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.

Mecanismos de compensación en la enfermedad de Parkinson / No disponible

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Isthmus organizer for mesencephalon and metencephalon.

The vertebrate central nervous system is elaborated from a simple neural tube. Brain vesicles formation is the first sign of regionalization. Classical transplantation using quail and chick embryos revealed that the mesencephalon-metencephalon boundary (isthmus) functions as an organizer of the mesencephalon and metencephalon. Fgf8 is accepted as a main organizing molecule of the isthmus. Strong Fgf8 signal activates the Ras-ERK signaling pathway to differentiate the cerebellum. In this review, the historical background of the means of identifying the isthmus organizer and the molecular mechanisms of signal transduction for tectum and cerebellum differentiation is reviewed.

Analysis and visualization of cell movement in the developing zebrafish brain.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Regulation of isthmic Fgf8 signal by sprouty2.

Fgf8 functions as an organizer at the mes/metencephalic boundary (isthmus). We showed that a strong Fgf8 signal activates the Ras-ERK signaling pathway to organize cerebellar differentiation. Sprouty2 is expressed in an overlapping manner to Fgf8, and is induced by Fgf8. Its function, however, is indicated to antagonize Ras-ERK signaling. Here, we show the regulation of Fgf8 signaling in relation to Sprouty2. sprouty2 expression was induced very rapidly by Fgf8b, but interfered with ERK activation. sprouty2 misexpression resulted in a fate change of the presumptive metencephalon to the mesencephalon. Misexpression of a dominant negative form of Sprouty2 augmented ERK activation, and resulted in anterior shift of the posterior border of the tectum. The results indicate that Fgf8 activates the Ras-ERK signaling pathway to differentiate the cerebellum, and that the hyper- or hypo-signaling of this pathway affects the fate of the brain vesicles. Sprouty2 may regulate the Fgf8-Ras-ERK signaling pathway for the proper regionalization of the metencephalon and mesencephalon.

Involvement of actin in the electrotonic conductivity of mixed synapses in Mauthner neurons in the goldfish.

The effects of phalloidin, a preperation which highly specifically and selectively polymerizes actin and which binds to actin, on the electrotonic conductivity and structure of mixed synapses were studied in goldfish Mauthner neurons (MN). These experiments showed that paired subthreshold electrical stimulation of the afferent input in the presence of phalloidin led to increases in the amplitude of MN responses to the second stimulus by an average of 80%. In controls, this amplitude increased by only 10% and only when suprathreshold stimuli were used, while subthreshold stimuli were ineffective. We regard these results as demonstrating increases in the conductivity of mixed synapses, this being induced by polymerization of actin. At the ultrastructural level, application of phalloidin to MN and their mixed synapses induced increases in the sizes and numbers of actin-containing desmosome-like contacts, and in the numbers of fibrillar bridges in the clefts of these contacts. Use of colloidal gold as a label for phalloidin demonstrated that bridges were made of actin. The interdependent morphofunctional changes seen in mixed synapses provide grounds for suggesting a role for actin in the conduction of the electrotonic signal through mixed synapses. The structural substrate for this process may be provided by bridges in the clefts of desmosome-like contacts.

Ultrastructure of desmosome-like contacts of mixed synapses of Mauthner neurons in long-term potentiation.

Electron microscopic morphometry was used to study the effects of long-term potentiation on the structure of fibrillar "cross-bridges" in the clefts of desmosome-like contacts in mixed synapses in Goldfish Mauthner neurons. These experiments showed that the number of bridges increased as the level of potentiation of electrotonic transmission increased. The structure of bridges changed after potentiation, which did not occur in controls (incubation). Double bridges appeared, which could have an altered (from control) organization within the cleft. The results obtained here and previously suggest that the bridges may be made of actin. Bridges are evidently a channel in which actin is organized as in nanotubules or plasmodesmata, and this may explain the stability of the bridge structure to treatment with cytochalasin and other external damaging factors which we have observed.

Functional circuitry involved in the regulation of whisker movements.

Neuroanatomical tract-tracing methods were used to identify the oligosynaptic circuitry by which the whisker representation of the motor cortex (wMCx) influences the facial motoneurons that control whisking activity (wFMNs). Injections of the retrograde tracer cholera toxin subunit B into physiologically identified wFMNs in the lateral facial nucleus resulted in dense, bilateral labeling throughout the brainstem reticular formation and in the ambiguus nucleus as well as predominantly ipsilateral labeling in the paralemniscal, pedunculopontine tegmental, Kölliker-Fuse, and parabrachial nuclei. In addition, neurons in the following midbrain regions projected to the wFMNs: superior colliculus, red nucleus, periaqueductal gray, mesencephalon, pons, and several nuclei involved in oculomotor behaviors. Injections of the anterograde tracer biotinylated dextran amine into the wMCx revealed direct projections to the brainstem reticular formation as well as multiple brainstem and midbrain structures shown to project to the wFMNs. Regions in which retrograde labeling and anterograde labeling overlap most extensively include the brainstem parvocellular, gigantocellular, intermediate, and medullary (dorsal and ventral) reticular formations; ambiguus nucleus; and midbrain superior colliculus and deep mesencephalic nucleus. Other regions that contain less dense regions of combined anterograde and retrograde labeling include the following nuclei: the interstitial nucleus of medial longitudinal fasciculus, the pontine reticular formation, and the lateral periaqueductal gray. Premotoneurons that receive dense inputs from the wMCx are likely to be important mediators of cortical regulation of whisker movements and may be a key component in a central pattern generator involved in the generation of rhythmic whisking activity.

Crossing the floor plate triggers sharp turning of commissural axons.

During development of the vertebrate CNS, commissural axons initially grow circumferentially toward the ventral midline floor plate. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. Although recent studies have unraveled the mechanisms that control navigation of these axons along the circumferential axis, those that result in the transition from circumferential to longitudinal trajectory remain unknown. Here, we examined whether an interaction with the floor plate is a prerequisite for the initiation of trajectory transition of commissural axons, using in vitro preparations of the rat metencephalon. We found that commissural axons in the metencephalon, once having crossed the floor plate, turned sharply to grow longitudinally. In contrast, axons extending in floor plate-deleted preparations, continued to grow circumferentially, ignoring the hypothetical turning point. These results suggest that a prior interaction of commissural axons with floor plate cells is a key step for these axons to activate a navigation program required for their change in axonal trajectory from the circumferential to the longitudinal axis.

Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo.

We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 microM), kynurenic acid (1 mM), and bicuculline (10 microM) and were found to be sensitive to strychnine (1 microM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of approximately 0.04 Hz, which increased to approximately 0.5 Hz by 30-40 hpf, and was approximately 10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar ( approximately 100 pA), suggesting rapid formation of the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (approximately 0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (tau(off1) and tau(off2)) time courses with a half-width in 26-29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, tau(off1) was approximately 15 ms and tau(off2) was approximately 60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau(off1) was approximately 40 ms and tau(off2) was approximately 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) approximately 9 ms and tau(off2) approximately 40 ms, and in larvae, events declined somewhat further to tau(off1) approximately 4 ms and tau(off2) approximately 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as approximately 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3-10 mM) to outside-out patches obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.

LHRH neuronal migration: heterotypic transplantation analysis of guidance cues.

During embryonic development, the olfactory placode (OP) differentiates into the olfactory epithelium (OE). Luteinizing hormone-releasing hormone (LHRH) neurons migrate out of the OE in close association with the olfactory nerve (ON) to the telencephalon. LHRH neuronal migration and ON extension to the telencephalon may be independent events which are correlated but do not represent a causal relationship. However, we hypothesize that LHRH neurons are dependent on ON axons to migrate to the brain. To test this hypothesis, we ablated the right trigeminal placode and replaced it with an OP from another chick embryo. After several days' additional incubation, the embryos were fixed, sectioned, and immunostained with antibodies against LHRH or N-CAM. The ectopic OPs were well integrated into the host and developed into relatively normal appearing OEs. The ONs extended from the OE to several different sites: the lateral rectus of the eye, the ciliary ganglion, and the trigeminal ganglion. In all cases, LHRH neurons were found in the OE and ON, regardless of where the ON terminated. When the ON extended to the trigeminal ganglion, LHRH neurons could clearly be seen entering the metencephalon. Our results support the idea that LHRH neurons are dependent on the ON for guidance as they appear to follow the nerve even when it extends away from the brain. The cues which direct the ON and LHRH neurons to the telencephalon do not appear to be unique to this brain region.