A. Porteros

A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25

Myopia and hyperopia are at opposite ends of the continuum of refraction, the measure of the eye′s ability to focus light, which is an important cause of visual impairment (when aberrant) and is a highly heritable trait. We conducted a genome-wide association study for refractive error in 4,270 individuals from the TwinsUK cohort. We identified SNPs on 15q25 associated with refractive error (rs8027411, P = 7.91 × 10−8). We replicated this association in six adult cohorts of European ancestry with a combined 13,414 individuals (combined P = 2.07 × 10−9). This locus overlaps the transcription initiation site of RASGRF1, which is highly expressed in neurons and retina and has previously been implicated in retinal function and memory consolidation. Rasgrf1−/− mice show a heavier average crystalline lens (P = 0.001). The identification of a susceptibility locus for refractive error on 15q25 will be important in characterizing the molecular mechanism responsible for the most common cause of visual impairment.

Cholinergic elements in the zebrafish central nervous system: Histochemical and immunohistochemical analysis

Recently, the zebrafish has been extensively used for studying the development of the central nervous system (CNS). However, the zebrafish CNS has been poorly analyzed in the adult. The cholinergic/cholinoceptive system of the zebrafish CNS was analyzed by using choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry in the brain, retina, and spinal cord. AChE labeling was more abundant and more widely distributed than ChAT immunoreactivity. In the telencephalon, ChAT‐immunoreactive (ChAT‐ir) cells were absent, whereas AChE‐positive neurons were observed in both the olfactory bulb and the telencephalic hemispheres. The diencephalon was the region with the lowest density of AChE‐positive cells, mainly located in the pretectum, whereas ChAT‐ir cells were exclusively located in the preoptic region. ChAT‐ir cells were restricted to the periventricular stratum of the optic tectum, but AChE‐positive neurons were observed throughout the whole extension of the lamination except in the marginal stratum. Although ChAT immunoreactivity was restricted to the rostral tegmental, oculomotor, and trochlear nuclei within the mesencephalic tegmentum, a widespread distribution of AChE reactivity was observed in this region. The isthmic region showed abundant AChE‐positive and ChAT‐ir cells in the isthmic, secondary gustatory and superior reticular nucleus and in the nucleus lateralis valvulae. ChAT immunoreactivity was absent in the cerebellum, although AChE staining was observed in Purkinje and granule cells. The medulla oblongata showed a widespread distribution of AChE‐positive cells in all main subdivisions, including the octavolateral area, reticular formation, and motor nuclei of the cranial nerves. ChAT‐ir elements in this area were restricted to the descending octaval nucleus, the octaval efferent nucleus and the motor nuclei of the cranial nerves. Additionally, spinal cord motoneurons appeared positive to both markers. Substantial differences in the ChAT and AChE distribution between zebrafish and other fish species were observed, which could be important because zebrafish is widely used as a genetic or developmental animal model. J. Comp. Neurol. 474:75–107, 2004.

TOPOGRAPHICAL DISTRIBUTION OF NADPH-DIAPHORASE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM OF THE FROG, RANA PEREZI

The distribution of NADPH‐diaphorase (ND) activity was histochemically investigated in the brain of the frog Rana perezi. This technique provides a highly selective labeling of neurons and tracts. In the telencephalon, labeled cells are present in the olfactory bulb, pallial regions, septal area, nucleus of the diagonal band, striatum, and amygdala. Positive neurons surround the preoptic and infundibular recesses of the third ventricle. The magnocellular and suprachiasmatic hypothalamic nuclei contain stained cells. Numerous neurons are present in the anterior, lateral anterior, central, and lateral posteroventral thalamic nuclei. Positive terminal fields are organized in the same thalamic areas but most conspicuously in the visual recipient plexus of Bellonci, corpus geniculatum of the thalamus, and the superficial ventral thalamic nucleus. Labeled fibers and cell groups are observed in the pretectal area, the mesencephalic optic tectum, and the torus semicircularis. The nuclei of the mesencephalic tegmentum contain abundant labeled cells and a conspicuous cell population is localized medial and caudal to the isthmic nucleus. Numerous cells in the rhombencephalon are distributed in the octaval area, raphe nucleus, reticular nuclei, sensory trigeminal nuclei, nucleus of the solitary tract, and, at the obex levels, the dorsal column nucleus. Positive fibers are abundant in the superior olivary nucleus, the descending trigeminal, and the solitary tracts. In the spinal cord, a large population of intensely labeled neurons is present in all fields of the gray matter throughout its rostrocaudal extent. Several sensory pathways were heavily stained including part of the olfactory, visual, auditory, and somatosensory pathways. The distribution of ND‐positive cells did not correspond to any single known neurotransmitter or neuroactive molecule system. In particular, abundant codistribution of ND and catecholamines is found in the anuran brain. Double labeling techniques have revealed restricted colocalization in the same neurons and only in the posterior tubercle and locus coeruleus. If ND is in amphibians a selective marker for neurons containing nitric oxide synthase, as generally proposed, with this method the neurons that may synthesize nitric oxide would be identified. This study provides evidence that nitric oxide may be involved in novel tasks, primarily related to forebrain functions, that are already present in amphibians.

Nitric oxide synthase in the brain of a urodele amphibian (Pleurodeles waltl) and its relation to catecholaminergic neuronal structures

The neuronal structures with NADPH-diaphorase activity and nitric oxide synthase (NOS) immunoreactivity have been studied in the brain of the urodele amphibian Pleurodeles waltl by means of histochemical and immunocytochemical techniques. Both approaches resulted in the selective labeling of the same neurons and fiber tracts in the brain, except for the primary olfactory fibers that did not stain for NOS but were positive for NADPH-diaphorase. NOS-containing neurons were found in the olfactory bulbs, pallial regions, septum, caudal striatum, amygdala and preoptic area. Only a few diencephalic cells were labeled in the posterior tubercle and ventral hypothalamus. In the brainstem, abundant cells were labeled in the tectum, mesencephalic tegmentum and isthmic region. The most conspicuous cell population was found in the isthmic-pretrigeminal region. Particularly well stained cells were distributed throughout the rhombencephalon in areas related to the descending trigeminal tract, solitary tract, raphe nucleus and the mid-caudal reticular formation. In the cervical spinal cord, NOS-containing cells were present in the dorsal, intermediate and ventral grey fields. Cells in the preoptic, postotic and dorsal root ganglia were also labeled. Double labeling techniques revealed an extensive codistribution of neurons with NOS and catecholamines in the urodele brain but actual colocalization in the same cells was never observed. The organization of the central systems in urodeles with NOS appears to share many features not only with other anamniotes but also with amniotes.

Calbindin D-28K and NADPH-diaphorase activity are localized in different populations of periglomerular cells in the rat olfactory bulb

Calbindin D-28k (CaBP) immunocytochemistry and NADPH-diaphorase (ND) histochemistry have been combined in the rat olfactory bulb by successive incubations of the same sections. The outer strata showed a similar neuronal staining pattern for both markers with positive periglomerular neurons (although the CaBP-stained periglomerular cells were six-fold more abundant than the ND-active ones) and larger neurons scattered in the glomerular and external plexiform layers. Both populations of periglomerular cells were distinct but they did not show specific morphological characteristics nor a predominant distribution around ND-positive and negative glomeruli. The colocalization study demonstrates that the larger ND and CaBP-stained juxtaglomerular cells, identified according to their size, location and processes branching patterns as two types of short axon cells (superficial short-axon and Van Gehuchten Cells) were also independent populations.

Development of the cholinergic system in the brain and retina of the zebrafish

We have analyzed the distribution pattern of choline acetyltransferase (ChAT) in the zebrafish brain and retina during ontogeny. ChAT-immunoreactive (ChAT-ir) neurons are observed in the prosencephalon from 60 h postfertilization (hpf) onwards, exclusively in the preoptic area (basal plate of p6) derived from the secondary prosencephalon. In the mesencephalon, ChAT-ir cells are observed in both the optic tectum and the tegmentum. Stained cells in the tegmentum are observed from 60 hpf onwards, while in the optic tectum they appear after hatching. In the rhombencephalon, ChAT-ir cells are first observed in the isthmic region (rh1) and in the medulla oblongata (rh5-rh7) at the end of embryonic life. The rhombencephalic cholinergic cell groups develop in a gradual caudorostral sequence. Motoneurons of the spinal cord are ChAT-ir from 48 hpf onwards. The retina displays ChAT-ir neuropil in both the inner and outer plexiform layers from embryonic life, whereas stained amacrine cells are only observed after hatching. The staining in the outer plexiform layer gradually decreases during juvenile development. The optic nerve axons show a transient expression of ChAT at the end of embryonic development. The early presence of ChAT immunolabeling suggests an important neuromodulator role for acetylcholine in the first developmental stages.

Laser microdissection and microarray analysis of the hippocampus of Ras-GRF1 knockout mice reveals gene expression changes affecting signal transduction pathways related to memory and learning

We used manual macrodissection or laser capture microdissection (LCM) to isolate tissue sections of the hippocampus area of Ras-GRF1 wild type and knockout mice brains, and analyzed their transcriptional patterns using commercial oligonucleotide microarrays. Comparison between the transcriptomes of macrodissected and microdissected samples showed that the LCM samples allowed detection of significantly higher numbers of differentially expressed genes, with higher statistical rates of significance. These results validate LCM as a reliable technique for in vivo genomic studies in the brain hippocampus, where contamination by surrounding areas (not expressing Ras-GRF1) increases background noise and impairs identification of differentially expressed genes. Comparison between wild type and knockout LCM hippocampus samples revealed that Ras-GRF1 elimination caused significant gene expression changes, mostly affecting signal transduction and related neural processes. The list of 36 most differentially expressed genes included loci concerned mainly with Ras/G protein signaling and cytoskeletal organization (i.e. 14-3-3gamma/zeta, Kcnj6, Clasp2) or related, cross-talking pathways (i.e. jag2, decorin, strap). Consistent with the phenotypes shown by Ras-GRF1 knockout mice, many of these differentially expressed genes play functional roles in processes such as sensory development and function (i.e. Sptlc1, antiquitin, jag2) and/or neurological development/neurodegeneration processes affecting memory and learning. Indeed, potential links to neurodegenerative diseases such as Alzheimer disease (AD) or Creutzfeldt-Jacobs disease (CJD), have been reported for a number of differentially expressed genes identified in this study (Ptma, Aebp2, Clasp2, Hebp1, 14-3-3gamma/zeta, Csnk1delta, etc.). These data, together with the previously described role of IRS and insulin (known Ras-GRF1 activators) in AD, warrant further investigation of a potential functional link of Ras-GRF1 to neurodegenerative processes.

Targeted Disruption of Ras-Grf2 Shows Its Dispensability for Mouse Growth and Development

ABSTRACT The mammalian Grf1 and Grf2 proteins are Ras guanine nucleotide exchange factors (GEFs) sharing a high degree of structural homology, as well as an elevated expression level in central nervous system tissues. Such similarities raise questions concerning the specificity and/or redundancy at the functional level between the two Grf proteins. grf1-null mutant mice have been recently described which showed phenotypic growth reduction and long-term memory loss. To gain insight into the in vivo function of Grf2, we disrupted its catalytic CDC25-H domain by means of gene targeting. Breeding among grf2+/− animals gave rise to viable grf2 −/− adult animals with a normal Mendelian pattern, suggesting that Grf2 is not essential for embryonic and adult mouse development. In contrast to Grf1-null mice, analysis of grf2 −/− litters showed similar size and weight as their heterozygous or wild-type grf2 counterparts. Furthermore, adult grf2 −/− animals reached sexual maturity at the same age as their wild-type littermates and showed similar fertility levels. No specific pathology was observed in adult Grf2-null animals, and histopathological studies showed no observable differences between null mutant and wild-type Grf2 mice. These results indicate that grf2 is dispensable for mouse growth, development, and fertility. Furthermore, analysis of double grf1/grf2 null animals did not show any observable phenotypic difference with single grf1 −/− animals, further indicating a lack of functional overlapping between the two otherwise highly homologous Grf1 and Grf2 proteins.

Calretinin immunoreactivity in the developing olfactory system of the rainbow trout

The distribution of calretinin immunoreactivity in the developing olfactory system of the rainbow trout was studied by using an indirect immunocytochemical method. Calretinin immunoreactivity was firstly detected at 150 day-degrees in the olfactory placode, where labeled primordial cells were observed. At 250 day-degrees, precursor cells of the olfactory receptor neurons located in the olfactory pit were calretinin-immunoreactive. At 300 day-degrees, recognizable olfactory receptor neurons displayed calretinin immunoreactivity in the olfactory epithelium, and calretinin-immunopositive olfactory axons reached the presumptive olfactory bulb. After hatching (400 day-degrees) and during the subsequent development and maturation of the olfactory system, the number of calretinin-immunopositive olfactory receptor cells increased and distributed homogeneously throughout the olfactory epithelium. Accordingly, new positive olfactory fibers arrived to the olfactory bulb arborizing in olfactory glomeruli distributed in nine different terminal fields. Six days after hatching, calretinin-immunopositive interneurons within the olfactory bulb were also observed. The size and number of calretinin-immunoreactive interneurons increased from this stage to adulthood. The adult pattern demonstrated both similarities and differences with the distribution of calretinin immunoreactivity previously described in the olfactory system of mammals.

Expression of neuronal nitric oxide synthase/NADPH‐diaphorase during olfactory deafferentation and regeneration

Neuronal nitric oxide synthase (nNOS) expression can be regulated under natural or experimental conditions. This work aims at elucidating whether the expression of nNOS or its related NADPH‐diaphorase (ND) activity are modified by manipulation of the normal inputs to neurons. We used the olfactory bulbs from two mouse strains, BALB and CD1, because they show divergences in their synapse patterns, and these differences affect periglomerular cells, interneurons expressing tyrosine hydroxylase or nNOS/ND. The olfactory inputs to these neurons can be disrupted by inhalation of methyl bromide. The effect of this gas on olfactory axons, as well as the synaptic features in both mouse strains, were studied using electron microscopy. The changes in expression were analysed qualitatively and quantitatively at different times after lesion to nine topographical regions of the olfactory bulb. Methyl bromide inhalation induced a degeneration of olfactory axons in both strains, but had different effects on the expression of nNOS/ND and tyrosine hydroxylase. In BALB mice, where periglomerular cells do not receive direct inputs from olfactory axons, no changes were detected in tyrosine hydroxylase or in ND expression. In CD1 periglomerular cells, where olfactory axons establish direct synapses, a significant down‐regulation of both markers was observed. These changes were observed differentially across the olfactory bulb, being more pronounced in rostral regions and more acute for ND than for tyrosine hydroxylase. Our results indicate that the synaptic inputs influence the expression of ND activity related to nNOS and that the activation of the enzyme is more severely affected than its protein expression.