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. 2008 Nov 12;28(46):12062-70.
doi: 10.1523/JNEUROSCI.4134-08.2008.

Calcium-sensing receptor activation depresses synaptic transmission

Cecilia G Phillips  1 , Mark T Harnett, Wenyan Chen, Stephen M Smith
Affiliations

Calcium-sensing receptor activation depresses synaptic transmission

Cecilia G Phillips et al. J Neurosci. .

Abstract

At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca](o)) and thus may impair neurotransmission. Increases in [Ca](o) activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits nonselective cation channels at the majority of cortical nerve terminals. This pathway may modulate synaptic transmission by attenuating the impact of decreases in [Ca](o) on synaptic transmission. Using patch-clamp recording from isolated cortical terminals, cortical neuronal pairs and isolated neuronal soma we examined the modulation of synaptic transmission by CaSR. EPSCs were increased on average by 88% in reduced affinity CaSR-mutant (CaSR(-/-)) neurons compared with wild-type. Variance-mean analysis indicates that the enhanced synaptic transmission was due largely to an increase in average probability of release (0.27 vs 0.46 for wild-type vs CaSR(-/-) pairs) with little change in quantal size (23 +/- 4 pA vs 22 +/- 4 pA) or number of release sites (11 vs 13). In addition, the CaSR agonist spermidine reduced synaptic transmission and increased paired-pulse depression at physiological [Ca](o). Spermidine did not affect quantal size, consistent with a presynaptic mechanism of action, nor did it affect voltage-activated Ca channel currents. In summary, reduced CaSR function enhanced synaptic transmission and CaSR stimulation had the opposite effect. Thus CaSR provides a mechanism that may compensate for the fall in release probability that accompanies decreases in [Ca](o).

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Figures

Figure 1.
Figure 1.
EPSC increased in CaSR−/− neurons. a, Photomicrograph and inset diagram of cell-attached recording from isolated synaptosome. Calibration bar is 20 μm in length. b, Currents activated by 150 mV step depolarizations in synaptosome recording at range of steady state bath [Ca] (6 μm–6 mm). c, Concentration-effect relationship for outward current and bath [Ca]. Average normalized data from 9 recordings made using Tyrode in pipette (2 mm Mg and Ca). Data from each recording were fit to Equation 1 (see Materials and Methods), where I was outward current at end of depolarizing step, Imax was I with 6 μm Ca in bath, and n was set to 1.3 (Smith et al., 2004). The curve was redrawn using the average value for IC50. d, Photomicrograph of whole-cell recording from two adjacent cortical neurons. e, Inset indicating paired recording and exemplar presynaptic action potential and postsynaptic current in neuron clamped at −70 mV. Broken line indicates 0 mV. f, Exemplars of EPSCs close to median values recorded from CaSR+/+ (n = 36) and −/− (n = 28) neuronal pairs. g, Histogram of mean ± SEM. EPSC for CaSR+/+ and −/− neuronal pairs. *p < 0.05.
Figure 2.
Figure 2.
Pooled V–M for CaSR+/+ and −/− neuronal pairs. a, b, Plot of pooled average EPSC versus pooled average variance for CaSR+/+ (n = 36) and CaSR−/− (n = 28) neuronal pairs. Data are plotted as pooled mean ± SEM and is linear over lower range and described by lines with slopes of 16.9 pA and 11.9 pA for the CaSR+/+ and CaSR−/− neuronal pairs respectively.
Figure 3.
Figure 3.
Spermidine reduces NSCC currents in synaptosomes and EPSC amplitude in cortical cultures. a, Outward currents activated with 60 μm bath Ca and 150 mV depolarizing step in a cell-attached synaptosome recording were decreased by bath application of spermidine (100 μm). Inset shows cell-attached configuration. b, Diary plot of NSCC amplitude versus time in same recording as a. Currents activated by 150 mV depolarization every 5 s were decreased by increases in bath [Ca] (60 μm–6 mm; upper unbroken trace) and bath spermidine concentration (10–300 μm; indicated by middle broken trace) reaching new steady states in ∼10 s. The Ca and spermidine axes are logarithmic and the absence of the broken trace indicates spermidine concentration is zero. c, Exemplar presynaptic action potentials and EPSCs before, during, and after spermidine application (100 μm) to paired recording from wild-type neurons. Average EPSCs in bold overlay the individual EPSCs (gray) recorded at steady state. d, Diary plot of EPSC amplitudes evoked by injection of depolarizing current into presynaptic neuron every 10 s. Broken line shows average EPSC values recorded during each phase of experiment. e, Histogram of mean effect of spermidine on EPSC amplitude (n = 8). f, Histogram of the effect of spermidine (100 μm) on mEPSC amplitude. *p < 0.05.
Figure 4.
Figure 4.
Low-dose spermidine does not inhibit VACC in SCG neurons. a, VACC currents activated by ramp depolarizations in an exemplar neuron were inhibited by spermidine (in mm). b, Diary plot of peak inward VACC current versus time and concentration of spermidine. c, Concentration-effect relationship for spermidine on peak VACC currents normalized against current in absence of polyamine. The curve was drawn using Equation 1, using the mean values and given IC50 of 9.5 ± 0.9 mm and n of 0.82 ± 0.06.
Figure 5.
Figure 5.
Spermidine enhances paired pulse depression. a, Average EPSC evoked after presynaptic action potential triggering with a 100 ms interstimulus interval and duty cycle of 10 s before, during, and after spermidine bath application (100 μm). Spermidine reduced P1 by ∼20% but reduced P2 by more, and the effect was reversible. b, The histogram of P2/P1, normalized for the PPR before spermidine application, was reduced by bath spermidine (n = 10).
Figure 6.
Figure 6.
CaSR activation decreases synaptic transmission in polysynaptic networks and in current clamped neurons. a, Single brief stimulation of the presynaptic neuron every 10 s resulted in three action potentials and three EPSCs in the postsynaptic neuron under control conditions in these superimposed traces. The multiple firing presumably reflected synaptic feedback onto the neuron as it was prevented by VACC block. EPSC and action potential number were reversibly reduced by bath application of spermidine. b, Stimulation of the presynaptic neuron at 1 Hz resulted in a presynaptic action potential and EPSP or action potential. c, Spermidine (100 μm) reversibly decreased the probability of postsynaptic action potential firing from ∼0.5 to 0. d, Spermidine also reduced the amplitude of subthreshold EPSPs as shown in these superimposed traces (mean trace in bold).
Figure 7.
Figure 7.
Diagram of proposed model for CaSR modulation of neurotransmitter release. a, Model depicts nerve terminal membrane containing CaSR (yellow oval) which is activated by extracellular Ca (red). A proportion of the NSCC are closed (gray) by the CaSR mediated signal (yellow circle). At physiological [Ca]o, CaSR is not fully activated and some active NSCC (yellow) facilitate vesicle fusion and transmitter release. b, At reduced [Ca]o, CaSR is no longer activated (gray) and in the absence of this signal NSCC may be activated (yellow) by depolarization. Because reduced [Ca]o will also decrease Ca entry through VACC and the probability of exocytosis, the increase in NSCC activity may partially compensate by facilitating vesicle fusion. c, At physiological [Ca]o reduced affinity CaSR−/− (gray hexagon) is less likely to be activated than CaSR+/+. Consequently, there is reduced signaling to NSCC, which remains active increasing the average probability of release. d, Application of spermidine (Spd; purple circles) and Ca to wild-type neurons fully activates CaSR reducing NSCC activity and transmitter release. Unlike Ca, spermidine does not enter the terminal and trigger exocytosis directly. Active molecules are colored yellow and inactive molecules are colored gray. The second messenger mediating signaling between NSCC and CaSR has not been identified and is denoted by a small intracellular circle.
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