Plasticity of the dopaminergic phenotype and of locomotion in larval zebrafish induced by changes in brain excitability 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 Neuronal Excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs but the global consequences for the developing organisms are not all understood. Here we monitored calcium transients in 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 for 24 hours at 2 days post fertilization (dpf), at the end of the embryonic period, promoted respectively an increase or a decrease in the numbers of dopamine (DA) neurons in the telencephalon and in the olfactory bulb of zebrafish larvæ at 6 dpf. This plasticity of dopaminergic specification occurs within a stable population of vMAT2-positive cells, hence identifying an unanticipated biological marker for this reserve pool of of DA neurons that can be recruited by increasing 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 larvæ at 6 dpf, reminiscent of endophenotypes reported for Attention Deficit with Hyperactivity Disorders and schizophrenia in zebrafish. These results provide a convenient framework to identify environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification, with relevance to the pathogenesis of neurodevelopmental disorders. Significance Statement - Spontaneous calcium transients, used as a proxy for Embryonic Neuronal Excitability (ENE), are detected in the forebrain of embryonic zebrafish. - Short-term pharmacological treatments by bath application could increase or decrease ENE. - The post-mitotic differentiation of the dopaminergic phenotype is modulated by ENE in the zebrafish forebrain. - The plasticity of the dopaminergic specification occurs within a reserve pool of vMAT2-positive cells. - Transient increase of ENE at the end of the embryonic period induces hyperlocomotion, a phenotype associated with ADHD and schizophrenia in this model. - Our results open clinically relevant perspectives to study the pathogenesis of neurodevelopmental disorders in zebrafish.

here as Ca 2+ transients, are sporadic, long lasting global increases of intracellular 48 concentration of Ca 2+ that occurs during restricted developmental windows called "critical present in zebrafish and respond to psychoactive drugs (Gawel et al., 2019). In addition, 73 the monoaminergic system has been extensively studied in zebrafish (Schweitzer & 74 Driever, 2009; Schweitzer et al., 2012). In vertebrates, DA modulates executive functions, 75 such as working memory and decision making, through innervation of specific regions of 76 the telencephalon. In the zebrafish brain, most DA neurons innervating the telencephalon 77 have their cell bodies located within the telencephalon itself (SP-DA cells) (Tay et al.,78 2011; Yamamoto et al., 2011). To the best of our knowledge, plasticity of the 79 neurotransmitter phenotype in these cells has not been studied so far. 80 To perturb ENE in conditions close to physiological exposure and ecotoxicology, we   Zebrafish were raised according to standards procedures (Westerfield, 2000). Briefly, 96 for breeding, male and female zebrafish were placed overnight, in different compartments 97 of a tank with a grid at the bottom that allows the eggs to fall through. The next morning 98 the separation was removed and after few minutes, the eggs were collected, rinsed and 99 placed in a Petri dish containing embryo medium (EM). Embryos were kept at 28 C, then 100 staged according to standard criteria. The number of animals used for each experiment 101 is indicated in the corresponding figure legends.

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Wild-type zebrafish were of AB background . at RT and were washed overnight in PBST.

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Timing of experiments 246 To study the contribution of Embryonic Neuronal Excitability (ENE) to zebrafish larval channels. We refer to this cocktail as TCNF.

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Spontaneous calcium transients in the brain of zebrafish embryos. 259 To evaluate the level of Embryonic ENE in the embryonic zebrafish brain, we used Ca 2+ 260 transients as a proxy. We followed the dynamics of intracellularCa 2+ concentration using To assess the ability of the pharmacological treatments to modify ENE, we measured 288 their impact on Ca 2+ signaling. We performed time-lapse recordings two to ten hours after 289 the addition of the treatments to the embryo medium. Here again, the results obtained 290 were similar in both genetic backgrounds. Following veratridine treatment, the four 291 parameters analyzed were increased ( Figure 1D-K, green symbols). In contrast, following 292 TCNF treatments, three of the analyzed parameters were decreased, frequency, 293 incidence, and amplitude while the duration was increased ( Figure 1D-K, red symbols).

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These results demonstrate that bath-mediated pharmacological treatments were able 295 to change the incidence, frequency and amplitude of spontaneous Ca 2+ transients in the 296 embryonic zebrafish forebrain. When analyzing the late consequences of these transient 297 treatments, we refer to the veratridine treatment as "increased ENE", and to TCNF 298 treatment as "decreased ENE". Effects of pharmacological treatments on the locomotion of 6-7 dpf larvae. 371 In order to analyze the consequence of the modifications of ENE on behaviors in 372 zebrafish larvae, we focused our analysis on spontaneous locomotion.

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During locomotion, zebrafish larvae display successive turn and swim bouts called 374 episodes, interleaved with resting periods. This behavior can be analyzed by semi-375 automated methods, detecting 4 parameters, the number of episodes, the average 376 duration of swimming, the distance covered and the average speed. 377 We compared these 4 locomotion parameters recorded at 6-7 dpf, more than 3 days 378 after the end of ENE manipulation ( Figure 1A). We recorded spontaneous locomotion

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The number of cruises was reduced following either the increase or the decrease of 393 ENE ( Figure 4F). The three other parameters, duration, distance and speed, were 394 increased in both conditions ( Figure 4G-J).

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By contrast, increase and decrease of ENE had opposite effects on burst episodes.

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Increased ENE resulted in more frequent, faster and longer bursts than in control 397 conditions, while they were scarcer, shorter and slower after ENE decrease 398 ( Figure 4J-M).

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To rule out the possibility that the differences observed following modifications of ENE

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For the present study, zebrafish embryos were exposed to pharmacological of these candidates will be tested in the future.

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Indirect mechanisms may also be involved. Ca 2+ transients have been shown to     A. Maximum projection of confocal z series of the brain of 6-7 dpf Et(VMAT2:eGFP) larvae, following ENE increase (left column, boxed in green), in control conditions (middle column, boxed in blue), and following ENE decrease (right column, boxed in red). Immunostaining to TH (magenta) and GFP (cyan) and DAPI (yellow) are shown as composite image (top) as well as for each color channels. Scale bars = 50 µm.

B-C. Boxplots showing the number of cells IR + for dopaminergic markers in the SP (B)
and the OB (C) from 6 independent experiments, in control conditions (blue), following ENE increase (green) and following ENE decrease (red  A. Maximum projection of confocal z series of the brain of 6-7 dpf Et(VMAT2:eGFP) larvae, following ENE increase (left image, boxed in green), in control conditions (middle image, boxed in blue), and following ENE decrease (right image, boxed in red).

B.
Boxplots showing the number of the caspase-3 + IR cells counted in the telencephalonfollowing ENE increase (green), in control conditions (blue) and following ENE decrease (red). KW test, 23<n<26 telencephalon from 4 independent experiments.