Endogenous dopamine suppresses initiation of swimming in pre - feeding zebrafish

Dopamine is a key neuromodulator of locomotory circuits, yet, the role that dopamine plays during development of these circuits is less well understood. Here, we describe a suppressive effect of dopamine on swim circuits in larval zebrafish. Zebrafish larvae exhibit marked changes in swimming behavior between 3 days post fertilization (dpf) and 5dpf. We found that swim episodes were fewer and of longer durations at 3dpf than at 5dpf. At 3dpf, application of dopamine as well as bupropion, a dopamine reuptake blocker, abolished spontaneous fictive swim episodes. Blocking D2 receptors increased frequency of occurrence of episodes and activation of adenylyl cyclase, a downstream target inhibited by D2-receptor signaling, blocked the inhibitory effect of dopamine. Dopamine had no effect on motor neuron firing properties, input impedance, resting membrane potential or the amplitude of spike after-hyperpolarization. Application of dopamine either to the isolated spinal cord or locally within the cord does not decrease episode frequency, whereas dopamine application to the brain silences episodes suggesting a supraspinal locus of dopaminergic action. Treating larvae with 10 M 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine hydrochloride (MPTP) reduced catecholaminergic innervation in the brain and increased episode frequency. These data indicate that dopamine inhibits the initiation of fictive swimming episodes at 3dpf. We found that at 5dpf, exogenously applied dopamine inhibits swim episodes yet the dopamine reuptake blocker or the D2 receptor antagonist have no effect on episode frequency. These results lead us to propose that endogenous dopamine release transiently suppresses swim circuits in developing zebrafish. Introduction Locomotion is achieved by the rhythmic activity of motor-pattern generating circuits (Grillner 2003; Kiehn 2006). Descending projections to these pattern generating circuits regulate their activation through the release of fast-acting neurotransmitters and slower acting neuromodulators (Barriere et al. 2005; El Manira et al. 1997; Li et al. 2006; Marder and Bucher 2001; McLean and Sillar 2003; Nishimaru et al. 2000; Roberts et al. 1998). As an animal develops, its locomotory behavior becomes more flexible and mature (Clarac et al. 2004; Saint-Amant and Drapeau 1998; Sillar et al. 1991) and in some cases, even undergoes dramatic changes (Combes et al. 2004). Proper maturation of locomotory behavior requires maturational changes in the neural circuits generating motor commands. Neuromodulators have been implicated in triggering the developmental maturation of pattern generating circuits (Branchereau et al. 2002; Brustein et al. 2003a; Fenelon et al. 2003; Sillar et al. 1995; Straus et al. 2000) and they may achieve this by affecting neurogenesis (Marsh-Armstrong et al. 2004), synaptogenesis (Niitsu et al. 1995), synaptic strength (McDearmid et al. 1997), intrinsic membrane properties of individual neurons within the network (Han et al. 2007; Sillar et al. 1995) or by changing the influence of other neuromodulators on target networks (McLean and Sillar 2004). Dopamine is a key neuromodulator involved in the control of motor systems in both invertebrates and vertebrates (Crisp and Mesce 2004; Kiehn and Kjaerulff 1996; Marder and Eisen 1984; Schotland et al. 1995). Loss of brain stem dopaminergic neurons leads to movement disorders in humans as well as non-human primates, rodents, and fish (Bretaud et al. 2004; Dauer and Przedborski 2003; Lam et al. 2005; McKinley et al. 2005). Furthermore, dopamine receptor blocking agents prescribed as anti-psychotics induce movement disorders (Dauer and Przedborski 2003). The effect of dopamine on the initiation (Kiehn and Kjaerulff 1996; Madriaga et al. 2004; Whelan et al. 2000) and frequency of motor patterns (Schotland et al. 1995; Svensson et al. 2003b) has been well-studied. Given the importance of dopamine in the initiation and control of locomotory behavior in established neural circuits, we tested whether dopamine controls the initiation of swimming in a developing vertebrate, i.e., the larval zebrafish. Locomotion in larval zebrafish evolves from slow tail flips at 18 hours postfertilization (hpf), to escape swimming at 28 hpf to robust spontaneous swimming at 5 days post fertilization (dpf) (Brustein et al. 2003b; Buss and Drapeau 2001). As late as 3dpf, larvae show very little spontaneous swimming but by 5dpf, larvae swim actively for foraging. In zebrafish, dopaminergic neurons are seen as early as 24hpf (McLean and Fetcho 2004a). By 3dpf, dopaminergic neurons are seen in the ventral diencephalon, the hypothalamus, the preoptic region and the pretectum (McLean and Fetcho 2004a; Rink and Wullimann 2002). Also, putative dopaminergic fibers densely innervate the mesencephalon, rhombencephalic reticulospinal neurons and the spinal cord (McLean and Fetcho 2004a; b). Here, we show that motor patterns generated by larval zebrafish at 3dpf are vastly different from those at 5dpf. The spinal cord in 3dpf zebrafish larvae is capable of initiating a high frequency of spontaneous fictive swimming episodes, but dopamine, acting via D2 receptors, selectively suppresses the initiation of spontaneous fictive swimming episodes. However, at 5dpf, endogenous release of dopamine does not suppress spontaneous swimming episodes, suggesting differential dopamine modulation of circuits involved in the initiation of spontaneous swimming at these two stages. Methods Adult wildtype zebrafish were obtained from a commercial supplier (Scientific Hatcheries, Huntington Beach, CA) and maintained in aquarium tanks at 28oC. Embryos were collected in a trap every morning and maintained in clean fish water in a water bath at 28oC. Larval swimming behavior A single larva was placed in a shallow translucent plastic dish filled with fish water. Larvae swam in a 5cm wide circular arena. Swimming behavior was recorded for fifteen minutes using a Hamamatsu ORCA ER CCD camera fitted with a Nikon 50mm zoom lens at 20 frames per second. The position of the larva in each frame was detected by background subtraction and the displacement was calculated from previous frame. The total displacement for fifteen minutes was calculated by adding the displacements in each frame. Extracellular Suction Recordings Recordings were performed as described in Masino and Fetcho (Masino and Fetcho 2005) with minor modifications (see Fig. 2A). Briefly, larvae were anaesthetized in 0.1% Tricaine (MS222) and pinned laterally through their notochord onto Sylgard using fine tungsten wire (California Fine Wire, Grover Beach, CA). We then paralyzed the larvae by replacing the MS222 with Danio external saline containing curare (in mM: 134 NaCl; 2.9 KCl; 1.2 MgCl2; 10 HEPES; 10 Glucose; 0.01 d-Tubocurarine; 2.1 CaCl2; pH 7.8; 290 mmol/Kg). Using fine tungsten wire, we peeled the skin to expose the musculature and the brain. Using thin-walled borosilicate capillaries with no filament (Sutter Instruments, Novato CA), we pulled large-tipped pipettes and filled them with Danio external saline. We positioned these close to the muscles and aspirated the fibers one by one to expose the spinal cord in two or three segments so that bathapplied drugs would permeate easily into the spinal cord. A micropipette filled with Danio saline (0.7 to 1.5 MΩ) was positioned very close to the myotomal boundary of one of the anterior segments and mild suction was applied. This resulted in the muscles, as well as the axons innervating them to be drawn up into the micropipette and the action potentials traveling down these axons could be recorded. We recorded mostly from muscle segments in the rostral one-third of the animal except during the local dopamine application experiments (see below). Multi-unit spiking activity was recorded using Multiclamp 700A amplifier and digitized using Digidata 1320 and pClamp 9.0 suite of software. For local application of dopamine, patch pipettes were filled with 10mM dopamine containing sulforhodamineB (Invitrogen, Carlsbad, CA). The third or fourth segment from the caudal end of the spinal cord was exposed by aspirating muscle fibers as explained above and the patch pipette containing dopamine was gently inserted into the cord. Mild positive pressure was applied while monitoring the extent of fluorescence. Pressure was applied until the solution traveled at least six segments rostrally. Recordings were made from segments within the extent of dopamine injection. Whole-cell Patch Clamp Recording The larva was pinned out and the spinal cord exposed as described above. Patch pipettes were pulled from borosilicate glass (Sutter Instruments, Novata, CA) and filled with internal solution (in mM: K Gluconate 115; KCl 15; MgCl2 2; HEPES 10; EGTA 10; Mg-ATP 3.94; pH 7.2; 290 mOsm; Pipette resistance 10-14 M ). The patch pipette was placed in the bath and in current clamp mode, the pipette offset and capacitance were calculated. Then the amplifier was switched to voltage-clamp mode and a giga-seal was formed with a ventrally located cell body. After adjusting for pipette capacitance, the seal was broken to achieve whole-cell configuration. The amplifier was switched to current clamp mode and DC current was injected to keep the cell membrane potential near –65mV. The bridge resistance and pipette capacitance were compensated for. Current pulses of varying amplitudes and about 1s duration were injected and the resulting membrane potential was recorded. The cell was filled with fluorescent dye included in the internal solution and at the end of the recording motor neuronal identity was confirmed from the morphology of the cell. Drugs Saline containing drugs at stated concentrations were bath-applied using a switching manifold and drugs tended to have an effect within 10 to 15 minutes of bath application. Some drugs were dissolved in 0.1% Dimethyl sulfoxide (DMSO) prior to further dilution in saline. 0.1% DMSO by itself did not have an effect on motor pattern activity (data not shown). Dosages for all drugs used were determined in preliminary experiments. Drugs were obtained from Sigma Chemical Company, St. Louis, MO (dopamine, bupropion hydrochloride, N-methyl d-aspartic acid, L741,626) and Tocris Inc, Ballwin, MO (S(-) sulpiride). Data Analysis Spikes were extracted offline using Spike2 software (Cambridge Electronic Design) and spike times were sorted into bursts, episodes and bouts using custom scripts written in Neuroexplorer (Nex Technologies, Littleton, MA) and Matlab (The Mathworks Inc, Natick, MA). Bouts were defined as intervals during which three or more spikes occurred and after which there was an inter-spike interval of at least 10s.Episodes were defined as time intervals during which three or more spikes occurred and after which there was an inter-spike interval (ISI) of at least 100ms. Bursts were defined as time intervals during which one or more spikes occurred and after which there was an ISI of at least 10ms. Burst period and episode period were calculated as the time between successive burst and episode start times respectively. Episode duration was calculated as the time between the start and end of an episode. Parameters such as episode period, duration and burst period were calculated using custom scripts written in Matlab. Swim episodes were counted in a 10-minute window and plotted. For the firing rate vs. current injected plots, the instantaneous firing rate was calculated as the inverse of the first ISI evoked by current of certain amplitude. The slope of the linear part of the firing rate vs. current curve was calculated to obtain gain. The input impedance and resting membrane potential were calculated as the slope and the y-intercept respectively of the voltage vs. current plot. Data were plotted using Microsoft Excel and SigmaPlot. Statistical testing was performed with Statview. In general the Mann-Whitney U test was used to test for significant differences in episode periods and durations because these data were non-normally distributed. The Student‟s T-test was used for burst period data because these passed the test for normality. Where present, error bars indicate standard error of the mean (SEM).