Synaptic Drive to Motoneurons During Fictive Swimming in the Developing Zebrafish

Abstract

The development of swimming behavior and the correlated activity patterns recorded in motoneurons during fictive swimming in paralyzed zebrafish larvae were examined and compared. Larvae were studied from when they hatch (after 2 days) and are first capable of locomotion to when they are active swimmers capable of capturing prey (after 4 days). High-speed (500 Hz) video imaging was used to make a basic behavioral characterization of swimming. At hatching and up to day 3, the larvae swam infrequently and in an undirected fashion. They displayed sustained bursts of contractions ('burst swimming') at an average frequency of 60-70 Hz that lasted from several seconds to a minute in duration. By day 4 the swimming had matured to a more frequent and less erratic "beat-and-glide" mode, with slower (approximately 35 Hz) beats of contractions for approximately 200 ms alternating with glides that were twice as long, lasting from just a few cycles to several minutes overall. In whole cell current-clamp recordings, motoneurons displayed similar excitatory synaptic activity and firing patterns, corresponding to either fictive burst swimming (day 2-3) or beat-and-glide swimming (day 4). The resting potentials were similar at all stages (about -70 mV) and the motoneurons were depolarized (to about -40 mV) with generally non-overshooting action potentials during fictive swimming. The frequency of sustained inputs during fictive burst swimming and of repetitive inputs during fictive beat-and glide swimming corresponded to the behavioral contraction patterns. Fictive swimming activity patterns were eliminated by application of glutamate antagonists (kynurenic acid or 6-cyano-7-nitroquinoxalene-2,3-dione and DL-2-amino-5-phosphonovaleric acid) and were modified but maintained in the presence of the glycinergic antagonist strychnine. The corresponding synaptic currents underlying the synaptic drive to motoneurons during fictive swimming could be isolated under voltage clamp and consisted of cationic [glutamatergic postsynaptic currents (PSCs)] and anionic inputs (glycinergic PSCs). Either sustained or interrupted patterns of PSCs were observed during fictive burst or beat-and-glide swimming, respectively. During beat-and-glide swimming, a tonic inward current and rhythmic glutamatergic PSCs (approximately 35 Hz) were observed. In contrast, bursts of glycinergic PSCs occurred at a higher frequency, resulting in a more tonic pattern with little evidence for synchronized activity. We conclude that a rhythmic glutamatergic synaptic drive underlies swimming and that a tonic, shunting glycinergic input acts to more closely match the membrane time constant to the fast synaptic drive.

Neurogenic Role of the Depolarizing Chloride Gradient Revealed by Global Overexpression of KCC2 from the Onset of Development

“…Therefore, we measured the amplitude and polarity of SBs to detect changes in the chloride gradient after overexpression of KCC2. Note that no significant GABAergic activity is observed at this stage of zebrafish development (Triller et al, 1997;Buss and Drapeau, 2001). In neurons from control embryos (n ϭ 4) and embryos expressing KCC2-C568A (n ϭ 3), SBs were always depolarizing from rest (Fig.…”

“…However, the swimming activity in response to touch (Fig. 3B), which depends on integration of glycinergic and glutamatergic synaptic activity (Buss and Drapeau, 2001;Saint-Amant and Drapeau, 2001), failed to develop in embryos overexpressing KCC2. Our results thus indicate that, although some synapses were functional in KCC2 embryos, many aspects of synaptic integration failed to develop normally during reversal of the chloride gradient, which may underlie the loss of both glycine and glutamate synapses attributable to the loss of neurons, as observed previously during knockdown of chloride-permeant glycine receptors in zebrafish embryos (McDearmid et al, 2006).…”

“…1997; Buss and Drapeau, 2001). Thus, potential homeostatic effects of compensatory transmitter systems may not occur in zebrafish and could explain the strong impact of interfering with glycinergic transmission on neuronal development in this species.…”

Synaptic Scaling and the Development of a Motor Network

“…Swimming was measured at either 3 or 4 dpf to parallel different experimental paradigms that elicited synaptic scaling. Swimming behavior in larval zebrafish transitions rapidly from sustained burst swimming at 2-3 dpf to a more mature beat-and-glide pattern by 4 dpf (Buss and Drapeau, 2001). Therefore it was most informative to measure tail contraction frequency and free-swimming velocity at 3 dpf, when burst swimming is still observed, and to measure tail contraction frequency and beat duration at 4 dpf, when discrete "beat" periods are present (Buss and Drapeau, 2001).…”

“…To measure tail contraction frequency (Buss and Drapeau, 2001), larvae were embedded in 1% low-melting-point agarose (Invitrogen) dorsal side up with the tail free to move. Episodes of tail alternations representative of swimming were elicited by a light tap on the tail with forceps.…”