How long does a photoreceptor cell take to die? Implications for the causative cell death mechanisms.

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity.

Apgar score and perinatal death after one previous caesarean delivery.

OBJECTIVE: To assess the impact of the indication for a previous caesarean section on the outcome of a subsequent delivery. DESIGN: Population-based cohort study. SETTING: Sweden. POPULATION: Women with two deliveries between 1987 and 2007 identified using the Swedish Medical Birth Registry. METHODS: The outcome of 69 133 pregnancies after one caesarean section was compared with the outcome of 487 610 pregnancies following one vaginal delivery. The indication for the first caesarean section was estimated using a new hierarchical system based on information from birth records. MAIN OUTCOME MEASURES: Perinatal death, low Apgar score (less than seven at 5 minutes). RESULTS: Infants of women with one previous caesarean section were at increased risk of low Apgar score compared with infants of women with one previous vaginal delivery (OR, 2.0; 95% CI, 1.9-2.1). The risk estimate was reduced when adjustment for maternal and fetal/infant characteristics was made (OR, 1.6; 95% CI, 1.5-1.8). The corresponding crude and adjusted odds ratios for perinatal death were 1.6 (95% CI, 1.4-1.7) and 1.1 (95% CI, 1.0-1.2), respectively. The infant outcome of the delivery after one caesarean section was mainly dependent on the indication for the first-delivery caesarean section and, when no medical indication was present, no increase in risk was detected. CONCLUSIONS: Infants of women with one previous caesarean section were at increased risk of low Apgar score and/or perinatal death compared with infants of women with one previous vaginal delivery. The results suggest that medical conditions, not the previous caesarean section per se, contributed to the increase in risk.

Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum.

Neural processes from mammalian pinealocytes have been discovered in several brain areas. These processes were visualized immunocytochemically in the Djungarian hamster, Phodopus sungorus, with an antiserum against bovine retinal S-antigen and traced as far as the region of the posterior commissure and habenular nuclei. This result indicates that pineal-to-brain connections exist in the mammal, and that the mammalian pineal gland, currently thought of only as a neuroendocrine organ, may communicate directly with select brain regions by way of these projections. The existence of mammalian pinealocyte projections is consistent with the view that these cells are not of glial origin but are derivatives of photoreceptor cells of the pineal complex of lower vertebrates that transmit signals to the brain by neural projections.

Significant photoreceptor rescue by treatment with a combination of antioxidants in an animal model for retinal degeneration.

The purpose of this study was to investigate the presence of oxidative DNA damage in the photoreceptors of the rd1 mouse, an animal model for retinitis pigmentosa, and to determine if antioxidants could delay the progress of photoreceptor cell death. Retinas of rd1 mice and congenic wild type controls were examined for DNA oxidation and fragmentation. To study the rescue effect of antioxidants on retinal degeneration, rd1 retinas were studied in vitro and in vivo using lutein, zeaxanthin, alpha lipoic acid and reduced l-glutathione. For the in vitro studies, antioxidants were added to the culture medium. For the in vivo studies, postnatal day (PN3) pups of rd1 mice were fed antioxidants either individually or in combination and control rd1 animals received vehicle alone. Histological evaluation was performed using hematoxylin/eosin and avidin staining, as well as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Many of the rd1 rod photoreceptors at PN11 displayed oxidative DNA damage and TUNEL positive reaction which co-localized in a subset of rod photoreceptors. Avidin-labeled rod photoreceptors were more abundant than the TUNEL positive photoreceptors of the rd1 mouse, indicating that oxidative DNA damage precedes fragmentation. The number of TUNEL positive and avidin positive cells was considerably decreased upon treatment with the combination of the antioxidants. Rescue of rd1 photoreceptors was significant at PN18 and PN17, respectively, in the in vitro and in vivo studies. In conclusion individual antioxidants had no significant rescue effect but the combination slowed down the rd1 rod photoreceptor degeneration, indicating an additive or synergistic effect.

Bilaterally symmetrical rhopalial nervous system of the box jellyfish Tripedalia cystophora.

Cubomedusae, or box jellyfish, have the most elaborate visual system of all cnidarians. They have 24 eyes of four morphological types, distributed on four sensory structures called rhopalia. Box jellyfish also display complex, probably visually guided behaviors such as obstacle avoidance and fast directional swimming. Here we describe the strikingly complex and partially bilaterally symmetrical nervous system found in each rhopalium of the box jellyfish, Tripedalia cystophora, and present the rhopalial neuroanatomy in an atlas-like series of drawings. Discrete populations of neurons and commissures connecting the left and the right side along with two populations of nonneuronal cells were visualized using several different histochemical staining techniques and electron microscopy. The number of rhopalial nerve cells and their overall arrangement indicates that visual processing and integration at least partly happen within the rhopalia. The larger of the two nonneuronal cell populations comprises approximately 2,000 likely undifferentiated cells and may support a rapid cell turnover in the rhopalial nervous system.

Use of explant cultures of peripheral nerves of adult vertebrates to study axonal regeneration in vitro.

Explanted preparations of peripheral nerves with attached dorsal root ganglia of adult mammals and amphibia survive for several days in serum-free medium and can be used to study axonal regeneration in vitro. This review outlines the methods which we routinely use and how they may be applied to study different aspects of axonal regeneration. When the peripheral nerves are crushed in vitro, axons regenerate through the crush site into the distal stump within 1 day (mouse) or 3 days (frog). The outgrowth distance of the leading sensory axons can be determined with the use of a simple method based on axonal transport of labelled proteins. A compartmentalised system permits selective application of drugs and other agents to either ganglia or peripheral nerve containing the regenerating axons and has been used to study selected aspects of regeneration including influence of non-neuronal cells, retrograde signalling, axonal release of proteins during regeneration and the role of phospholipase A2 activity. Explanted preparations may also be cultured in a layer of extracellular matrix material (matrigel), in which spontaneous outgrowth of a large number of naked axons from the cut ends of nerves starts within 1 day and continues for several days. This provides an opportunity to study the direct effects of different agents on axonal elongation. Preparations cultured in collagen gels show sparse spontaneous axonal growth, but this can be increased by addition of certain growth factors. The phenotype of the regenerating axons can be studied using immunohistochemical methods.

Effect of estramustine phosphate on the assembly of isolated bovine brain microtubules and fast axonal transport in the frog sciatic nerve.

Estramustine phosphate (0.01 to 0.5 mM), an estradiol mustard derivative used in the therapy of prostatic carcinoma, inhibited the assembly of brain microtubules proteins in vitro and disassembled preformed microtubules. In the presence of estramustine phosphate, the minimum microtubule-protein concentration sufficient for the assembly of microtubules was increased. Low concentrations of taxol (20 microM) completely reversed the inhibition of assembly by estramustine phosphate. The effects were specific to estramustine phosphate since neither estradiol 17 beta-phosphate, the hormonal moiety of the drug, nor nornitrogen mustard, the alkylating moiety, had any effect on assembly. Estramustine phosphate (0.1 to 0.5 mM) was also found to reversibly inhibit fast axonal transport in the frog sciatic nerve. The nerve content of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate was not significantly affected by estramustine phosphate. Our results suggest that the cytotoxic action of estramustine phosphate could be dependent partially on an interaction with microtubules, probably via the microtubule-associated proteins.

Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6a (V685M/R562W) mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

The effect of gossypol on fast axonal transport and microtubule assembly.

Gossypol at micromolar concentrations (2 microM) was found to inhibit axonal transport and a microsomal ATPase activity in the frog sciatic nerve, although axonal microtubules and the neuronal content of AMP, ADP and ATP were not affected. At slightly higher concentrations (30-40 microM), gossypol also inhibited microtubule assembly and neuronal energy metabolism. Gossypol accumulated in the nerve and the results indicate that gossypol may act as a potent neurotoxin.

PIP: The effect of gossypol on axonal transport, the translocation of substances along microtubules in neurites, was investigated in an experiment on sciatic nerves and 8th and 9th dorsal ganglia dissected from frogs. This effect was defined as the ratio between the radioactivity in front of the ligature in the experimental and the control nerve. When gossypol was present in the nerve compartment, axonal transport decreased from 52% at 2 mcgM to 13% at 30 mcgM. On the other hand, when the drug was present only in the ganglionic compartment, the inhibition was less marked, and at 30 mcgM axonal transport was still 70% of control. Since gossypol is lipid-soluble, its action may be related to its accumulation in myelin, which is more abundant in the nerve compartment than the ganglion. Support for this hypothesis was provided by the finding that, when frog nerves were incubated for 17 hours in Ringer solution containing 30 mcgM of gossypol, the concentration of gossypol in the nerve was 100-times higher than in the surrounding Ringer solution. Nerves incubated for 17 hours with 2 mcgM of gossypol did not show any significant changes in their content of ATP, ADP, or AMP. Moreover, the axonal morphology and the density of the microtubules were normal after treatment of the nerve with gossypol concentrations at or below 10 mcgM. At concentrations of 30-40 mcgM, gossypol did inhibit microtubule assembly and neuronal energy metabolism. The finding that gossypol accumulates in the nerve suggests a need for attention to the possible neurotoxicity of this male contraceptive.

Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release: a biochemical, immunohistochemical, and confocal microscopic study.

Carbon monoxide (CO) has been suggested as a novel messenger molecule in the brain. We now report on the cellular localization and hormone secretory function of a CO-producing constitutive heme oxygenase (HO-2) in mouse islets. Islet homogenates produced large amounts of CO which were suppressed dose-dependently by the HO inhibitor zincprotoporphyrin-IX (ZnPP-IX). We also show, for the first time, that glucose markedly stimulates the HO activity (CO production) in intact islets. A further potentiation was induced by the HO substrate hemin. Western blot showed that islet tissue expressed HO-2, and confocal microscopy revealed that HO-2 resided in insulin, glucagon, somatostatin, and pancreatic polypeptide cells. ZnPP-IX dose-dependently inhibited, whereas hemin enhanced, both insulin and glucagon secretion from glucose-stimulated islets. Stimulation or inhibition of CO production was accompanied by corresponding changes in islet cGMP levels. Exogenously applied CO stimulated insulin and glucagon release from isolated islets, whereas exogenous nitric oxide (NO) inhibited insulin and stimulated glucagon release. Islets stimulated by glucose or L-arginine displayed a marked increase in their NO-synthase (NOS) activity. Such an increase was suppressed by hemin, conceivably because NOS activity was inhibited by hemin-derived CO. Consequently, hemin enhanced L-arginine-induced insulin secretion. Insulin release stimulated by either hemin-derived CO or exogenous CO was strongly inhibited by the guanylate cyclase inhibitor ODQ, but it was unaffected by ZnPP-IX. Glucagon release induced by CO (but not by hemin) was inhibited by ODQ and partly inhibited by ZnPP-IX. We propose that the islets of Langerhans are equipped with a heme oxygenase-carbon monoxide pathway, which constitutes a novel regulatory system of physiological importance for the stimulation of insulin and glucagon release. This pathway is stimulated by glucose, is at least partly dependent on the cGMP system, and displays interaction with islet NOS activity.

Decreased glutathione transferase levels in rd1/rd1 mouse retina: replenishment protects photoreceptors in retinal explants.

Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.

Role of the pineal organ in the photoregulated hatching of the Atlantic halibut.

The timing of hatching in the Atlantic halibut (Hippoglossus hippoglossus) has been suggested to be regulated by environmental light conditions. However, the photosensory organ that perceives the triggering light has not been identified. In the present study, we investigated the morphogenesis of the pineal organ and the neurochemical differentiation of photoreceptors in the pineal organ and the retina of the Atlantic halibut during embryonic development. Immunocytochemical techniques were used for detection of integral protein components of the phototransduction process: opsins, arrestin (S-antigen) and alpha-transducin. We also studied the expression of serotonin (5-HT), a precursor of the neurohormone melatonin known to be synthesized by pineal photoreceptors. In the pineal anlage, opsin immunoreactive (ir) cells appear at 11 days post-fertilization (pf), arrestin, alpha-transducin and serotonin ir cells appear at 14 days pf; hatching took place 15 days pf. The retina contained no immunoreactive cells in embryos or in newly hatched larva. During this period, the pineal anlage is morphologically discernible only as a wedge-shaped region in the diencephalic roof, where elongated cells are aligned with their long axes converging toward a centrally located presumptive pineal lumen. The results show that the pineal photoreceptors contain serotonin and molecules involved in the phototransduction cascade before hatching. We suggest that the pineal organ has the capacity to perceive and mediate photic information before hatching in halibut embryos, and may thereby influence the timing of hatching.

Distribution of choline acetyltransferase-immunoreactive neurons in the brain of a cyprinid teleost (Phoxinus phoxinus L.).

The distribution of putative cholinergic neurons in the brain of a cyprinid teleost was investigated by immunocytochemistry, with well-characterized polyclonal antibodies to porcine choline acetyltransferase (ChAT), correlated with acetylcholinesterase (AChE) histochemistry. AChE-positive neurons were more numerous than ChAT-immunoreactive (ChAT-IR) neurons. Regions with ChAT-IR neurons generally also contained AChE-positive ones, but regions with AChE-positive neurons often did not contain (or contained only small numbers of) ChAT-IR neurons. ChAT-IR neurons were located in the brainstem cranial nerve motor nuclei, in the brainstem reticular formation, in the nucleus lateralis valvulae and an adjacent subnucleus "a," in the nucleus isthmi, and in the stratum griseum periventriculare of the tectum opticum. All neurons in these areas were AChE positive. ChAT-IR neurons were also observed within the boundaries of the nucleus sensibilis nervi trigemini and the n. descendens nervi trigemini. The periventricular hypothalamus and the paraventricular organ, the pineal organ, and (possibly) the nucleus suprachiasmaticus also contained ChAT-IR neurons. In these areas, AChE activity was either low or located mainly in neurons other than the ChAT-IR ones. A small population of ChAT-IR neurons was observed in area ventralis telencephali pars lateralis. This was the only telencephalic ChAT-IR cell group. Furthermore, some previously unrecognized cell groups were observed. A small number of ChAT-IR neurons, located on the dorsal aspect of the fasciculus longitudinalis medialis (caudal to n. raphe dorsalis), emitted axons that passed caudally along the raphe midline and innervated some of the large reticular neurons. Another group of ChAT-IR neurons was observed caudal to the thalamic nucleus centralis posterior and was tentatively designed n. tractus rotundus, on the basis of the neuronal morphology. The almost Golgilike staining of some of the ChAT-IR cell groups permitted the identification of their efferent connections and the areas covered by their dendrites.

Central neural connections of the pineal organ and retina in the teleost Gasterosteus aculeatus L.

The relations of the central neural connections of the pineal organ to those of the retinae of the lateral eyes were investigated in the three-spined stickleback, Gasterosteus aculeatus L. (Teleostei), by anterograde and retrograde transport of horseradish peroxidase (HRP). HRP was applied to the crushed pineal stalk and/or injected into the left or the right eye. Both pineal and retinal efferents project to area praetectalis, dorsal and ventral thalamic areas, and dorsal tegmentum. The most notable overlapping occurs in nucleus commissurae posterioris of area praetectalis. Pineal efferents also innervate the habenular nuclei and dorsal hypothalamus, while retinal efferents innervate rostral hypothalamus, ventrolateral thalamus, and tectum opticum. A small number of retinofugal axons recross and innervate the ipsilateral nucleus anterioris periventricularis and area praetectalis. After intraocular HRP injections, labeled perikarya were located both in retinofugal terminal areas and in areas not receiving direct retinal input, such as the telencephalic nucleus olfactoretinalis, deep tectal layers, and an area rostroventral to nucleus dorsolateralis thalami. No neurons afferent to the pineal organ were demonstrated. The close association of pineal efferents with retinofugal and possible retinopetal elements is in accordance with the view that both systems are potential neural mediators of photoperiodic events in the teleostean circadian system.

Distribution of Met-enkephalin, Leu-enkephalin, substance P, neuropeptide Y, FMRFamide, and serotonin immunoreactivities in the optic tectum of the Atlantic salmon (Salmo salar L.).

The distribution of the neuropeptides methionine- and leucine-enkephalins, substance P, FMRFamide, neuropeptide Y, and vasoactive intestinal peptide, as well as the biogenic amine serotonin was studied in the optic tectum of the Atlantic salmon by means of immunocytochemistry. Peroxidase-antiperoxidase and indirect immunofluorescence methods were used to compare the differential laminar distribution of each of these substances. Nine parts of the optic tectum were selected for analysis on frontal sections: median, dorsolateral, and ventrolateral areas at rostral, medial, and caudal levels. Methionine- and leucine-enkephalin immunoreactive fibers were found in discrete sublayers in the following strata: stratum opticum, stratum fibrosum et griseum superficiale, stratum griseum centrale, stratum, and album centrale. Most of the substance P-, serotonin-, and vasoactive intestinal peptide-immunoreactive fibers were found in the stratum album centrale, whereas the FMRFamide- and neuropeptide Y-immunoreactive fibers were more or less randomly distributed within most of the strata of the optic tectum. Neuropeptide Y-immunoreactive cell bodies were located in the stratum periventriculare. We suggest an extrinsic origin for most of the immunoreactive fibers observed in the optic tectum, except for the neuropeptide Y-immunoreactive fibers that probably originate in the periventricular neurons. Although retinal peptidergic input to the optic tectum has been proposed in other vertebrates, there is no evidence that any of the neuropeptidelike or serotonin immunoreactive fibers in the optic tectum of the salmon should be of retinal origin. Differences and similarities with the distribution of neuropeptides in the optic tectum in representatives of other vertebrate classes are discussed.

Electron microscopic analysis of S-antigen- and serotonin-immunoreactive neural and sensory elements in the photosensory pineal organ of the salmon.

Photoreceptor cells in the pineal complex of poikilothermic vertebrates are regarded as homologous with the neuroendocrine pinealocytes in the mammalian pineal organ. They possess an indolamine metabolism, and they contain a number of substances that are immunochemically similar to photo-transduction-related proteins otherwise found in photoreceptors of the lateral eye retina. Using correlative light and electron microscopic pre-embedding immunocytochemistry, we have identified photosensory and neural elements that are immunoreactive with specific antisera against serotonin (5-hydroxy-tryptamine) and the 48 kDa soluble protein S-antigen (arrestin). One type of serotonin-immunoreactive (5HTir) photoreceptor cell was identified. This was characterized by a short basal pole, into which an immunonegative (post-synaptic?) element protruded. Two types of S-antigen-immunoreactive (SAir) photoreceptor cells were observed, one characterized by a short basal pole, similar to that of the 5HTir photoreceptors and the other characterized by a long, extensively branching basal pole. In addition, two types of neurons bearing no morphological specializations typical of photoreceptor cells were SAir: bipolar neurons and multipolar neurons. These were often situated dorsally in the pineal organ. The results indicate an emergence of multiple lines of photoreceptor-derived "pinealocytes" either early in phylogeny, or independently in different taxa. The results are discussed in relation to current theories of pineal evolution.

An immunocytochemical study of the olfactory projections in the three-spined stickleback, Gasterosteus aculeatus, L.

The distribution of olfactory fibers in the brain of the three-spined stickleback was visualized by means of immunohistochemistry. The labeling of the olfactory fibers was produced by serum containing antibodies against somatostatin-14. Olfactory fibers were observed entering the olfactory bulbs, where they terminated in the glomerular layer or collected into fascicles that coursed through the bulbs into the telencephalon without participating in the formation of the glomerules. In the telencephalon the fascicles, which belonged to the medial olfactory tract, formed two fiber systems: ventral descending fibers and dorsal descending fibers. The ventral descending fibers could be followed through the ventral telencephalon to the vicinity of the lateral tuberal nucleus. The dorsal descending fibers coursed via the anterior commissure to the posterior part of the telencephalon. Part of the postcommissural fibers of the dorsal descending system coursed to the posterior zone of the area dorsalis telencephali while others left the telencephalon via the medial forebrain bundle and could be followed to the periventricular hypothalamus. Some axons formed synaptic contacts with unlabeled cell bodies in the nucleus of the terminal nerve which, in this species, is situated immediately behind the bulbs. In addition, an extensive terminal field associated with the dorsal descending fibers was found in the ventromedial aspects of the telencephalon. It is unlikely that the labeling represents immunoreactive somatostatin-14 because: 1) the labeling persisted after the absorption of the antiserum with synthetic somatostatin-14; 2) antiserum against somatostatin-14 from another manufacturer did not have this labeling property; and 3) the production of the absorbable labeling depended on the choice of fixative whereas the production of the unabsorbable labeling did not.

Distribution of 5-hydroxytryptamine (serotonin) in the brain of the teleost Gasterosteus aculeatus L.

The distributions of serotoninergic neurons in the brain of the three-spined stickleback was demonstrated with the indirect peroxidase-antiperoxidase (PAP) immunohistochemical method with antibodies against serotonin. Serotoninergic perikarya were demonstrated in the brainstem reticular formation (nucleus raphe dorsalis, nucleus raphe medialis, and nucleus tegmenti dorsalis lateralis) and in the periventricular ventral thalamus and hypothalamus (nucleus ventromedialis thalami, nucleus posterioris periventricularis, nucleus recessus lateralis, and nucleus recessus posterioris). After pharmacological pretreatment of the animals with a monoamine oxidase inhibitor, serotoninergic perikarya were also visualized in area praetectalis and in the medial brainstem, caudal to nucleus raphe medialis. Whereas the cell groups of the brainstem give rise to both ascending and descending pathways, it was not possible to analyze the distribution of efferent projections from the diencephalic cell groups. Distribution of serotoninergic axons showed marked regional differences. Only scattered varicose fibers were demonstrated in the cerebellum, the facial lobes, and the lateral line lobes. In the mesencephalon, the dorsal periventricular tegmentum and the central gray receive only small numbers of serotoninergic axons, while torus semicircularis and the visual layers of tectum opticum are profusely innervated. In the diencephalon, the hypothalamus and ventral thalamus generally display the highest density of serotoninergic axons. Exceptions are found in nucleus glomerulosus and the ventromedial portion of lobus inferioris, where densities are low. In the telencephalon, the density of serotoninergic axons is very high in area dorsalis pars medialis and pars lateralis dorsalis, but low in area dorsalis pars dorsalis and pars lateralis ventralis, and intermediate in area ventralis.

Galanin-like immunoreactivity in the brain of teleosts: distribution and relation to substance P, vasotocin, and isotocin in the Atlantic salmon (Salmo salar).

The presence of galanin-like substances and their relation to substance P-, vasotocin-, and isotocin-immunoreactive neurons and fibers in the brain of teleosts was investigated with immunohistochemical methods. Two specific antisera against synthetic porcine galanin (GAL) revealed cell bodies and fibers in the brain of four different teleost species (Salmo salar, Carassius carassius, Gasterosteus aculeatus, and Anguilla anguilla). In all four species the main location of galanin immunoreactivity was in the hypothalamo-pituitary region. A detailed study of the distribution of galanin immunoreactivity in S. salar showed that galanin immunoreactive (GALir) perikarya were present in the nucleus preopticus periventricularis, an area that may be compared to the supraoptic nucleus in mammals, and in the nucleus lateralis tuberis, a nucleus involved in pituitary control in fishes that may be compared with the arcuate nucleus in mammals. GALir perikarya were found also in the nucleus recessus lateralis and in the nucleus recessus posterior. Numerous GALir fibers were present in the telencephalon and diencephalon, whereas only small numbers of fibers were found in the brainstem. In contrast to the situation in mammals, no GALir perikarya were observed in the brainstem areas corresponding to the noradrenergic locus coeruleus and serotonergic raphe nuclei in S. salar. We did not find any coexistence of GALir substances with arginine vasotocin or isotocin in neurosecretory neurons, as has been shown for galanin with the mammalian counterparts vasopressin and oxytocin. Also, the galanin-like substance(s) and their structurally closest related peptide family, the tachykinins, belong to separate neuronal systems in teleosts. The presence of GALir neurons in brain areas known to be involved in pituitary control, and a massive GALir innervation of the pituitary, strongly indicate a role for galanin-like substances in pituitary control also in teleosts. Furthermore, the presence of extrahypothalamic GALir fibers suggests involvement of galanin-like substances in other brain functions in teleosts. In conclusion, there are general similarities between teleosts and mammals concerning the distribution of galanin-like substances. However, there seem to be substantial differences in their distribution relative to functionally related peptides within the hypothalamo-pituitary system. Whereas galanin appears to be colocalized and released together with vasopressin and oxytocin in mammals, in teleosts the homologous substances are contained within different sets of neurons that innervate the same target, the pituitary.

Ventricular proliferation zones in the brain of an adult teleost fish and their relation to neuromeres and migration (secondary matrix) zones.

Zones containing actively dividing cells (proliferation zones: PZs), in the brain of adult three-spined sticklebacks, were identified by autoradiographic detection of (3)H-thymidine and immunocytochemical detection of the thymidine analogue 5'-bromodeoxyuridine (BrdU), singly or in combination, and by immunocytochemical detection of proliferating cell nuclear antigen (PCNA) by monoclonal antibodies. The PZs are associated with boundaries between adult brain regions, as well as with defined morphofunctional subdivisions. PZs are located at the border between the telencephalon and diencephalon, and at the border between the mesencephalon and the rhombencephalon. In the midbrain, the PZ follows the dorsomedial, caudal, and ventrolateral aspects of each tectal hemisphere, extending over the caudal aspect of the torus semicircularis to the nucleus lateralis valvulae. In the hindbrain, the major PZ apparently represents the persisting embryonic secondary matrix layer of the developing cerebellum. In the forebrain, the PZs are associated with the ventricular zones of the olfactory bulbs and ventral telencephalic area ("subpallium"), dorsal telencephalic area ("pallium"), preoptic region, ventral thalamus, dorsal thalamus, epithalamus, pretectum, posterior tuberculum, and the hypothalamus. The diencephalic PZs are parcellated according to a neuromeric organisation (a synencephalic, a posterior, and an anterior parencephalic neuromere: p1, p2, and p3). The PZs of the secondary prosencephalon (telencephalon and hypothalamus) thus would belong to neuromeres p4-6, but do not form an immediately recognised serial pattern. The prosencephalic PZs correlate well with parts of embryonic migration areas as defined by Bergquist and Källén ([1954] J. Comp. Neurol. 100:627-659), morphogenetic fields from which postmitotic neurones migrate to their final destination.