Protein kinase C acts as a molecular detector of firing patterns to mediate sensory gating in Aplysia

Abstract

Habituation of a behavioral response to a repetitive stimulus enables animals to ignore irrelevant stimuli and focus on behaviorally important events. In Aplysia, habituation is mediated by rapid depression of sensory synapses, which could leave an animal unresponsive to important repetitive stimuli, making it vulnerable to injury. We identified a form of plasticity that prevents synaptic depression depending on the precise stimulus strength. Burst-dependent protection from depression is initiated by trains of 2-4 action potentials and is distinct from previously described forms of synaptic enhancement. The blockade of depression is mediated by presynaptic Ca2+ influx and protein kinase C (PKC) and requires localization of PKC via a PDZ domain interaction with Aplysia PICK1. During protection from depression, PKC acts as a highly sensitive detector of the precise pattern of sensory neuron firing. Behaviorally, burst-dependent protection reduces habituation, enabling animals to maintain responsiveness to stimuli that are functionally important.

Figure 1

Bursts of 2 to 4 spikes in presynaptic sensory neurons protect against development of HSD and partially reverse previously developed HSD. a. Sensory neuron (SN)-motor neuron (MN) EPSPs when sensory neurons were activated with single stimuli per trial or two to four stimuli per trial at 20 Hz. Intertrial interval (ITI) was 15 sec. In quantifying BDP, only the amplitude of the first EPSP during each burst is measured, which can be compared with the EPSP when sensory neurons are activated by single stimuli. In these experiments, the amplitude of the second EPSP within a burst is largely independent of the amount of depression of the first EPSP . [Arrow and arrowhead highlight the transient paired-pulse facilitation during the burst, which disappears with repeated testing (as discussed in Results.] Amplitude calibration bars apply to postsynaptic records. b. Combined data for experiments when sensory neurons fired 1, 2 or 4 spikes per trial (n = 39, 26 and 18, respectively). Both 2 spikes per trial and 4 spikes per trial resulted in significant reduction in HSD. [(see Supplementary Table 1 for all repeated measures ANOVA results); both 2 spikes per trial and 4 spikes per trial differed significantly from 1 spike per trial, p < 0.001 for both comparisons, but not from each other, p = 0.113. With one spike per trial, trial #1 differed significantly from all subsequent trials, p < 0.001, indicating that significant HSD was induced by trial #2.] All data plotted are mean ± SEM. c, d. Reversal of HSD by bursts of spikes. sensory neurons were stimulated to fire a single spike per trial for 15 trials; the stimulation pattern was then changed to 4 spikes per trial on trial #16. c. Example of reversal of HSD by bursts. In this synapse, the reversal of HSD was unusually effective. Arrows, as in panel a. d. Group data for experiments on reversal of HSD by bursts of spikes (n = 11). e. Bursts are less effective in restoring transmission at depressed synapses than in protecting sensory neuron synapses from undergoing HSD. Depressed (“Depr”) is the mean EPSP amplitude on trials #13–15 (with single spikes). “Recovery” is the mean EPSP amplitude on trials #19–21 (with bursts). (Data for 4 spikes per trial are from panel b.) Both bursts of 2 and 4 spikes produced significant reversal of synaptic depression (p < 0.001 for both, n = 18 and 11 respectively). BDP is the mean EPSP amplitude on trials #13–15 from experiments in Fig. 1b. Bursts are significantly less effective in restoring transmission at depressed synapses than in protecting sensory neuron synapses from undergoing HSD (F_1,70 = 15.3, p <0.001; with both 2 spikes and 4 spikes, reversal of depression was less effective than protection of naïve synapses, p = 0.002 and 0.017, respectively).

Figure 2

Effect of BDP on sensory neuron synapses is stable, distinguishing BDP from PTP. a. Examples of EPSPs from sensory neuron-motor neuron synapses activated with either one spike per trial or 4 spikes per trial at a 1 min ITI. EPSPs are shown for trials #1 and #15, and for a posttest 20 min after trial #15. b. BDP persists after induction for tens of minutes. Group data from experiments in a (n = 6). Amplitudes of EPSPs with BDP protocol were significantly greater than with HSD protocol on trials #13–15 (p < 0.001) and at 20 min posttest (p = 0.017). For HSD or BDP, there was no significant difference between the EPSP amplitudes at the posttest and during trials #13–15 (p = 1.0 for both); at the 1 min posttest, mean EPSP = 98.0 ± 10.0% of initial. (Thus, there was neither recovery from HSD nor waning of BDP after 20 min.)

Figure 3

Initiation of BDP involves presynaptic Ca²⁺ influx. a. Examples of EPSPs when sensory neurons fired bursts of 4 spikes in normal high-divalent saline or high-divalent saline with a 60% reduction in Ca²⁺. b. BDP was eliminated in low Ca²⁺ saline. Group data for BDP experiments as in a. BDP in low Ca²⁺ was significantly different than in normal Ca²⁺ (p <0.001), but was not significantly different from normal HSD (p = 0.691, pairwise comparisons). In contrast, the induction of HSD with 1 spike per trial was unaffected by this reduction in extracellular Ca²⁺ (p = 0.234), which resulted in a 4.5-fold decrease in initial EPSP amplitude. c. Presynaptic injection of EGTA eliminated BDP elicited with 4 spikes per trial, whereas postsynaptic EGTA or BAPTA did not affect BDP. When sensory neurons were injected with EGTA (50 mM in pipette, n = 7), synapses showed significantly less protection from depression than controls (n = 4, p = 0.009 for pairwise comparison) or than when motor neurons were injected with EGTA (100 mM in pipette, n = 3, p = 0.011) or BAPTA (200 mM in pipette, n = 4, p < 0.001). Postsynaptic injection of either EGTA or BAPTA did not affect induction of BDP, as compared with vehicle-injected controls (p = 1.00 and 0.72, respectively). [There was no significant difference during the BDP protocol between synapses with postsynaptic EGTA or postsynaptic BAPTA (p = 0.907); therefore these two sets of results have been combined and plotted as a single set of data.]

Figure 4

Initiation of BDP involves PKC, but not CaMKII. a. Examples of synapses, activated with 4 spikes per trial, after presynaptic injection of autoinhibitory domain peptides from either CaMKII or PKC. The concentration in the pipette was 1 mM for PKC(19-31) and 20 mM for CaMKII(281-302). b. PKC(19-31) in sensory neurons blocks BDP, but does not affect HSD. (n = 8 and 12, for BDP and HSD, respectively). BDP was significantly reduced by presynaptic PKC(19-31) (p < 0.001), whereas there was not a significant effect on depression with single spikes (p = 0.69). [For PKC(19-31), the small residual difference between 1 spike and 4 spikes per trial was still significant (p = 0.001).] PKC(19-31) did not significantly affect the amplitude of EPSP #1 compared with pre-injection amplitude. c. BDP was not affected by CaMKII(281-302) (n = 9, p = 0.97). Average of EPSP amplitudes on trials 13–15 is expressed as a percent of initial EPSP amplitude. For comparison, EPSPs #13–15 from BDP experiments in b are plotted; BDP was effectively inhibited by PKC(19-31) (n = 8, p = 0.005). d. PTP was blocked by presynaptic injection of CaMKII(281-302) (n = 5, p = 0.003). PKC(19-31) resulted in a marginally significant, partial decrease in PTP (n = 8, p = 0.051). A small contribution of PKC could reflect activation of the BDP mechanism by the train of spikes that was used to induce PTP. PTP was measured 1 min after stimulation of sensory neurons at 20 Hz for 2 sec.

Figure 5

The Ca2+-activated PKC Apl-I interacts with the PDZ domain protein Aplysia PICK1. To test whether PKC Apl-I binds to Apl-PICK1, CFP-tagged PKC Apl-I was co-expressed with FLAG-tagged Apl-PICK1 in HEK 293 cells. Alternatively, GFP-tagged PKC Apl-II was co-expressed with FLAG-tagged Apl-PICK1. Agarose beads coupled to anti-FLAG antibody were used to immunoprecipitate FLAG-Apl-PICK1. PKC Apl-I co-immunoprecipitated with anti-FLAG beads, but only in the presence of FLAG-Apl-PICK1. (Very faint bands for PKC Apl-II are visible; unlike Apl-I, this binding does not require FLAG-Apl-PICK1.) Immunoblots were probed with anti-GFP or anti-FLAG antibodies; the anti-GFP antibody also recognizes CFP. (Blots shown have been cropped to show the relevant bands. Full-length blots/gels are presented in Supplementary Figure 11.)

Figure 6

BDP depends upon PDZ domain interactions. Presynaptic sensory neurons were injected with either a 10 AA peptide corresponding to the C terminus of PKC Apl-I, which ends in a PDZ binding motif, a 10 AA peptide corresponding to the C terminus of PKC Apl-II, or an antibody against the PDZ domain of Aplysia PICK1. a. Example of synapses activated with 4 spikes per trial from sensory neurons injected with either vehicle (Control), Apl-I C terminus peptide, Apl-II C terminus peptide (3 mM peptide in pipette), affinity purified antibody against the PDZ domain of Aplysia PICK1 or control antibody (affinity purified antibody against Aplysia adenylyl cyclase AC-AplB, which is not expressed in sensory neurons (see Methods). Note, BDP was eliminated by both the Apl-I C terminus peptide and by the anti-Apl-PICK1 antibody. b. Group data for sensory neurons injected with Apl-I C terminus peptide (n = 8), Apl-II C terminus peptide (n = 7) or vehicle (n = 15). Apl-I C terminus peptide significantly reduced BDP (p = 0.001, pairwise comparison). BDP was unaffected by Apl-II peptide [p = 0.474, pairwise comparison; the effect of the two peptides was significantly different, p = 0.006]. c. Group data for sensory neurons injected with anti-Aplysia PICK1 antibody (n = 7), control (anti-AC-AplB) antibody (n = 8), or vehicle (n = 12). The anti-Apl-PICK1 antibody significantly reduced BDP compared with either vehicle or the control antibody (p < 0.001 and p = 0.001, respectively, pairwise comparisons), whereas the control antibody had no effect (p = 0.96).

Figure 7

Bursts of spikes in siphon sensory neurons reduce habituation of siphon withdrawal response via a homosynaptic mechanism. Two 5HT receptor antagonists, 150 μM methiothepin and 100 μM spiperone, were used to block effects of any 5HT released by sensory neuron activity. a. To confirm blockade, the siphon nerve was stimulated at intensity that activated all LE sensory neuron neurons with 6 shocks at 20 Hz; no increase in sensory neuron excitability occurred (p = 0.197). 5HT (5 μM), in the absence of receptor antagonists, substantially increased sensory neuron excitability (p = 0.001) in same ganglia. b. Examples of siphon withdrawal response elicited by one shock per trial or 4 shocks per trial at 20 Hz, delivered focally to 2 mm diameter area of siphon skin (ITI = 1 min). To deliver methiothepin and spiperone, abdominal ganglion was superfused independently from the siphon (Supplementary Fig. 8). c. Group data from experiments in b. With bursts of 4 skin shocks per trial, the siphon withdrawal response decremented more gradually than with 1 shock per trial (F_14,126 = 4.66; p < 0.001). After 15 trials with bursts of 4 stimuli, the habituated response amplitude was 3.5 times greater than with single stimuli. There was no difference in average peak amplitude of the response on trial #1 for single stimuli and bursts of 4 stimuli (776 ± 164 vs. 784 ± 64, respectively, arbitrary units), suggesting the brief bursts of sensory neuron spikes did not dramatically enhance the synaptic input to interneurons and motor neurons compared with single sensory neuron spikes.