Neurons In Fish Research Articles

Pecularities of the structure of glycogen as an indicator of the functional state of mauthner neurons in fish Percсottus glehni during wintering.

Mauthner neurons (MN)*, specific multifunctional neurons of fish, are a unique object for investigating the adaptive potentialities of the cell. The goal of the work was to study the structure of MN of fish Percсottus glehni during wintering under the conditions of hypothermia and deficit of oxygen and energy substrates. It was shown using a light microscope that the volume of the somatic moiety of neurons changes slightly at the beginning of wintering and is significantly reduced by the termination of wintering. It was found on the ultrastructural level that, at the beginning of wintering, glycogen exists in the central cytoplasm of MN in the form of large concentrated fields consisting of separate granules, whereas in the summer period the granules are distributed diffusely throughout the cell. In the vicinity of glycogen fields, lamellar structures of the smooth reticulum were seen. At the boundaries of glycogen fields, the aggregation of mitochondria and their active intrusion into glycogen fields were evident. By the end of wintering, the amount of glycogen significantly decreased, which was accompanied by a reduction of the smooth reticulum and rare contacts of mitochondria with glycogen fields. We assume that, because MN have a broader range of metabolic and functional possibilities than usual neurons in which glycogen is lacking or its content is negligible, they possess their own systems of glycolysis, glyconeogenesis, and deposition of glycogen. This allows them to maintain a sufficient level of energy supply under anaerobic conditions for performing the function of "guard neurons" during wintering.

An optimized method for counting dopaminergic neurons in zebrafish.

In recent years, considerable effort has been devoted to the development of a fish model for Parkinson’s disease (PD) to examine the pathological mechanisms of neurodegeneration. To effectively evaluate PD pathology, the ability to accurately and reliably count dopaminergic neurons is important. However, there is currently no such standardized method. Due to the relatively small number of dopaminergic neurons in fish, stereological estimation would not be suitable. In addition, serial sectioning requires proficiency to not lose any sections, and it permits double counting due to the large size of some of the dopaminergic neurons. In this study, we report an optimized protocol for staining dopaminergic neurons in zebrafish and provide a reliable counting method. Finally, using our optimized protocol, we confirmed that administration of 6-hydroxydopamine (a neurotoxin) or the deletion of the PINK1 gene (one of the causative genes of familiar PD) in zebrafish caused significant reduction in the number of dopaminergic and noradrenergic neurons. In summary, this method will serve as an important tool for the appropriate evaluation and establishment of fish PD models.

Visual Threat Assessment and Reticulospinal Encoding of Calibrated Responses in Larval Zebrafish

All visual animals must decide whether approaching objects are a threat. Our current understanding of this process has identified a proximity-based mechanism where an evasive maneuver is triggered when a looming stimulus passes a subtended visual angle threshold. However, some escape strategies are more costly than others, and so it would be beneficial to additionally encode the level of threat conveyed by the predator's approach rate to select the most appropriate response. Here, using naturalistic rates of looming visual stimuli while simultaneously monitoring escape behavior and the recruitment of multiple reticulospinal neurons, we find that larval zebrafish do indeed perform a calibrated assessment of threat. While all fish generate evasive maneuvers at the same subtended visual angle, lower approach rates evoke slower, more kinematically variable escape responses with relatively long latencies as well as the unilateral recruitment of ventral spinal projecting nuclei (vSPNs) implicated in turning. In contrast, higher approach rates evoke faster, more kinematically stereotyped responses with relatively short latencies, as well as bilateral recruitment of vSPNs and unilateral recruitment of giant fiber neurons in fish and amphibians called Mauthner cells. In addition to the higher proportion of more costly, shorter-latency Mauthner-active responses to greater perceived threats, we observe a higher incidence of freezing behavior at higher approach rates. Our results provide a new framework to understand how behavioral flexibility is grounded in the appropriate balancing of trade-offs between fast and slow movements when deciding to respond to a visually perceived threat.

MASH1/Ascl1a leads to GAP43 expression and axon regeneration in the adult CNS.

Unlike CNS neurons in adult mammals, neurons in fish and embryonic mammals can regenerate their axons after injury. These divergent regenerative responses are in part mediated by the growth-associated expression of select transcription factors. The basic helix-loop-helix (bHLH) transcription factor, MASH1/Ascl1a, is transiently expressed during the development of many neuronal subtypes and regulates the expression of genes that mediate cell fate determination and differentiation. In the adult zebrafish (Danio rerio), Ascl1a is also transiently expressed in retinal ganglion cells (RGCs) that regenerate axons after optic nerve crush. Utilizing transgenic zebrafish with a 3.6 kb GAP43 promoter that drives expression of an enhanced green fluorescent protein (EGFP), we observed that knock-down of Ascl1a expression reduces both regenerative gap43 gene expression and axonal growth after injury compared to controls. In mammals, the development of noradrenergic brainstem neurons requires MASH1 expression. In contrast to zebrafish RGCs, however, MASH1 is not expressed in the mammalian brainstem after spinal cord injury (SCI). Therefore, we utilized adeno-associated viral (AAV) vectors to overexpress MASH1 in four month old rat (Rattus norvegicus) brainstem neurons in an attempt to promote axon regeneration after SCI. We discovered that after complete transection of the thoracic spinal cord and implantation of a Schwann cell bridge, animals that express MASH1 exhibit increased noradrenergic axon regeneration and improvement in hindlimb joint movements compared to controls. Together these data demonstrate that MASH1/Ascl1a is a fundamental regulator of axonal growth across vertebrates and can induce modifications to the intrinsic state of neurons to promote functional regeneration in response to CNS injury.

Proliferation potential of Müller glia after retinal damage varies between mouse strains.

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors.

Improved tools for the Brainbow toolbox

In a recently introduced transgenic multicolor labeling strategy called “Brainbow,” Cre-lox recombination is used to create a stochastic choice of expression among fluorescent proteins (XFPs), resulting in the indelible marking of mouse neurons with multiple, distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but has yet to realize its full potential in the mouse brain. Here, we present several new lines of mice that overcome limitations of the initial lines and an adaptation of the method for use in adeno-associated viral (AAV) vectors. We also provide technical advice about how best to image Brainbow transgenes.

Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube.

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.

Increased Mauthner cell activity and escaping behaviour in seabream fed long-chain PUFA

There is limited information on the specific effects of long-chain PUFA (LCPUFA) on neuron development and functioning. Deficiency of those essential fatty acids impairs escape and avoidance behaviour in fish, where Mauthner cells (M-cells) play a particularly important role in initiating this response. Gilthead seabream larvae fed two different LCPUFA profiles were challenged with a sonorous stimulus. Feeding n-3 LCPUFA increased the content of these fatty acids in fish tissues and caused a higher number of larvae to react to the stimulus with a faster burst swimming speed response. This faster startle response in fish fed n-3 LCPUFA was also associated with an increased immune-positive neural response, particularly in M-cells, denoting a higher production of acetylcholine. The present study shows the first evidence of the effect of n-3 LCPUFA on the functioning of particular neurons in fish, the M-cells and the behaviour response that they modulate to escape from a sound stimulus.

Nemo through the looking-glass: a commentary on Desjardins & Fernald

Mirror-elicited aggression has been widely used in animal behaviour research. Recently, Desjardins & Fernald [[1][1]] reported that males of the cichlid fish Astatotilapia burtoni fighting a mirror had higher expression of immediate early genes in brain areas homologous to the amygdala and the

Endoreplication: A Molecular Trick during Animal Neuron Evolution

The occurrence of endoreplication has been repeatedly reported in many organisms, including protists, plants, worms, arthropods, molluscs, fishes, and mammals. As a general rule, cells possessing endoreplicated genomes are large-sized and highly metabolically active. Endoreplication has not been frequently reported in neuronal cells that are typically considered to be fully differentiated and non-dividing, and which normally contain a diploid genome. Despite this general statement, various papers indicate that giant neurons in molluscs, as well as supramedullary and hypothalamic magnocellular neurons in fishes, contain DNA amounts larger than 2C. In order to study this issue in greater detail here, we review the available data about endoreplication in invertebrate and vertebrate neurons, and discuss its possible functional significance. As a whole, endoreplication seems to be a sort of molecular trick used by neurons in response to the high functional demands that they experience during evolution.

Thermal activation of escape swimming in post-hatching Xenopus laevis frog larvae

Survival requires the selection of appropriate behavioural responses in the face of danger. With respect to the threat of predation, both the decision to escape and the underlying neuronal mechanisms have been extensively studied, but processes that trigger evasion of abiotic stressors, which are potentially hazardous to survival, are less well understood. Here, we document the interplay between rhythmic locomotory and 'C-start' escape swimming in Xenopus frog larvae when exposed to hyperthermic conditions. As temperature rises, swim cycle frequency increases while swim bout duration decreases, until swimming can no longer be initiated by sensory stimuli. Above a critical higher temperature, more intense sequences of spontaneous high amplitude C-start escape activity occur. Each C-start is followed by a few cycles of fast rhythmic swimming in which activity alternates between the two sides. The initial, high amplitude ventral root burst of an escape sequence propagates rostrocaudally approximately threefold faster than subsequent cycles. The high conduction velocity of this initial burst is consistent with the activation of a Mauthner neuron, one of a pair of giant reticulospinal neurons in fish and amphibians. In support of the involvement of a Mauthner neuron, unilateral lesions of the caudal hindbrain eliminated escape activity on the operated side, but activity remained on the un-operated side. Behaviourally, tadpoles responded to temperature ramps with a sequence of C-start responses in which the body arced through approximately 130 degrees in 22 ms, followed by high frequency swimming. These results suggest that high temperature activates the Mauthner neurons to trigger C-start escape behaviour.

Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

Uptake, tissue distribution and excretion of domoic acid after oral exposure in coho salmon ( Oncorhynchus kisutch)

Domoic acid (DA) is a potent neurotoxin naturally produced by some pennate diatom species of the genus Pseudo-nitzschia. It is well known that during harmful algal blooms fish can accumulate DA in the gastrointestinal (GI) tract and act as vectors of the toxin to higher trophic level piscivores, often with severe neurotoxic consequences to the predators. Although neurotoxicity and mass mortality have been observed in vertebrates (i.e. marine mammals and sea birds) feeding on contaminated fish, to date there has been no evidence of neurobehavioral toxicity in the fish vectors themselves. It has been hypothesized that fish may not absorb DA from the digestive tract, thus making them insensitive to dietary consumption of DA. To test this hypothesis, we performed oral gavage exposures followed by a time series of tissue dissections to characterize uptake, depuration, and tissue distribution of DA in fish. Intracoelomic (IC) injection exposures (which bypass the GI tract) were also performed to determine if coho neurons are neurologically susceptible to DA. Excitotoxic symptoms were observed in fish via IC injection at similar toxin levels that have been reported to induce excitotoxic symptoms in intraperitoneal (IP) exposures with mammalian models such as mice, suggesting that fish neurons have a similar sensitivity to DA as other vertebrates. Surprisingly, after oral gavage with ecologically relevant doses of DA, the toxin was detected in plasma collected from the dorsal aorta via a permanent intraarterial catheter within 15 min, yet excitotoxic symptoms were not observed. Additionally, DA was detected in liver, heart, spleen, kidney, muscle, brain and bile. These data indicate that although DA is absorbed from the gut, fish do not exhibit neuroexcitatory effects at maximum ecologically relevant oral doses of DA. Tissue distribution and DA uptake and depuration patterns suggest that a majority of the absorbed toxin is excreted via the kidneys and bile, thereby preventing toxic levels of DA from reaching sensitive nervous tissue. Additionally, greater than 20% of total IC administered DA doses were sequestered in bile within 1 h of injection in five symptomatic fish, providing evidence for biliary sequestration of the toxin from blood. Here, we comprehensively describe the uptake, depuration, and tissue distribution patterns of DA and propose that renal and biliary processes may serve as primary routes of toxin clearance in fish.

Early coevolution of adhesive but not antiadhesive tenascin-R ligand-receptor pairs in vertebrates: A phylogenetic study

Axon growth inhibitory CNS matrix proteins, such as tenascin-R (TN-R), have been supposed to contribute to the poor regenerative capacity of adult mammalian CNS. With regard to TN-R function in low vertebrates capable of CNS regeneration, questions of particular interest concern the (co)evolution of ligand-receptor pairs and cellular response mechanisms associated with axon growth inhibition and oligodendrocyte differentiation. We address here these questions in a series of comparative in vivo and in vitro analyses using TN-R proteins purified from different vertebrates (from fish to human). Our studies provide strong evidence that unlike TN-R of higher vertebrates, fish TN-R proteins are not repellent for fish and less repellent for mammalian neurons and do not interfere with F3/contactin- and fibronectin-mediated mammalian cell adhesion and axon growth. However, axonal repulsion is induced in fish neurons by mammalian TN-R proteins, suggesting that the intracellular inhibitory machinery induced by TN-R-F3 interactions is already present during early vertebrate evolution. In contrast to TN-R-F3, TN-R-sulfatide interactions, mediating oligodendrocyte adhesion and differentiation, are highly conserved during vertebrate evolution. Our findings thus indicate the necessity of being cautious about extrapolations of the function of ligand-receptor pairs beyond a species border and, therefore, about the phylogenetic conservation of a molecular function at the cellular/tissue level.

Olfactory Receptor Neurons in Fish: Structural, Molecular and Functional Correlates

Olfactory Receptor Neurons in Fish: Structural, Molecular and Functional Correlates

Activation of Mauthner neurons during prey capture

The Mauthner (M-) cells, a bilateral pair of medullary neurons in fish, initiate the characteristic “C-start” predatory escape response of teleosts. Similar movements have been described during hatching, social interactions, and feeding. M-cell firing, however, has not been correlated directly with these other behaviors. The objective of this study was to determine whether the M-cell, in addition to escape, plays a role in feeding. 1. Goldfish were chronically implanted with electrodes positioned near the axon cap of one of the two M-cells. Subsequently, M-cell activity was monitored for up to 8 days while fish were surface feeding on live crickets. 2. The M-cell fires and the fish performs a C-shaped flexion in association with the terminal phase of prey capture. Thus, the M-cell is active in the context of at least two behaviors, predator escape and prey capture, and may be considered a part of behaviorally shared neural circuitry. 3. For the goldfish, Mauthner-initiated flexions during feeding rapidly remove the prey from the water's surface and minimizes the fish's own susceptibility to surface predation. Other species may possess a diverse repertoire of Mauthner-mediated feeding behaviors that depend on their adaptive specializations for predation. Moreover, group competition between predators and their prey may have facilitated a “neural arms race” for M-cell morphology and physiology.

L1 immunoreactivity in the developing fish visual system

Previous studies have suggested that L1, the cell adhesion molecule, is present in the regenerating fish optic nerve. The present study was undertaken in order to determine whether L1 is expressed by fish neurons and specifically by non-myelinated axons, using fish retinal explants in vitro and the developing fish visual system in vivo. In vitro, the nonmyelinated axons emerging from retinal explants showed L1 immunoreactivity and in vivo, L1 immunoreactive sites were found to be associated with areas rich in non-myelinated axons. At embryonic stage 23, as the eye developed and optic nerve axons began to elongate towards the tectum, L1-like immunoreactivity was seen both in optic nerve and in plexiform layers of the retina. At this stage and until hatching, the cellular layers within the retina showed little or no staining relative to the layers containing axons or dendrites. After hatching, L1 immunoreactivity was also observed in the ganglion cell layer, but upon maturation both the retina and the optic nerve lost most of their L1 immunoreactivity. We therefore suggest that non-myelinated axons of the fish visual system express L1 during development, which is lost after myelination and presumably reappears during regeneration.

Swimbladder acoustic pressure transduction initiates Mauthner-mediated escape

THE bilateral Mauthner neurons initiate the characteristic 'C-start'1,2 escape of fishes when they respond to underwater sounds accompanying attacks by predators3–6. Either of the two sound components, particle motion or pressure change can initiate Mauthner-mediated escape. Particle motion can be detected by inner ear sensory receptors and used for locating a sound source7, but sound pressure requires a transduction mechanism to stimulate the same receptors8–10. For fishes, sound pressure is a nondirec-tional scalar signal that is transduced by the swimbladder11,12. It might be assumed that particle motion, because of its directional nature, triggers Mauthner-initiated predator evasion2, but we demonstrate here that for acoustically mediated escape, sound pressure is the salient stimulus for activating the Mauthner neurons in fishes with swimbladders. This implies that displacement determines which of the two Mauthner neurons is activated, so that the fish turns away from the direction of the predatory attack.

Early development of spiny neurons in fish and mouse: Morphometric measures of dendritic spine formation pattern

Positions of spines on apical dendrites were evaluated using 4 pattern analysis techniques: spine counts, variance/mean ratio, Lloyd's patchiness index, and nearest neighbor distance matrix. Spiny tectal interneurons from jewel fish (100, 130, 160 and 1550 days old), and layer V pyramidal cells in layer IV auditory cortex of CBA/J mice (100 and 450 days old) were studied. Fish's spines became more numerous and more clumped on distal dendritic strata during development, while mice lost dendritic spines with age. Both species developed a significantly regular spacing pattern between neighboring spines during development. These changes are explained in the context of spine function and a biophysical model of dendritic spine patterns.

Characteristics of electrical activity of olfactory bulb neurons of fish

It was shown by intracellular recording of the activity of olfactory bulb neurons of the carp that their dendrites are excited both by synaptic activation and by direct stimulation with an electric current. The dendrites generate an action potential and probably conduct it for some distance toward the soma. The neurons can be divided into two groups: one responds to ortho- and antidromic stimuli with one, rarely with two peaks, the other responds with a rhythmical discharge. The presence of early and late IPSP is characteristic of neurons of both groups. Rhythmical variations in potential with a frequency of 26–33/sec, so-called oscillations, are recorded; they may be excitatory (in secondary neurons they correspond to EPSP) or inhibitory (they correspond to IPSP). Possible mechanisms of the excitatory oscillations and the rhythmical discharge in olfactory bulb neurons of the carp are discussed.