyr) in the glutamate receptor ionotropic kainate-1 (Grik1) gene and its implications for the GASH/Sal seizure susceptibility. Using a protein 3D structure prediction, we showed a potential effect of this sequence variation, located in the amino-terminal domain, on the stability and/or conformation of the kainate receptor subunit-1 protein (GluK1). We further employed a multi-technique approach, encompassing gene expression analysis (RT-qPCR), Western blotting, and immunohistochemistry in bright-field and confocal fluorescence microscopy, to investigate critical seizure-associated brain regions in GASH/Sal animals under seizure-free conditions compared to matched wild-type controls. We detected disruptions in the transcriptional profile of the Grik1 gene within the audiogenic seizure-associated neuronal network. Alterations in GluK1 protein levels were also observed in various brain structures, accompanied by an unexpected lower molecular weight band in the inferior and superior colliculi. This correlated with substantial disparities in GluK1-immunolabeling distribution across multiple brain regions, including the cerebellum, hippocampus, subdivisions of the inferior and superior colliculi, and the prefrontal cortex. Notably, the diffuse immunolabeling accumulated within perikarya, axonal fibers and terminals, exhibiting a prominent concentration in proximity to the cell nucleus. This suggests potential disturbances in the GluK1-trafficking mechanism, which could subsequently affect glutamate synaptic transmission. Overall, our study sheds light on the genetic underpinnings of seizures and underscores the importance of investigating the molecular mechanisms behind synaptic dysfunction in epileptic neural networks, laying a crucial foundation for future research and therapeutic strategies targeting GluK1-containing kainate receptors.

Molecular tools for the characterization of seizure susceptibility in genetic rodent models of epilepsy.

Epilepsy is a chronic neurological disorder characterized by abnormal neuronal activity that arises from imbalances between excitatory and inhibitory synapses, which are highly correlated to functional and structural changes in specific brain regions. The difference between the normal and the epileptic brain may harbor genetic alterations, gene expression changes, and/or protein alterations in the epileptogenic nucleus. It is becoming increasingly clear that such differences contribute to the development of distinct epilepsy phenotypes. The current major challenges in epilepsy research include understanding the disease progression and clarifying epilepsy classifications by searching for novel molecular biomarkers. Thus, the application of molecular techniques to carry out comprehensive studies at deoxyribonucleic acid, messenger ribonucleic acid, and protein levels is of utmost importance to elucidate molecular dysregulations in the epileptic brain. The present review focused on the great diversity of technical approaches available and new research methodology, which are already being used to study molecular alterations underlying epilepsy. We have grouped the different techniques according to each step in the flow of information from DNA to RNA to proteins, and illustrated with specific examples in animal models of epilepsy, some of which are our own. Separately and collectively, the genomic and proteomic techniques, each with its own strengths and limitations, provide valuable information on molecular mechanisms underlying seizure susceptibility and regulation of neuronal excitability. Determining the molecular differences between genetic rodent models of epilepsy and their wild-type counterparts might be a key in determining mechanisms of seizure susceptibility and epileptogenesis as well as the discovery and development of novel antiepileptic agents. This article is part of the Special Issue "NEWroscience 2018".

Overexpression of the immediate-early genes Egr1, Egr2, and Egr3 in two strains of rodents susceptible to audiogenic seizures.

Genetic animal models of epilepsy are an important tool for further understanding the basic cellular mechanisms underlying epileptogenesis and for developing novel antiepileptic drugs. We conducted a comparative study of gene expression in the inferior colliculus, a nucleus that triggers audiogenic seizures, using two animal models, the Wistar audiogenic rat (WAR) and the genetic audiogenic seizure hamster (GASH:Sal). For this purpose, both models were exposed to high intensity auditory stimulation, and 60min later, the inferior colliculi were collected. As controls, intact Wistar rats and Syrian hamsters were subjected to stimulation and tissue preparation protocols identical to those performed on the experimental animals. Ribonucleic acid was isolated, and microarray analysis comparing the stimulated Wistar and WAR rats showed that the genomic profile of these animals displayed significant (fold change, |FC|≥2.0 and p<0.05) upregulation of 38 genes and downregulation of 47 genes. Comparison of gene expression profiles between stimulated control hamsters and stimulated GASH:Sal revealed the upregulation of 10 genes and the downregulation of 5 genes. Among the common genes that were altered in both models, we identified the zinc finger immediate-early growth response gene Egr3. The Egr3 protein is a transcription factor that is induced by distinct stress-elicited factors. Based on immunohistochemistry, this protein was expressed in the cochlear nucleus complex, the inferior colliculus, and the hippocampus of both animal models as well as in lymphoma tumors of the GASH:Sal. Our results support that the overexpression of the Egr3 gene in both models might contribute to neuronal viability and development of lymphoma in response to stress associated with audiogenic seizures. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

The noradrenergic projection from the locus coeruleus to the cochlear root neurons in rats.

The cochlear root neurons (CRNs) are key components of the primary acoustic startle circuit; mediating auditory alert and escape behaviors in rats. They receive a great variety of inputs which serve to elicit and modulate the acoustic startle reflex (ASR). Recently, our group has suggested that CRNs receive inputs from the locus coeruleus (LC), a noradrenergic nucleus which participates in attention and alertness. Here, we map the efferent projection patterns of LC neurons and confirm the existence of the LC-CRN projection using both anterograde and retrograde tract tracers. Our results show that each LC projects to the CRNs of both sides with a clear ipsilateral predominance. The LC axons terminate as small endings distributed preferentially on the cell body and primary dendrites of CRNs. Using light and confocal microscopy, we show a strong immunoreactivity for tyrosine hydroxylase and dopamine ß-hydroxylase in these terminals, indicating noradrenaline release. We further studied the noradrenergic system using gene expression analysis (RT-qPCR) and immunohistochemistry to detect specific noradrenergic receptor subunits in the cochlear nerve root. Our results indicate that CRNs contain a noradrenergic receptor profile sufficient to modulate the ASR, and also show important gender-specific differences in their gene expression. 3D reconstruction analysis confirms the presence of sexual dimorphism in the density and distribution of LC neurons. Our study describes a coerulean noradrenergic projection to the CRNs that might contribute to neural processes underlying sensory gating of the ASR, and also provides an explanation for the gender differences observed in the behavioral paradigm.

Whole-genome expression profile in zebrafish embryos after chronic exposure to morphine: identification of new genes associated with neuronal function and mu opioid receptor expression.

BACKGROUND: A great number of studies have investigated changes induced by morphine exposure in gene expression using several experimental models. In this study, we examined gene expression changes during chronic exposure to morphine during maturation and differentiation of zebrafish CNS. RESULTS: Microarray analysis showed 254 genes whose expression was identified as different by at least 1.3 fold change following chronic morphine exposure as compared to controls. Of these, several novel genes (grb2, copb2, otpb, magi1b, grik-l, bnip4 and sox19b) have been detected for the first time in an experimental animal model treated with morphine. We have also identified a subset of genes (dao.1, wls, bnip4 and camk1γb) differentially expressed by chronic morphine exposure whose expression is related to mu opioid receptor gene expression. Altered expression of copb2, bnip4, sox19b, otpb, dao.1, grik-l and wls is indicative of modified neuronal development, CNS patterning processes, differentiation and dopaminergic neurotransmission, serotonergic signaling pathway, and glutamatergic neurotransmission. The deregulation of camk1γb signaling genes suggests an activation of axonogenesis and dendritogenesis. CONCLUSIONS: Our study identified different functional classes of genes and individual candidates involved in the mechanisms underlying susceptibility to morphine actions related to CNS development. These results open new lines to study the treatment of pain and the molecular mechanisms involved in addiction. We also found a set of zebrafish-specific morphine-induced genes, which may be putative targets in human models for addiction and pain processes.

Chronic administration of risperidone in a rat model of schizophrenia: a behavioural, morphological and molecular study.

In the present work we analyzed the effect of the chronic administration of risperidone (2mg/kg over 65 days) on behavioural, morphological and molecular aspects in an experimental model of schizophrenia obtained by bilateral injection of ibotenic acid into the ventral hippocampus of new-born rats. Our results show that during their adult lives the animals with hippocampal lesions exhibit different alterations, mainly at behavioural level and in the gene expression of dopamine D(2) and 5-HT(2A) receptors. However, at morphological level the study performed on the prefrontal cortex did not reveal any alterations in either the thickness or the number of cells immunoreactive for c-Fos, GFAP, CBP or PV. Overall, risperidone administration elicited a trend towards the recovery of the values previously altered by the hippocampal lesion, approaching the values seen in the animals without lesions. It may be concluded that the administration of risperidone in the schizophrenia model employed helps to improve the altered functions, with no significant negative effects.

Pax2+ astrocytes in the fish optic nerve head after optic nerve crush.

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2(+) astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2(+) astrocytes in this region. These Pax2(+) astrocytes show a higher number of Cytokeratin (Ck)(+)/GFAP(+) processes compared with control animals. In contrast, a different S100(+) astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2(+)/PCNA(+) that is activated after the injury. We conclude that Pax2(+) astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2(+) astrocytes are probably directly involved in the process of axonal regeneration.

Cortical Auditory Deafferentation Induces Long-Term Plasticity in the Inferior Colliculus of Adult Rats: Microarray and qPCR Analysis.

The cortico-collicular pathway is a bilateral excitatory projection from the cortex to the inferior colliculus (IC). It is asymmetric and predominantly ipsilateral. Using microarrays and RT-qPCR we analyzed changes in gene expression in the IC after unilateral lesions of the auditory cortex, comparing the ICs ipsi- and contralateral to the lesioned side. At 15 days after surgery there were mainly changes in gene expression in the IC ipsilateral to the lesion. Regulation primarily involved inflammatory cascade genes, suggesting a direct effect of degeneration rather than a neuronal plastic reorganization. Ninety days after the cortical lesion the ipsilateral IC showed a significant up-regulation of genes involved in apoptosis and axonal regeneration combined with a down-regulation of genes involved in neurotransmission, synaptic growth, and gap junction assembly. In contrast, the contralateral IC at 90 days post-lesion showed an up-regulation in genes primarily related to neurotransmission, cell proliferation, and synaptic growth. There was also a down-regulation in autophagy and neuroprotection genes. These findings suggest that the reorganization in the IC after descending pathway deafferentation is a long-term process involving extensive changes in gene expression regulation. Regulated genes are involved in many different neuronal functions, and the number and gene rearrangement profile seems to depend on the density of loss of the auditory cortical inputs.

Improved limit on direct α decay of the Hoyle state.

The current evaluation of the triple-α reaction rate assumes that the α decay of the 7.65 MeV, 0+ state in 12C, commonly known as the Hoyle state, proceeds sequentially via the ground state of 8Be. This assumption is challenged by the recent identification of two direct α-decay branches with a combined branching ratio of 17(5)%. If correct, this would imply a corresponding reduction in the triple-α reaction rate with important astrophysical consequences. We have used the 11B(3He,d) reaction to populate the Hoyle state and measured the decay to three α particles in complete kinematics. We find no evidence for direct α-decay branches, and hence our data do not support a revision of the triple-α reaction rate. We obtain an upper limit of 5×10(-3) on the direct α decay of the Hoyle state at 95% C.L., which is 1 order of magnitude better than a previous upper limit.

Characterization of Pax2 expression in the goldfish optic nerve head during retina regeneration.

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2(+) astrocytes located in the ONH and S100(+) astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2(+) and S100(+) astrocytes have different responses. At 7 days after injury the number of Pax2(+) cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100(+) astrocytes in the retina surrounding the ONH and S100(+) processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100(+) astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2(+) cells increase. At the same time, S100(+)/GFAP(+)/GS(+) astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2(+)/PCNA(+) astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2(+) astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.

Effects on soil organic matter mineralization and microbiological properties of applying compost to burned and unburned soils.

This study was undertaken in the context of a project of reclamation of a burned forest area applying municipal waste compost (MWC) and it consisted of an incubation experience carried out under laboratory conditions. The objectives of this research were to asses the effect of three doses of MWC added to burned and unburned calcareous soils on a) SOM mineralization and b) soil microbiological parameters. The laboratory incubation experience was carried out with three compost doses (1, 2 and 4% w/w) on a burned soil and another unburned one from an adjacent plot, besides the corresponding control samples. The mineralization kinetics of the organic matter was studied for 92 days. The kinetics data were adjusted to a double exponential model, showing two C pools of different degrees of resistance to mineralization and concentration, with half-life times of 1.9-4.9 and 34-76 days, respectively. In the unburned soil, the initial potential mineralization rate of the labile and stable C pools showed an opposed behavior, increased and decreased with the MWC dose, respectively. However in the burned soil no significant tendencies were observed. Although applying compost tended to increase the size of more labile pool with respect to total mineralizable C, however most of the soil or compost OM did not result mineralizable in the short and medium term. The compost amendment did not increase soil microbial activity.

Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).

The nociceptin receptor (NOP) and its endogenous ligand, nociceptin/orphanin FQ (OFQ), are involved in a wide range of biological functions, such as pain, anxiety, learning, and memory. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs of abuse and to discover new pharmacological targets. We report the cloning, expression, and pharmacological characterization of a NOP receptor from zebrafish (drNOP). The full-length cDNA codes a protein of 363 residues, which shows high sequence similarity to other NOPs. Phylogenetic analysis indicates that NOPs are broadly conserved during vertebrate evolution, and that they stand for the most divergent clade of the opioid/OFQ receptor family. Expression studies have revealed that drNOP mRNA is highly expressed in the central nervous system, and low expression levels are also found in peripheral tissues such as gills, muscle, and liver. Pharmacological analysis indicates that drNOP displays specific and saturable binding for [Leucyl-3,4,5-(3)H]nociceptin, with a K(d)=0.20 ± 0.02 nM and a B(max)=1703 ± 81 fmol/mg protein. [(3)H]Nociceptin binding is displaced by several opioid ligands such as dynorphin A (DYN A), naloxone, bremazocine, or the κ-selective antagonist nor-binaltorphimine. [(35)S]GTPγS stimulation studies showed that drNOP receptor is functional, as nociceptin is able to fully activate the receptor and DYN A behaves as a partial agonist (50% stimulation). Our results indicate that drNOP receptor displays mixed characteristics of both NOP and κ opioid receptors. Hence, drNOP, which has retained more of the likely ancestral features, bridges the gap between nociceptin and opiate pharmacology.

El proceso de absorción del Real Tribunal del Protoalbeitarato por la Escuela de Veterinaria de Madrid (1792-1855) / The takeover process of the Real Tribunal del Protoalbeitarato by the Faculty of Veterinary of Madrid (1792-1855)

La absorción del Real Tribunal del Protoalbeitarato1 por la Escuela de Veterinaria de Madrid presenta ciertas singularidades respecto al proceso equivalente en medicina y farmacia. La pugna y hegemonía del Real Tribunal durante casi sesenta años desde la creación de la Escuela de Veterinaria (1792), ha sido estudiada por la historiografía tradicional con una visión centrada fundamentalmente en aspectos veterinarios. En el presente artículo se investigan en detalle los procesos conducentes a la absorción y se pone de manifiesto la relevancia de las motivaciones políticas y su predominio sobre las de carácter técnico, sanitario o profesional. Por ello, se analiza el juego de instituciones y de agentes externos a la veterinaria implicados, en especial destacamos el papel desempeñado por el XV Marqués de Cerralbo (AU)

The absorption of the Real Tribunal del Protoalbeitarato by the Veterinary Science School of Madrid shows several singularities in relation to the equivalent process in medicine and pharmacy.The conflict and the hegemony of the Real Tribunal during almost sixty years from the creation of the Veterinary Science School (1792), has been studied by the traditional historiography with a view fundamentally focused on veterinary aspects. In this article, the process of absorption is investigated in full detail. Also, the relevance of political motivations and their predominance over technical, sanitary or professional motivations are shown. So, the game of institutions and agents which are external to the veterinary science are analysed; specially, the out standing role performed by the XV Marquis of Cerralbo(AU)

Characterisation and differential expression during development of a duplicate Disabled-1 (Dab1) gene from zebrafish.

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five different isoforms. Both drDab1a and drDab1b show differential gene expression levels in distinct adult tissues and during development. drDab1b is expressed in peripheral tissues (gills, heart, intestine, muscle), the immune system (blood, liver) and the central nervous system (CNS), whereas drDab1a is only expressed in gills, muscle and the CNS, suggesting a division of functions for two Dab1 genes in zebrafish adult tissues. RT-PCR analysis also reveals that both drDab1 genes show distinct developmental-specific expression patterns throughout development. drDab1b expression was higher than that of drDab1a, suggesting a major role of drDab1b in comparison with drDab1a during development and in different adult tissues. In addition, new putative Dab1 (a and/or b) from different teleost species were identified in silico and predicted protein products are compared with the previously characterised Dab1, demonstrating that the Dab1b group is more ancestral than their paralogue, the Dab1a group.

[The takeover process of the "Real Tribunal del Protoalbeitarato" by the faculty of Veterinary of Madrid (1792-1855)]. / El proceso de absorción del Real Tribunal del Protoalbeitarato por la Escuela de Veterinaria de Madrid (1792-1855).

The absorption of the "Real Tribunal del Protoalbeitarato" by the Veterinary Science School of Madrid shows several singularities in relation to the equivalent process in medicine and pharmacy. The conflict and the hegemony of the "Real Tribunal" during almost sixty years from the creation of the Veterinary Science School (1792), has been studied by the traditional historiography with a view fundamentally focused on veterinary aspects. In this article, the process of absorption is investigated in full detail. Also, the relevance of political motivations and their predominance over technical, sanitary or professional motivations are shown. So, the game of institutions and agents which are external to the veterinary science are analysed; specially, the outstanding role performed by the XV Marquis of Cerralbo.

Pax2 in the optic nerve of the goldfish, a model of continuous growth.

Pax2 is a well known transcription factor which participates in optic nerve development. It assures the correct arrival and package of the newly formed retinal axons and the adequate differentiation of the newly formed glial cells. Pax2 protein expression is continuous throughout adult life in the goldfish optic nerve. We have found two populations of astrocytes in the optic nerve: Pax2(+) and Pax2(-). Moreover, we have observed that the Pax2(+) astrocytes from the optic nerve head present differences in number and organization to those of the rest of the optic nerve. In the optic nerve head some Pax2(+) astrocytes, principally localized in the glia limitans, have thin GFAP(+) processes and weak cytokeratin and ZO1 immunolabeling. Several Pax2(+) astrocytes are in close association with the GFAP(+)/GS(+) Müller cell vitreal processes and with the growing Zn8(+) retinal ganglion cell axons. However, in the intraorbital segment, Pax2(+) astrocytes are more numerous and they have strongly cytokeratin(+)/ZO1(+) processes and form part of the reticular astrocytes and the glia limitans. We also found Pax2(-) astrocytes in both the optic nerve head and the intraorbital segment. In the intraorbital segment there are GS(+)/Pax2(-) cells which are absent from the optic nerve head. We propose that the maintenance of Pax2 protein expression in adult goldfish optic nerve could be related to the continuous addition of new ganglion cell axons and new glial cells.

Bioinformatic analysis of the origin, sequence and diversification of mu opioid receptors in vertebrates.

Opioid receptors are a class of G protein-coupled receptors that mediate the effects of the different families of endogenous opioid peptides and natural alkaloid drugs such as morphine and its synthetic derivatives. In particular, the mu opioid receptor (MOR) represents the principal molecular target for morphine and it plays key roles in opioid analgesia and addiction. In this work, new putative MORs from different vertebrate species were identified in silico and their gene organization and predicted protein products are compared with the previously characterized MORs. Also, for the first time a new genomic organization in euteleleostei teleosts has been identified. Moreover, we suggest that MORs may be specific to craniate lineage. The analysis of functional mapping of MORs we present is an important contribution to the identification of their evolutionarily conserved regions.

Rab27a regulates exocytosis of tertiary and specific granules in human neutrophils.

The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinase-enriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca(2+) and GTP-gamma-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation.