Postsynaptic current bursts instruct action potential firing at a graded synapse

Abstract

Nematode neurons generally produce graded potentials instead of action potentials. It is unclear how the graded potentials control postsynaptic cells under physiological conditions. Here we show that postsynaptic currents frequently occur in bursts at the neuromuscular junction of Caenorhabditis elegans. Cholinergic bursts concur with facilitated action potential firing, elevated cytosolic [Ca(2+)] and contraction of the muscle whereas GABAergic bursts suppress action potential firing. The bursts, distinct from artificially evoked responses, are characterized by a persistent current (the primary component of burst-associated charge transfer) and increased frequency and mean amplitude of postsynaptic current events. The persistent current of cholinergic postsynaptic current bursts is mostly mediated by levamisole-sensitive acetylcholine receptors, which correlates well with locomotory phenotypes of receptor mutants. Eliminating command interneurons abolishes the bursts whereas mutating SLO-1 K(+) channel, a potent presynaptic inhibitor of exocytosis, greatly increases the mean burst duration. These observations suggest that motoneurons control muscle by producing postsynaptic current bursts.

Figure 1. Bursts of postsynaptic currents (PSC burstss) occurred concurrently in a pair of neighboring body-wall muscle cells

A. Diagrams showing the anatomy of ventral body-wall muscles. The left panel is a crosssection of the muscles shown on the right. Two muscle strips known as quadrants run beside the ventral nerve cord. Muscle cells in the right quadrant are designated as R1 and R2 whereas those in the left quadrant as L1 and L2. One L2R2 cell pair was highlighted. B. Voltage-clamp recording showing that PSC bursts happened concurrently in the L2R2 cell pair. The holding potential was −60 mV. Extracellular solution I and pipette solution I were used. Only those PSC bursts meeting our criterion (an apparent increase in PSC frequency with a baseline shift lasting > 3 sec)are labeled as bursts.

Figure 2. Postsynaptic current (PSC) bursts instructed muscle activities

A& B. Spontaneous cholinergic PSC bursts were associated with facilitated muscle action potential (AP) firing (A), increased cytosolic [Ca²⁺], and shortening of muscle cells (B). Cytosolic [Ca²⁺] was positively correlated with the total charge transfer of endogenous PSC bursts but negatively correlated with muscle cell length. A movie of this representative experiment is shown in the supplement (Supplementary Movie 1). C. Spontaneous GABAergic PSC bursts suppressed muscle AP firing. Cholinergic PSCs appeared as inward currents (downward deflections) whereas GABAergic PSCs appeared as outward currents (upward deflections). The traces were from a light-off period in a transgenic worm expressing channel rhodopsin-2 in GABAergic neurons. The holding potential was −60 mV in A and B but −10 mV in C. The experiments in A&B were performed with extracellular solution I and either pipette solution I (for APs) or pipette solution III (for PSCs). The experiment in C was performed with extracellular solution II and pipette solution II.

Figure 3. Postsynaptic current (PSC) bursts were are characterized by increased PSC frequency, increased PSC amplitude activity, and the appearance of a persistent current

Properties of cholinergic (A) and GABAergic (B) PSC bursts. Top: Comparisons of PSC frequency, mean amplitude, mean charge transfer, half-width, 10–90% rise time, and decay time between intra- and extra-burst periods. The frequency was quantified from all PSCs whereas the other parameters were quantified from only monophasic PSCs. Middle: Sample monophasic and multiphasic PSCs and comparison of their frequencies between intra- and extra-burst periods. Bottom: Sample PSC bursts, and comparisons of the mean charge transfer rate and persistent current amplitude between intra- and extra-burst periods. The quantification was performed with the same data represented in Figure 2. The asterisk indicates a significant difference compared with the extra-burst period (p< 0.05, paired t-test). n = 9 in both A and B. Data are shown as mean ± SE.

Figure 4. Optogenetically evoked cholinergic and GABAergic postsynaptic currents (PSCs)facilitated and inhibited muscle action potential (modulate AP)firing, respectively

Experiments were performed with transgenic worms expressing channel rhodopsin-2 in either cholinergic (A) or GABAergic (B) neurons. The worms were raised either in the presence or absence of all-trans retinal as indicated. PSCs and APs were recorded simultaneously from the L2R2 cell pair. Top: Temporal relationships between evoked PSCs and muscle APs. The horizontal blue lines represent blue light pulses. The arrows mark spontaneous GABAergic PSC bursts during light-off periods. The “^” sign indicates that the peak was cut off. Middle: A segment of the recording traces in the top panels (indicated by the dotted rectangle) shown at expanded scales. The initial transient current is displayed separately on the left. Bottom: Comparisons of PSC and AP frequencies between light-on and light-off periods (n = 8 in A; n = 9 in B) and representative traces from worms that had been not been raised in the presence of retinal (representative of 3 experiments in both A and B). The asterisk indicates a significant difference compared with the extra-burst period (p< 0.01, paired t-test). The data shown in A were performed with extracellular solution I and pipette solution I whereas those shown in B were performed with extracellular solution II and pipette solution II. Data are shown as mean ± SE.

Figure 5. The persistent current of Ooptogenetically evoked cholinergic and GABAergic postsynaptic current (PSC) bursts depended on specific postsynaptic receptors

A& B. Left: Sample traces from worms raised in the presence of all-trans retinal showing the effects of (+)-tubocurarine (TBC, 0.5 mM) and gabazine (0.5 mM) on optogenetically evoked cholinergic and GABAergic PSC bursts, respectively. The horizontal blue lines represent blue light pulses while the “^” sign indicates that the peak was cut off. Right: The persistent current of evoked cholinergic (n = 15)and GABAergic (n = 12) bursts were abolished by TBC and gabazine, respectively. C. The persistent current of evoked cholinergic PSC bursts was greatly decreased in unc-29(e1072) but unchanged in acr-16(ok789) compared with wild type (WT). D. The large initial transient of evoked cholinergic PSC bursts was greatly decreased in acr-16(ok789) but unchanged in unc-29(e1072) compared with WT. In C&D, the sample size (n) was 8 for every group. The WT group is the same as that shown in Figure 4. The asterisk (*) indicates a statistically significant difference (p< 0.01) compared with either the control period of A or B (paired t-test) or the WT group in C and D (one-way ANOVA followed by Bonferroni post hoc test). Extracellular solution I and pipette solution III were used to record cholinergic PSCs whereas extracellular solution II and pipette solution II were used to record GABAergic PSCs.

Figure 6. Optogenetic stimulation of command interneurons caused postsynaptic responses resembling spontaneous postsynaptic current (PSC) bursts

The experiments were performed with worms expressing channel rhodopsin-2 in command interneurons. The worms had been raised in either the presence or the absence of all-trans retinal as indicated. A. Sample traces showing PSCs and membrane potentials recorded simultaneously from a L2R2 cell pair of a worm raised in all-trans retinal. Photo-stimuli (marked by the blue horizontal line) evoked PSC bursts, which facilitated action potential (AP) firing. The “^” sign indicates that the peak was cut off. B. Comparisons of PSC and AP frequencies between light-on and light-off periods (n = 7). The asterisk indicates a significant difference (p< 0.01, paired t-test). Data are shown as mean ± SE. C. A sample trace from a worm raised in the absence of retinal showing that a photostimulus (marked by the blue horizontal line) had no effect on PSCs (representative of 3 experiments). The experiments were performed with extracellular solution I and either pipette solution I (for APs) or pipette solution III (for PSCs).

Figure 7. Cholinergic PSC bursts appeared to be dependent on inputs from command interneurons

A. Sample PSC traces representing wild type (WT) (n = 9), unc-7(e5) (n = 7), unc-9(fc16)(n = 9) and a worm with all command interneurons removed (Head & tail off) (n = 6). B. Comparisons of PSC burst frequency, duration, charge transfer rate, and persistent current amplitude. PSC bursts were completely absent in the Head & tail off group. The asterisk (*) indicates a statistically significant difference compared with WT (p< 0.05, one-way ANOVA). Extracellular solution I and pipette solution III were used in these experiments. Data are shown as mean ± SE.

Figure 8. Loss-of-function mutation of the SLO-1 K⁺ channels increases d the duration of cholinergic postsynaptic current (PSC) burst durations and the frequency of muscle Ca²⁺ transient frequency s

A. Sample traces of PSCs and muscle Ca²⁺ transients representing wild type (WT) (n = 9), slo-1(md1745) (n = 9), slo-1(md1745) rescued in cholinergic neurons using unc-17 promoter (Punc-17) (n = 7), and slo-1(md1745) rescued in command interneurons using glr-1 promoter (Pglr-1) (n = 7). Ca²⁺ transients of these representative experiments are shown in Supplementary Movies 2–5. B. Comparisons of the frequency and duration of PSC bursts, and the frequency of Ca²⁺ transients among the different groups. The asterisk (*) indicate a significant difference compared with WT whereas the pound sign (#) indicates a significant difference compared with slo-1 (p< 0.05, one-way ANOVA with Bonferroni post hoc tests). Quantitative comparisons for other parameters of intra- and extra-burst PSCs are shown in Supplementary Fig. S5. Extracellular solution I and pipette solution III were used in these experiments. Data are shown as mean ± SE.