Synaptic scaling and the development of a motor network

Abstract

Neurons respond homeostatically to chronic changes in network activity with compensatory changes such as a uniform alteration in the size of miniature postsynaptic current (mPSC) amplitudes termed synaptic scaling. However, little is known about the impact of synaptic scaling on the function of neural networks in vivo. We used the embryonic zebrafish to address the effect of synaptic scaling on the neural network underlying locomotion. Activity was decreased during development by TTX injection to block action potentials or CNQX injection to block glutamatergic transmission. Alternatively TNFalpha was chronically applied. Recordings from spinal neurons showed that glutamatergic mPSCs scaled up approximately 25% after activity reduction and fortuitously scaled down approximately 20% after TNFalpha treatment, and were unchanged following blockade of neuromuscular activity alone with alpha-bungarotoxin. Regardless of the direction of scaling, immediately following reversal of treatment no chronic effect was distinguishable in motoneuron activity patterns or in swimming behavior. We also acutely induced a similar increase of glutamatergic mPSC amplitudes using cyclothiazide to reduce AMPA receptor desensitization or decrease of glutamatergic mPSC amplitudes using a low concentration of CNQX to partially block AMPA receptors. Though the strength of the motor output was altered, neither chronic nor acute treatments disrupted the patterning of synaptic activity or swimming. Our results show, for the first time, that scaling of glutamatergic synapses can be induced in vivo in the zebrafish and that synaptic patterning is less plastic than synaptic strength during development.

Figure 1.

A significant increase in glutamatergic mPSC amplitude was only seen following chronic blockade of network activity with TTX from 1 or 2 dpf to 4 dpf. Boxed text refers to TTX treatment duration starting at time of injection and ending at time of recording, number of recordings in control and treated larvae, and significance of mPSC amplitude change.

Figure 2.

Chronic TTX or CNQX treatment results in a scaling up of glutamatergic mEPSC amplitudes at 4 dpf. A, Left, Cumulative histograms of glutamatergic mEPSC amplitudes from control (n = 24) and TTX-treated (n = 30) embryos (≥50 events per neuron). Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms of one neuron for the same conditions. B, Left, Cumulative histograms of glutamatergic mEPSC amplitudes from control (n = 17) and CNQX-treated (n = 22) embryos. Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms of one neuron for the same conditions. C, Left, Cumulative histograms of glutamatergic mEPSC amplitudes from control (n = 9) and AP-5-treated (n = 13) embryos. Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms of one neuron for the same conditions. All data here and below are reported as mean ± SEM for the number of neurons indicated; two-way ANOVA, *p < 0.05, **p < 0.01.

Figure 3.

Chronic TTX, CNQX, or AP-5 treatment does not scale glycinergic mIPSC amplitudes at 4 dpf. A, Left, Cumulative histograms of glycinergic mIPSC amplitudes from control (n = 11) and TTX-treated (n = 12) embryos. Inset, Average mIPSC amplitude for the same conditions. Right, Average mIPSC waveforms of one neuron for the same conditions. B, Left, Cumulative histograms of glycinergic mIPSC amplitudes from control (n = 11) and CNQX-treated (n = 16) embryos. Inset, Average mIPSC amplitude for the same conditions. Right, Average mIPSC waveforms of one neuron for the same conditions. C, Left, Cumulative histograms of glycinergic mIPSC amplitudes from control (n = 9) and AP-5-treated (n = 10) embryos. Inset, Average mIPSC amplitude for the same conditions. Right, Average mIPSC waveforms of one neuron for the same conditions.

Figure 4.

Chronic TNFα treatment results in a scaling down of glutamatergic mEPSC amplitudes at 3 dpf. A, RT-PCR for zebrafish tnfα at 18, 24, 48, 72, 96, and 120 hpf. tnfα is clearly expressed at 18 hpf and from 72 to 120 hpf, but expression may be reduced or absent from 24 to 48 hpf. Band identity was confirmed by sequencing. Primers for the ubiquitously expressed tnfb serve as a control. B, Cumulative histogram of glutamatergic mEPSC amplitudes from control (n = 27) and TNFα-treated (n = 32) embryos. Inset, Average mEPSC amplitude for the same conditions. C, Average mEPSC waveforms for the same conditions as B. D, Left, Average frequency of glutamatergic mEPSCs from same conditions as B. Center, Average number of GFP+ cells per somite from control (n = 7) and TNFα-treated (n = 6) embryos. Right, Example confocal image of one somite from an Isl-GFP transgenic fish spinal cord. E, Left, Cumulative histograms of glycinergic mIPSC amplitudes from control (n = 5) and TNFα-treated (n = 5) embryos. Inset, Average mIPSC amplitude for the same conditions. Right, Average mIPSC waveforms for the same conditions.

Figure 5.

Chronic CNQX or TNFα treatment does not alter cellular excitability. A, Rheobase current was not significantly different at 3 dpf between control (n = 11) and TNFα-treated (n = 10) larvae, at 4 dpf between control (n = 6) and CNQX-treated (n = 8) larvae. B, The average spike threshold (V_T − V_m) was also unchanged after chronic TNFα or CNQX treatment under same conditions as B. C, Representative spike trains evoked by somatic current injection steps (Δstep = 25 pA, 100 ms duration, 15 steps total) in control and treated motoneurons at 4 dpf. D, Average f–I curves were also unchanged after chronic TNFα or CNQX treatment under same conditions as B.

Figure 6.

Slow oscillation frequency underlying fictive swimming in motoneurons is unchanged following chronic CNQX or TNFα treatment. Top, An example whole-cell current-clamp trace showing the slow oscillations elicited in a motoneuron by the extracellular application of 200 μm NMDA. Bottom, Average frequency of NMDA-induced slow oscillations in motoneurons are not significantly different between control (n = 4) and TNFα-treated (n = 3) larvae at 3 dpf, and control (n = 3) and CNQX-treated (n = 3) larvae at 4 dpf.

Figure 7.

Chronic TNFα or CNQX treatment does not significantly alter the patterning of motor input to muscle cells at 3 dpf or 4 dpf, respectively. A, Left, Example whole-cell current-clamp recording from a slow-twitch muscle fiber of a fictive swimming burst at 3 dpf. Analysis parameters are indicated. Right, Sample burst recordings from a control or TNFα-treated larvae at 3 dpf. B, Average burst duration, maximum synaptic amplitude, and synaptic frequency for slow-twitch muscle fibers at 3 dpf for control (n = 11) and TNFα-treated (n = 9) larvae; *p < 0.05. C, Left, Example whole-cell current-clamp recording from a slow-twitch muscle fiber of fictive beat-and-glide swimming at 4 dpf. Analysis parameters are indicated. Right, Sample beat-and-glide recordings from a control or CNQX-treated larvae at 4 dpf. D, Episode duration, burst duration, beat frequency, synaptic peaks per beat, synaptic peak frequency, maximum synaptic amplitude, and percentage time active for slow-twitch muscle fibers at 4 dpf for control (n = 6) and CNQX-treated (n = 8) larvae. None of the differences between conditions were significant (p > 0.05).

Figure 8.

Larval swimming behavior is not altered following chronic TNFα or CNQX treatment. A, Swimming activity at 3–4 dpf is composed of high-frequency (20–30 Hz), low-amplitude tail contractions. In this example, one full cycle of swimming is observed from 50 to 80 ms. The larva is immobilized in agarose, dorsal side up and anterior to the top of the image. Time of each frame is shown in the bottom right corner. B, Left, Average swimming frequency of control (n = 3) and TNFα-treated (n = 5) larvae at 3 dpf. Right, Average free-swimming velocity under same conditions (control n = 11, TNFα-treated n = 13). C, Left, Average swimming frequency of control (n = 8) and CNQX-treated (n = 9) larvae at 4 dpf. Right, Average duration of swimming bursts under same conditions (control n = 8, TNFα-treated n = 9).

Figure 9.

Acute CNQX or CTZ treatment significantly altered maximum synaptic amplitude (and contraction frequency of motor input—only in the second case) to muscle cells at 3 dpf or 4 dpf, respectively. A, Sample fictive swimming burst recordings at 3 dpf from a muscle fiber at baseline (control) and again 5 min after start of CNQX application. B, Average burst duration, maximum synaptic amplitude, and synaptic peak frequency at 3 dpf for slow-twitch muscle fibers at baseline and following CNQX application (n = 3). Paired Student's t test, *p < 0.05. C, Sample fictive beat-and-glide swimming recordings at 3 dpf from a muscle fiber at baseline (control) and again 5 min after start of CTZ application. D, Episode duration, burst duration, beat frequency, synaptic peaks per beat, synaptic peak frequency, maximum synaptic amplitude, and percentage time active at 4 dpf for slow-twitch muscle fibers at baseline and following CNQX application (n = 10). Paired Student's t test, **p < 0.01.