Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS

Abstract

Presynaptic group III metabotropic glutamate receptor (mGluR) activation by exogenous agonists (such as L-2-amino-4-phosphonobutyrate (L-AP4)) potently inhibit transmitter release, but their autoreceptor function has been questioned because endogenous activation during high-frequency stimulation appears to have little impact on synaptic amplitude. We resolve this ambiguity by studying endogenous activation of mGluRs during trains of high-frequency synaptic stimuli at the calyx of Held. In vitro whole-cell patch recordings were made from medial nucleus of the trapezoid body (MNTB) neurones during 1 s excitatory postsynaptic current (EPSC) trains delivered at 200 Hz and at 37 degrees C. The group III mGluR antagonist (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG, 300 microm) had no effect on EPSC short-term depression, but accelerated subsequent recovery time course (tau: 4.6 +/- 0.8 s to 2.4 +/- 0.4 s, P = 0.02), and decreased paired pulse ratio from 1.18 +/- 0.06 to 0.97 +/- 0.03 (P = 0.01), indicating that mGluR activation reduced release probability (P). Modelling autoreceptor activation during repetitive stimulation revealed that as P declines, the readily releasable pool size (N) increases so that the net EPSC (NP) is unchanged and short-term depression proceeds with the same overall time course as in the absence of autoreceptor activation. Thus, autoreceptor action on the synaptic response is masked but the synapse is now in a different state (lower P, higher N). While vesicle replenishment clearly underlies much of the recovery from short-term depression, our results show that the recovery time course of P also contributes to the reduced response amplitude for 1-2 s. The results show that passive equilibration between N and P masks autoreceptor modulation of the EPSC and suggests that mGluR autoreceptors function to change the synaptic state and distribute metabolic demand, rather than to depress synaptic amplitude.

Figure 1. Presynaptic group III mGluRs depress the calyx EPSC

EPSCs were recorded from MNTB neurones under whole-cell voltage clamp. A, the stimulus artefact (asterisk) precedes the EPSC (arrow). The control EPSC (black trace) was reduced by the application of 50 μm l-AP4 (grey trace) and reversed by 300 μm CPPG. B, EPSC magnitude plotted every 10 s during l-AP4 and CPPG bath application. C and D, bath application of CPPG (grey trace) alone had no effect on EPSC magnitude or time course. E, summary data: the asterisk indicates a significant reduction in the EPSC by l-AP4 (n = 3,P = 0.02). F, EPSC trains at 200 Hz were elicited for 1 s (stimulus artefacts have been erased for clarity). G, the same data on a faster time-scale, 1 s 200 Hz trains, control (black trace) and following CPPG (grey trace). The EPSCs are almost identical in control and CPPG. H, each EPSC from the 200 Hz train is shown normalized to the magnitude of the first EPSC in the train. Data from five cells are averaged and standard error bars are plotted on every 5th point.

Figure 2. Block of mGluRs accelerates recovery from synaptic depression

A, the time course of recovery from depression was assessed by eliciting a single EPSC at varying time intervals (Δt) following a conditioning train. Unitary EPSCs are overlaid to illustrate the recovery time course. B, the magnitude of the unitary EPSC is normalized to the first EPSC of the train and plotted against the recovery time interval for control (black symbols) and following CPPG (300 μm, grey symbols). Recovery time intervals of 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 s were used. Data from five cells were averaged and fitted with double exponential functions. C, sample data from the 2 s recovery time point in control (black trace) and following CPPG (grey trace). The first few EPSCs in the 200 Hz train are shown, which are unaffected by CPPG (as in Fig. 1G). The recovery EPSC is shown, significantly enhanced by CPPG. D, the recovery 2 s after the train was assessed every 4 min and is plotted as a function of time following CPPG application. Data from five cells were averaged.

Figure 3. Inhibition of mGluRs increases release probability

Changes in release probability following the train were assessed using paired pulses 1 s after the train. A, two EPSCs, 5 ms apart, are shown and normalized to the magnitude of the first EPSC, in the absence (black traces) and presence (grey traces) of 300 μm CPPG. The broken lines indicate the change from paired-pulse facilitation to paired-pulse depression following CPPG application. B, the paired-pulse ratio following the train is plotted as a function of time. Data are averaged from five cells. C, The paired-pulse ratio is significantly decreased following CPPG application (asterisk indicates P = 0.01).

Figure 4. mGluR activation reduces release probability during recovery from short-term depression

A, 200 Hz, 1 s trains were elicited for 1 s, followed 1 s later by a second train (200 Hz, 0.25 s). The resulting EPSCs are shown with stimulus artefacts removed for clarity. B, the cumulative amplitudes of the first 50 EPSCs in the first train are shown in the presence and absence of CPPG, with the control and CPPG data points overlapping. A straight line was fitted by linear regression to the last 15 EPSCs on the control graph, and back-extrapolated to time 0 to estimate the releasable pool size. The cumulative amplitudes of the EPSCs in the second train are also shown in control (black traces) and in the presence of CPPG (grey traces). Straight lines were also fitted to the last 15 EPSCs to estimate the pool size and release probability. All data are averages from five cells. C, The pool size and release probability are shown for the second train in the presence (grey) and absence (black) of CPPG. D, the time between the two trains was increased and the calculated release probability plotted as a function of the time interval, in the presence of CPPG (grey trace) and in control, with mGluRs being activated (black trace). Asterisks indicate statistical significance in C and D (P < 0.05).

Figure 5. A simple model reveals that depression is insensitive to changes in release probability

A, the presynaptic terminal contains a single pool of vesicles, a proportion (P) of which is released by each action potential, and it is replenished by a first-order process with an exponential time constant (τ). The resulting EPSC magnitude is proportional to the number of vesicles released. B, the model is driven with a 200 Hz 1 s train and P is kept constant (grey traces). The normalized EPSC and pool size decline to a steady state. P is changed to mimic presynaptic mGluR activation (black traces), with little effect on EPSC magnitude (arrow, middle panel), but causing an increase in pool size (right panel). C, if τ is changed by a similar proportion (black line), significant changes in the EPSC are observed (arrow, middle panel).

Figure 6. A more complex model fits the synaptic depression and EPSC recovery following a train

Adding facilitation and activity-dependent vesicle recycling to the model (see Methods) allows assessment of recovery following a conditioning train. A, the model is driven by a 200 Hz, 1 s train with mGluR activation mimicked by an activity-dependent decrease in release proportion (P; black traces) or with mGluR activation absent (grey traces). The resulting EPSC depression curve (black and grey overlapping lines, middle panel) fits well with sample data points from Fig. 1H. The pool size during the train is increased by mGluR activation (right panel). B, P recovers with a time constant of 7 s following the train (black trace, left panel). Data from Fig. 2B are plotted and fitted with the EPSC recovery from the model (middle panel). The paired-pulse ratio of two EPSCs 1 s following the train is increased by the lowered P (right panel).