Plasticity of Dopaminergic Phenotype and Locomotion in Larval Zebrafish Induced by Brain Excitability Changes during the Embryonic Period.

During the embryonic period, neuronal communication starts before the establishment of the synapses with alternative forms of neuronal excitability, called here embryonic neural excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs, but the global consequences for developing organisms are not all understood. Here, we monitored calcium (Ca2+) transients in the telencephalon of zebrafish embryos as a proxy for ENE to assess the efficacy of transient pharmacological treatments to either increase or decrease ENE. Increasing or decreasing ENE at the end of the embryonic period promoted an increase or a decrease in the numbers of dopamine (DA) neurons, respectively. This plasticity of dopaminergic specification occurs in the subpallium (SP) of zebrafish larvae at 6 d postfertilization (dpf), within a relatively stable population of vMAT2-positive cells. Nondopaminergic vMAT2-positive cells hence constitute an unanticipated biological marker for a reserve pool of DA neurons that can be recruited by ENE. Modulating ENE also affected larval locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted hyperlocomotion of larvae at 6 dpf, reminiscent of zebrafish endophenotypes reported for attention deficit hyperactivity disorders (ADHDs). These results provide a convenient framework for identifying environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification.

Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish.

The zebrafish is an important vertebrate model for disease, drug discovery, toxicity, embryogenesis, and neuroscience. In vivo fluorescence microscopy can reveal cellular and subcellular details down to the molecular level with fluorescent proteins (FPs) currently the main tool for zebrafish imaging. However, long maturation times, low brightness, photobleaching, broad emission spectra, and sample autofluorescence are disadvantages that cannot be easily overcome by FPs. Here, a bright and photostable terbium-to-quantum dot (QD) Förster resonance energy transfer (FRET) nanoprobe with narrow and tunable emission bands for intracellular in vivo imaging is presented. The long photoluminescence (PL) lifetime enables time-gated (TG) detection without autofluorescence background. Intracellular four-color multiplexing with a single excitation wavelength and in situ assembly and FRET to mCherry demonstrate the versatility of the TG-FRET nanoprobes and the possibility of in vivo bioconjugation to FPs and combined nanoprobe-FP FRET sensing. Upon injection at the one-cell stage, FRET nanoprobes can be imaged in developing zebrafish embryos over seven days with toxicity similar to injected RNA and strongly improved signal-to-background ratios compared to non-TG imaging. This work provides a strategy for advancing in vivo fluorescence imaging applications beyond the capabilities of FPs.

Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates).

Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.

Dopaminergic Neurons Controlling Anterior Pituitary Functions: Anatomy and Ontogenesis in Zebrafish.

Dopaminergic (DA) neurons located in the preoptico-hypothalamic region of the brain exert a major neuroendocrine control on reproduction, growth, and homeostasis by regulating the secretion of anterior pituitary (or adenohypophysis) hormones. Here, using a retrograde tract tracing experiment, we identified the neurons playing this role in the zebrafish. The DA cells projecting directly to the anterior pituitary are localized in the most anteroventral part of the preoptic area, and we named them preoptico-hypophyseal DA (POHDA) neurons. During development, these neurons do not appear before 72 hours postfertilization (hpf) and are the last dopaminergic cell group to differentiate. We found that the number of neurons in this cell population continues to increase throughout life proportionally to the growth of the fish. 5-Bromo-2'-deoxyuridine incorporation analysis suggested that this increase is due to continuous neurogenesis and not due to a phenotypic change in already-existing neurons. Finally, expression profiles of several genes (foxg1a, dlx2a, and nr4a2a/b) were different in the POHDA compared with the adjacent suprachiasmatic DA neurons, suggesting that POHDA neurons develop as a distinct DA cell population in the preoptic area. This study offers some insights into the regional identity of the preoptic area and provides the first bases for future functional genetic studies on the development of DA neurons controlling anterior pituitary functions.

Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain.

Regionalization is a critical, highly conserved step in the development of the vertebrate brain. Discrepancies exist in how regionalization of the anterior vertebrate forebrain is conceived since the "preoptic area" is proposed to be a part of the telencephalon in tetrapods but not in teleost fish. To gain insight into this complex morphogenesis, formation of the anterior forebrain was analyzed in 3D over time in zebrafish embryos, combining visualization of proliferation and differentiation markers, with that of developmental genes. We found that the region containing the preoptic area behaves as a coherent morphogenetic entity, organized around the optic recess and located between telencephalon and hypothalamus. This optic recess region (ORR) makes clear borders with its neighbor areas and expresses a specific set of genes (dlx2a, sim1a and otpb). We thus propose that the anterior forebrain (secondary prosencephalon) in teleosts contains three morphogenetic entities (telencephalon, ORR and hypothalamus), instead of two (telencephalon and hypothalamus). The ORR in teleosts could correspond to "telencephalic stalk area" and "alar hypothalamus" in tetrapods, resolving current inconsistencies in the comparison of basal forebrain among vertebrates.

Evolution of dopamine receptor genes of the D1 class in vertebrates.

The receptors of the dopamine neurotransmitter belong to two unrelated classes named D1 and D2. For the D1 receptor class, only two subtypes are found in mammals, the D1A and D1B, receptors, whereas additional subtypes, named D1C, D1D, and D1X, have been found in other vertebrate species. Here, we analyzed molecular phylogeny, gene synteny, and gene expression pattern of the D1 receptor subtypes in a large range of vertebrate species, which leads us to propose a new view of the evolution of D1 dopamine receptor genes. First, we show that D1C and D1D receptor sequences are encoded by orthologous genes. Second, the previously identified Cypriniform D1X sequence is a teleost-specific paralog of the D1B sequences found in all groups of jawed vertebrates. Third, zebrafish and several sauropsid species possess an additional D1-like gene, which is likely to form another orthology group of vertebrate ancestral genes, which we propose to name D1E. Ancestral jawed vertebrates are thus likely to have possessed four classes of D1 receptor genes-D1A, D1B(X), D1C(D), and D1E-which arose from large-scale gene duplications. The D1C receptor gene would have been secondarily lost in the mammalian lineage, whereas the D1E receptor gene would have been lost independently in several lineages of modern vertebrates. The D1A receptors are well conserved throughout jawed vertebrates, whereas sauropsid D1C receptors have rapidly diverged, to the point that they were misidentified as D1D. The functional significance of the D1C receptor loss is not known. It is possible that the function may have been substituted with D1A or D1B receptors in mammals, following the disappearance of D1C receptors in these species.

Neurotransmitter phenotype plasticity: an unexpected mechanism in the toolbox of network activity homeostasis.

The transmitter phenotype of a neuron has long been thought to be stable for the lifespan. Much as eyes have one color and do not change it over time, neurons have been thought to have one neurotransmitter and retain it for their lifetime. Both principles, exclusivity and stability, are challenged by recent data. More and more neurons in different regions of the brain appear to coexpress two or more neurotransmitters. Moreover, the profile of neurotransmitter expression of a given neuron has been shown to change over time, both during development and in response to changes in activity. The present review summarizes recent studies of this neurotransmitter phenotype plasticity (NPP). Homeostatic mechanisms of plasticity are aimed at maintaining the system within a functional range. They appear to be critical for optimal network operations and have been thought to operate largely by regulating intrinsic excitability, synapse number and synaptic strength. NPP provides a new and unexpected level of regulation of network homeostasis. We propose that it provides the basis for NT coexpression and discuss emerging issues and new questions for further studies in coming years.

Activity-dependent expression of Lmx1b regulates specification of serotonergic neurons modulating swimming behavior.

Genetic programs, environmental factors, and electrical activity interact to drive the maturation of the brain. Although the cascade of transcription factors that leads to specification of the serotonergic phenotype has been well characterized, its interactions with electrical activity are not known. Here we show that spontaneous calcium spike activity in the hindbrain of developing Xenopus laevis larvae modulates the specification of serotonergic neurons via regulation of expression of the Lmx1b transcription factor. Activity acts downstream of Nkx2.2 but upstream of Lmx1b, leading to regulation of the serotonergic phenotype. Using global manipulation of activity and targeted alteration of Lmx1b expression, we also demonstrate that changes in the number of serotonergic neurons change larval swimming behavior. The results link activity-dependent regulation of a transcription factor to transmitter specification and altered behavior.

A noncanonical release of GABA and glutamate modulates neuronal migration.

Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

Glutamate transporters prevent the generation of seizures in the developing rat neocortex.

Glutamate transporters are operative at an early developmental stage well before synapse formation, but their functional significance has not been determined. We now report that blockade of glutamate transporters in the immature neocortex generates recurrent NMDA receptor-mediated currents associated with synchronous oscillations of [Ca2+]i in the entire neuronal population. Intracerebroventricular injections of the blocker to pups generate seizures that are prevented by coinjections of NMDA receptor blockers. Therefore, the early expression of glutamate transporters plays a central role to prevent the activation by local glutamate concentrations of NMDA receptors and the generation of seizures that may alter the construction of cortical networks. A dysfunction of glutamate transporters may be a central event in early infancy epilepsy syndromes.

Paracrine intercellular communication by a Ca2+- and SNARE-independent release of GABA and glutamate prior to synapse formation.

GABA and glutamate receptors are expressed in immature "silent" CA1 pyramidal neurons prior to synapse formation, but their function is unknown. We now report the presence of tonic, spontaneous, and evoked currents in embryonic and neonatal CA1 neurons mediated primarily by the activation of GABA(A) receptors. These currents are mediated by a nonconventional release of transmitters, as they persist in the presence of calcium channel blockers or botulinium toxin and are observed in Munc18-1-deficient mice in which vesicular release is abolished. This paracrine communication is modulated by glutamate but not GABA transporters, which do not operate during this period of life. Thus, a Ca(2+)- and SNARE-independent release of transmitters underlies a paracrine mode of communication before synapse formation.