Cytoarchitectonic and neurochemical differentiation of the visual system in ethanol-induced cyclopic zebrafish larvae.

Embryonic exposure to ethanol leads to malformations such as cyclopia. Cyclopic embryos present fused eyes and lack of the ventral specification of the brain, with physiological and morphological defects in the visual system, which provides a useful model for teratology and neurotoxicity assessments. We analysed the differentiation of the visual areas in the ethanol-induced cyclopic animals. For this purpose we exposed zebrafish embryos to 1.5% ethanol from 4 hours post-fertilisation (hpf) to 24 hpf in order to get cyclopic embryos. We monitored cytoarchitecture and quantified both the proliferation rate and cell differentiation from 2 days post-fertilisation (dpf) onwards, focusing on the main components of the visual system (retina, optic nerve and optic tectum) of normal and cyclopic zebrafish embryos. The visual system of the zebrafish embryos is affected by exposure to ethanol; two optic nerves that fuse before leaving the eyes are present in cyclopic specimens but an optic chiasm is not evident. Cell differentiation is severely delayed throughout the visual system at 2 dpf. At 5 dpf, lamination in the cyclopic retina and optic tectum is completed, but they are filled with pyknotic nuclei demonstrating cell death. At this stage the proliferation rate and expression patterns are unaltered and glial and neuronal neurochemical differentiations are similar to untreated animals. We found that the alterations produced by exposure to ethanol are not only cell-selective, but also tissue-selective. Cyclopia is the most severe phenotype induced by ethanol, although cell differentiation and proliferation can reach normal patterns after a certain period of time, which points to a neural plasticity process. Zebrafish embryos may possess a compensation mechanism against the ethanol effect, which would account for their use for pharmacogenetic and chemical screenings in the analysis of new molecules that could improve visual problems.

Characterisation of neuronal and glial populations of the visual system during zebrafish lifespan.

During visual system morphogenesis, several cell populations arise at different time points correlating with the expression of specific molecular markers We have analysed the distribution pattern of three molecular markers (zn-1, calretinin and glial fibrillary acidic protein) which are involved in the development of zebrafish retina and optic tectum. zn-1 is a neural antigen expressed in the developing zebrafish central nervous system. Calretinin is the first calcium-binding protein expressed in the central nervous system of vertebrates and it is widely distributed in different neuronal populations of vertebrate retina, being a valuable marker for its early and late development. Glial fibrillary acidic protein (GFAP), which is an astroglial marker, is a useful tool for characterising the glial environment in which the optic axons develop. We describe the expression profile changes in these three markers throughout the zebrafish lifespan with special attention to ganglion cells and their projections. zn-1 is expressed in the first postmitotic ganglion cells of the retina. Calretinin is observed in the ganglion and amacrine cells of the retina in neurons of different tectal bands and in axons of retinofugal projections. GFAP is localised in the endfeet of Müller cells and in radial processes of the optic tectum after hatching. A transient expression of GFAP in the optic nerve, coinciding with the arrival of the first calretinin-immunoreactive optic axons, is observed. As axonal growth occurs in these regions of the zebrafish visual pathway (retina and optic tectum) throughout the lifespan, a relationship between GFAP expression and the correct arrangement of the first optic axons may exist. In conclusion we provide valuable neuroanatomical data about the best characterised sensorial pathway to be used in further studies such as teratology and toxicology.

Characterization of NADPH-diaphorase-positive glial cells of the tench optic nerve after axotomy.

NADPH-diaphorase (ND) positive cell types were characterized throughout the optic nerve of the tench in normal conditions and after optic nerve transection with survival periods of 1, 3, 7, 14, 30, 60, 120 and 180 days. Astrocytic markers (S100 and glutamine synthetase) and the microglial marker tomato lectin were employed. In the control prechiasmatic optic nerve two types (types I and II) of ND-positive glial cells appeared. All type I cells showed S100 immunoreactivity, whereas only a subpopulation of them were positive to glutamine synthetase. Type II cells only presented S100 immunoreactivity. In the control anterior optic tract, all ND-positive glial cells (type III) presented immunolabeling to S100 and glutamine synthetase. After transection, types I and II did not show any changes in the staining patterns for the glial markers when observed. Two new types of ND-positive glial cells (types IV and V) were observed after axotomy. All type IV cells were S100-immunopositive, and a subpopulation presented glutamine synthetase immunolabeling. Only a subpopulation of type V cells showed glutamine synthetase immunostaining. The presence of type IV or V cells in the lesioned optic nerve occurred simultaneously with significant decreases or absence of type I cells. These data suggest that type I and III cells are astrocytes and type II cells are oligodendrocytes. Types IV and V cells are the result of the activation of type I cells after optic nerve section. The polymorphism observed in ND-positive cells may reflect different cell functions during degenerative and regenerative processes.

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.

Teratogenic effects of ethanol exposure on zebrafish visual system development.

Ethanol intake during pregnancy can produce a wide range of adverse effects on nervous system development including fetal alcohol syndrome (FAS). The most severe congenital malformation observed in newborns with FAS is cyclopia. In this study, we have exposed zebrafish embryos to different ethanol concentrations (2.4%, 1.5% or 1.0%) during eye morphogenesis in four zebrafish strains (AB, EK, GL and TL). In addition, we have studied the survival rate of the cyclopic animals to the end of larval development. The zebrafish strains GL and AB generated the higher percentage of cyclopic animals after exposure to 2.4% ethanol, while EK showed the higher percent cyclopic animals using 1.5% and 1.0% ethanol. The EK strain showed the higher percent survival during the larval period at all ethanol concentrations (2.4%, 1.5% and 1.0%). Moreover, we have investigated cytoarchitectural alterations in the main components of the visual pathway-retina and optic tectum-and ethanol treatment affects both the retina and the optic tectum. The lamination of neural retina is clearly delayed in treated larvae 3 days postfertilization and the thickness of the pigmented epithelium is considerably reduced. With regard to the optic tectum, treatment with ethanol alters the normal pattern of tectal lamination. The use of zebrafish EK strain is a suitable in vivo vertebrate model system for analyzing the teratogenic effect of ethanol during vertebrate visual system morphogenesis as it relates to both cyclopia and FAS.

Tyrosine hydroxylase immunoreactivity in the developing visual pathway of the zebrafish.

We analyzed the distribution of tyrosine hydroxylase immunoreactivity in the central nervous zones involved in the processing of visual information during zebrafish ontogeny, employing a segmental approach. In the retina, we observed immunolabeled cells in the inner nuclear layer after hatching. From the juvenile stages onwards, some of these cells presented two immunolabeled processes towards the inner and outer plexiform layers of the retina, which are identified as interplexiform cells. In the adult zebrafish retina, we have identified two cellular types displaying immunoreactivity for tyrosine hydroxylase: interplexiform and amacrine cells. In the optic tectum, derived from the mesencephalon, no immunolabeled neurons were observed in any of the stages analyzed. The periventricular gray zone and the superficial white zone display immunostained neuropile from the end of fry life onwards. At the 30-day postfertilization, the tyrosine hydroxylase immunoreactive neuropile in the optic tectum presents two bands located within the retinorecipient strata and deeper strata, respectively. All diencephalic regions, which receive direct retinal inputs, show immunolabeled cells in the preoptic area, in the pretectum, and in the ventral thalamus from embryonic stages onwards. During the fry development, the immunolabeled neurons can be observed in the periventricular pretectum from 15-days postfertilization and in both the ventrolateral thalamic nucleus and suprachiasmatic nucleus from 30-days postfertilization. The transient expression of tyrosine hydroxylase is observed in fibers of the optic tract during fry and juvenile development. The existence of immunolabeled neuropile in the zebrafish retinorecipient strata could be related to the turnover of retinotectal projections.

Changes in immunoreactivity to calcium-binding proteins in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.

Effects of axotomy on the expression of NADPH-diaphorase in the visual pathway of the tench.

The distribution of NADPH-diaphorase (ND) positive elements was analyzed throughout the visual pathway of the tench in normal conditions and after optic nerve transection. In the control retina, ND-labeled elements were observed in the photoreceptor, inner nuclear, outer nuclear and ganglion cell layers. In the optic nerve of control animals, small and numerous ND-positive glial cells that were identified as presumably astrocyte-like cells were observed. In the optic tracts and optic tectum, a different type of ND-positive glial cell was detected. Axotomy induced severe changes in the ND staining pattern in the visual pathway. A decrease in the number of ND-stained cells was detected in the retina. In the optic nerve of lesioned animals, the number of small cells gradually decreased, whereas the number of large cells did not change. Two new ND-positive cell populations were observed after the lesion: microglial-like cells appeared close to the lesioned area from 24 h to 7 days after transection, and astrocyte-like cells were found throughout the optic nerve from 14 days up to at least 120 days. The total number of ND-stained glial cells increased at 30 and 60 days and returned to control parameters at 120 days. In addition, the number of ND-positive cells increased at the same survival times in the optic tracts and in the retinorecipient strata of the optic tectum with respect to control animals. Thus, degenerative/regenerative processes in the fish visual pathway are accompanied by an increase in the number of ND-positive cells. Synthesis of nitric oxide is elicited in microglial-like cells as a response to axon injury, whereas the expression in astrocyte-like cells seems to be associated with both normal processes under physiological conditions and with the regenerative phase after the lesion.

Volumetric changes in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effect of olfactory deprivation in the postnatal development of the anterior olfactory nucleus (AON) was studied in 60-day-old rats which underwent unilateral naris closure after birth (postnatal day 1). Volumetric and morphometric analyses of the AON ipsilateral and contralateral to the closed naris were performed and data were statistically compared among them and with those of control animals. The volumes of the AONs and those of their subdivisions were calculated by the Cavalieri method and the area of the subdivisions was measured at seven established rostrocaudal levels. Whereas no statistically significant differences were detected between the ipsilateral and the contralateral AONs, comparison of these with controls revealed significant reductions in the volumes and dimensions of most AON subdivisions. The reduction was larger in the ipsilateral than in the contralateral AON and more pronounced in the rostralmost subdivisions (external and lateral) than in the caudal ones, the dorsal subdivision not being affected. These data demonstrate that the disruption of the normal afferent activity to one olfactory bulb has effects on the postnatal development of both the ipsilateral and the contralateral AONs. In addition, the most affected subdivisions were those that develop later and that receive the bulk of projections from the olfactory bulb, suggesting that the degree of maturity is an important factor in susceptibility to changes induced by reduced afferent activity. Finally, the results indicate that, contrary to the olfactory bulb, the contralateral AON cannot be used as a control structure in deprivation studies.

Calretinin-, neurocalcin-, and parvalbumin-immunoreactive elements in the olfactory bulb of the hedgehog (Erinaceus europaeus).

The distribution pattern and morphology of calretinin-, neurocalcin-, and parvalbumin-immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. The detection of these markers was carried out by using monoclonal or polyclonal antibodies and the avidin-biotin-immunoperoxidase method. Specific neuronal populations were positive for these calcium-binding proteins in the hedgehog olfactory bulb, revealing both similarities to and differences from the data reported in the olfactory bulb of rodent species. The distribution pattern of each calcium-binding protein studied in the accessory olfactory bulb was highly similar to that described in other macrosmatic species. However, in the main olfactory bulb, the markers analyzed were expressed in similar interneuronal populations as they are in the rodent olfactory bulb, whereas cell groups categorized as projecting neurons demonstrated striking differences in the expression of these calcium-binding proteins. These results suggest that the expression of calcium-binding proteins in a given brain region is not a constant feature among species despite a similar organization but that different factors could influence their expression. Thus, the accessory olfactory system involved in the processing of specific and similar olfactory cues among species demonstrates a more constant organization among species. By contrast, the functionally important role of the main olfactory system in the hedgehog is accompanied by a more complex organization, which is reflected in an increased diversity of calcium-buffering systems.

A sexually dimorphic group of atypical glomeruli in the mouse olfactory bulb.

Atypical glomeruli (AtG) are clearly distinguishable from typical ones because of their strong cholinergic innervation. AtG are located in defined positions in the caudal half of the main olfactory bulb of rodents. The AtG partially overlap with other specialized olfactory subsystems, such as the modified glomerular complex, which is close to the accessory olfactory bulb. So far, possible sex differences in these specialised olfactory systems have not been investigated. In this work we have identified AtG in the mouse by means of acetylcholinesterase histochemistry and compared the number and size of these glomeruli between the sexes and also between the two strains that demonstrate intraglomerular synaptic differences, i.e. BALB/c and CD-1 mice. First, we divided the AtG into three types according to their position (I, rostral-most; II, around the accessory olfactory bulb; III, caudal-most) or their reactivity to acetylcholinesterase histochemistry (AtG type II being the least reactive glomeruli). ANOVA analyses revealed differences in the maximum diameter of glomeruli among the three types, but not in their sectional areas, indicating that all three types have different shapes. Moreover, both morphoplanimetric parameters were seen to be different between the two strains studied and also between the sexes: male mice and BALB/c animals had the largest glomeruli. The number of AtG was also significantly different between the sexes and strains, although these factors presented a strong interaction. Thus, the males had higher numbers of AtG in the CD-1 strain whereas in the BALB/c mice males demonstrated fewer AtG than females. These differences in number were largely due to AtG type II. The present work is evidence that AtG type II is a sexually dimorphic group of specialized glomeruli located in the main olfactory bulb.

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.

Distribution of parvalbumin immunoreactivity in the brain of the tench (Tinca tinca L., 1758).

The distribution of parvalbumin (PV) immunoreactivity in the tench brain was examined by using the avidin-biotin-peroxidase immunocytochemical method. This protein was detected in neuronal populations throughout all main divisions of the tench brain. In the telencephalic hemispheres, PV-immunopositive neurons were distributed in both the dorsal and ventral areas, being more abundant in the area ventralis telencephali, nucleus ventralis. In the diencephalon, the scarce distribution of PV-containing cells followed a rostrocaudal gradient, and the most evident staining was observed in the nucleus periventricularis tuberculi posterioris and in a few nuclei of the area praetectalis. In the mesencephalon, abundant PV-immunoreactive elements were found in the tectum opticum, torus semicircularis, and tegmentum. In the tectum opticum, PV-immunoreactivity presented a laminar distribution. Three PV-containing neuronal populations were described in the torus semicircularis, whereas in the tegmentum, the PV staining was mainly located in the nucleus tegmentalis rostralis and in the nucleus nervi oculomotorii. In the metencephalon, Purkinje cells were PV-immunopositive in the valvula cerebelli, lobus caudalis cerebelli, and in the corpus cerebelli. In the myelencephalon, PV immunoreactivity was abundant in the nucleus lateralis valvulae, in the nucleus nervi trochlearis, nucleus nervi trigemini, nucleus nervi abducentis, nucleus nervi glossopharyngei, and in the formatio reticularis. Mauthner cells were also PV immunostained. By contrast to other vertebrate groups, only a restricted population of PV-containing neurons was GABA-immunoreactive in the tench, demonstrating that this calcium-binding protein cannot be considered a marker for GABAergic elements in the teleost brain. This study demonstrates a low phylogenetic conservation of the distribution of PV comparing teleosts and tetrapods.

Expression of ZFOR1, a delta-opioid receptor, in the central nervous system of the zebrafish (Danio rerio).

Opioid receptors, besides mediating the effects of analgesic compounds, are involved in drug addiction. Although a large amount of work has been done studying these receptors in mammals, little information has been obtained from nonmammalian vertebrates. We have studied the regional distribution in the central nervous system (CNS) of the zebrafish of the recently cloned delta-opioid receptor homologue ZFOR1 using nonradioactive in situ hybridization. Our findings show that different nuclei within the main subdivisions of the brain displayed specific mRNA signal. The expression is widespread throughout the brain, but only specific cells within each nucleus displayed ZFOR1. Stained cells were abundant in the telencephalon, both in the olfactory bulb and telencephalic hemispheres, and in the diencephalon, where expression was observed in all the different subdivisions. In the mesencephalon, expression of ZFOR1 was abundant in the periventricular layer of the optic tectum. In the cerebellum, expression of ZFOR1 was detected in valvula cerebelli, corpus cerebelli, and lobus vestibulolateralis in both granule and Purkinje cells. In the myelencephalon, cells expressing ZFOR1 were also distributed in the octavolateralis area, the reticular formation, and the raphe nuclei, among others. Also, ZFOR1 was detected in cells of the dorsal and ventral horn of the spinal cord. This work presents the first detailed distribution of a delta-opioid receptor in the CNS of zebrafish. Distribution of ZFOR1 expression is compared with that of the delta-opioid receptor described in mammals.

Distribution of acetylcholinesterase and choline acetyltransferase in the main and accessory olfactory bulbs of the hedgehog (Erinaceus europaeus).

The distribution of cholinergic markers was studied in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) of the western European hedgehog (Erinaceus europaeus) by using choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. A dense network of AChE-containing and ChAT-immunoreactive fibers was observed innervating all layers of the MOB except the olfactory nerve layer, where neither AChE- nor ChAT-labeled elements were found. The highest density of AChE- and ChAT-positive axons was found in the glomerular layer (GL)/external plexiform layer (EPL) boundary, and in the internal plexiform layer. This general distribution pattern of ChAT- and AChE-stained axons resembled the distribution pattern found in rodents. Nevertheless, some interspecies differences, such as the lack of atypical glomeruli in the hedgehog, were also found. In addition to fibers, a population of noncholinergic and presumably cholinoceptive AChE-active neurons was observed in the hedgehog. All mitral and tufted cells of the hedgehog MOB showed a dark AChE staining unlike previous observations in the mitral and tufted cells of rodents. As in other species previously reported, subpopulations of external tufted cells and short-axon cells were also AChE-active. Finally, a population of small AChE-containing cells was observed in the EPL of the hedgehog MOB. The size, shape, and location of these cells coincided with those of satellite and perinidal cells, two neuronal types described previously in the EPL of the hedgehog and not present in the rodent MOB. The AOB of the hedgehog showed a distribution of AChE- and ChAT-positive fibers similar to the rodent AOB. Nevertheless, a heterogeneous innervation of vomeronasal glomeruli by bundles of AChE- and ChAT-labeled axons found in the hedgehog has not been previously found in any other species. As in the MOB, all mitral cells in the AOB showed a strong AChE activity. These results demonstrate some similarities but also important differences between the distribution of ChAT and AChE in the MOB and AOB of rodents and this primitive mammalian. These variations may indicate a different organization of the cholinergic modulation of the olfactory information in the insectivores.

Differential brain expression of a new beta-actin gene from zebrafish (Danio rerio).

It has been shown that actin genes exhibit distinct tissue and stage-specific patterns of expression. We have cloned a new beta-actin gene from the teleost zebrafish (Danio rerio), a well-established model for developmental studies, and analysed its expression by Northern blot and in situ hybridization studies. Our results suggest that in adult brain zebrafish, this new gene is expressed during neuronal cell proliferation.

Chemical anatomy of the macaque monkey olfactory bulb: NADPH-diaphorase/nitric oxide synthase activity.

The distribution and the morphology of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND)-active and neuronal nitric oxide synthase (NOS)-immunoreactive neurons and fibers were studied in the olfactory bulb of three species of primates, i.e., the cynomolgus macaque monkey (Macaca fascicularis), the Japanese macaque monkey (Macaca fuscata), and the pig-tail macaque monkey (Macaca nemestrina). The ND staining was carried out by means of a direct histochemical method with beta-NADPH as cosubstrate and nitro blue tetrazolium as chromogen. The NOS immunostaining was carried out by using a polyclonal antibody and the avidin-biotin peroxidase method. Similar results were found in the three species, where a distinct distribution pattern of ND/NOS-stained neurons and fibers was observed. All olfactory fibers demonstrated ND-positive labeling but they were NOS-immunonegative. In the superficial modulatory area of the olfactory bulb, a few weakly ND- and NOS-positive periglomerular cells, stellate cells, and darkly stained superficial short-axon cells were observed. In the inframitral layers, granule cells, deep stellate cells, and deep short-axon cells were distinguished. Short-axon cells had oriented morphologies and spiny dendrites. Many thick, varicose ND/NOS-stained fibers identified as centrifugal fibers were observed in the white matter, granule cell layer, internal plexiform layer, mitral cell layer, and external plexiform layer. This distribution of ND activity and NOS immunoreactivity showed similarities to and differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents (rat, mouse, and hamster) and insectivores (hedgehog). These data confirm that the complexity of the ND/NOS staining in the olfactory bulb of one species correlates with the importance of olfaction in the biology of such species.

Transient expression of calretinin in the trout habenulo-interpeduncular system during development.

Calcium-binding proteins control calcium homeostasis during neural development. The expression of one of these proteins, calretinin (CR), was monitored by immunohistochemistry in the developing habenulo-interpeduncular system of the rainbow trout, a conserved region of the brain along vertebrate phylogeny that undergoes a neurochemical reorganization in late development. No CR-immunoreactivity was observed in the habenulo-interpeduncular system during the embryonic development. CR-immunolabeling appeared in newly hatched fry and during the fry development the number of CR-immunostained elements increased progressively. During the juvenile stages (from 30 days post-hatching onwards) a gradual decrease in the number of CR-immunostained cells occurred, until its complete disappearance in adults. These variations in CR expression may represent the variable calcium-buffering needs during different developmental stages.

Parvalbumin immunoreactivity during the development of the cerebellum of the rainbow trout.

The distribution of parvalbumin immunoreactivity in the developing cerebellum of the rainbow trout was studied by using a specific monoclonal antibody and the avidin-biotin peroxidase method. Parvalbumin immunoreactivity was absent during the embryonic development of the cerebellum. The first immunoreactive elements, identified by their localization and posterior morphological evolution as immature Purkinje cells, appeared at 6 days posthatching in the presumptive corpus cerebelli and lobus vestibulolateralis. The labeling extended throughout the cerebellum following a caudorostral gradient, and in 21 days alevins, parvalbumin immunoreactive Purkinje cells were also observed in the valvula cerebelli. The appearance of parvalbumin-immunostaining in the Purkinje cells was not simultaneous; the labeling was observed initially in the cell body, extending gradually to the dendritic branches and finally to the axon. From 1 year onwards, parvalbumin immunoreactive terminal puncta from the Purkinje cell axons were observed surrounding the cell bodies of eurydendroid cells, that were parvalbumin immunonegative in all developmental stages studied. The spatio-temporal pattern of parvalbumin immunoreactivity in the rainbow trout cerebellum is different to previous observations in the cerebellum of amniotes.

Co-localization of calretinin and parvalbumin with nicotinamide adenine dinucleotide phosphate-diaphorase in tench Mauthner cells.

The co-localization of calretinin (CR) and parvalbumin (PV) immunoreactivity with nicotinamide adenine dinucleotide phosphate-diaphorase (ND) activity was analyzed in the Mauthner cells of the tench. Mauthner cells were ND active, and ND staining was observed in the soma, axon cap region, and axon of these neurons. CR co-localized with ND in the axon of the Mauthner cells but not in the cell body or in the dendrites, whereas PV immunoreactivity co-localized with ND in the soma, axon and dendrites. The presence of two different calcium-binding proteins in the Mauthner cells indicates that these neurons need complex calcium-buffering systems. The co-localization of these calcium-binding proteins with ND might suggests their involvement in nitric oxide-related events.