Vm regulates mGlu5-NMDA receptors crosstalk in
hippocampal neurons
The NMDA receptor conductance is well established to be regulated by
type I mGlu receptors . Specifically, NMDA receptor potentiation by
mGlu5 receptors involves the Gq-protein-coupled receptor
(GPCR) pathway, which includes protein kinase C (PKC) and Src signaling,
in various neuronal contexts . In this study, we aimed to investigate
whether Vm could regulate the crosstalk between mGlu5
and NMDA receptors in primary cell cultures of hippocampal neurons
(Figure 6 ). To avoid stimulation of mGlu1 receptors, we used
the mGlu1-specific negative allosteric modulator (NAM), CPCCOEt (100
µM), while stimulating mGlu5 receptors with DHPG. We recorded
NMDA-induced currents in the absence of magnesium
(Mg2+) to prevent voltage-dependent blockade of NMDA
receptors. When applied alone at a holding potential of -80 mV, DHPG (50
µM) had a negligible effect (1.046 ± 0.45 pA/pF). In contrast, NMDA (30
µM) induced an inward current of 23.4 ± 3.7 pA/pF (Figure 6A ),
which was potentiated by 39.5 ± 6.5% by DHPG when applied at -80 mV
(INMDA + DHPG/INMDA ratio measured just
before and after DHPG application, Figure 6B, 6C left ).
However, at a holding potential of -40 mV, DHPG only induced a 25.4 ±
5.2% potentiation of NMDA current density (Figure 6B, 6C
right ). Paired measurements of INMDA +
DHPG/INMDA performed subsequently at -80 mV or -40 mV
on the same neuron in a random order confirmed a reduction of
DHPG-induced NMDA receptor potentiation at -40 mV (+25 ± 4.8%) compared
to -80 mV (+38.11 ± 8.2%) (Figure 6D ). These results
demonstrate the crucial role of Vm in the control of
mGlu5-NMDA crosstalk in neurons, and they corroborate our previous
findings on the global voltage-dependence of mGlu5 receptor activity,
which is inhibited by depolarized membrane potentials.
Therefore, the optimal functioning of the mGlu5 receptor, which enhances
NMDA receptor activity in neurons, occurs at the resting potential of
neurons. This finding appears to conflict with the fact that NMDA
receptors are typically blocked by Mg2+ at resting
membrane potential. Activation of the NMDA receptor indeed requires
depolarization of the postsynaptic element to release magnesium from the
channel pore, and simultaneous binding of glutamate released from the
depolarized axon terminal. However, recent studies have shown that the
physiological concentration of Mg2+ in the
interstitial medium (0.7 mM, ) is lower than what is typically used
experimentally in the ACSF composition to record ex vivo neuronal
activity, suggesting that the NMDA receptor activity at resting
potential in physiological concentrations of Mg2+ may
have been underestimated . Furthermore, GluN2D-containing receptors may
exhibit reduced Mg2+ sensitivity compared to GluN2A-
or GluN2B-containing receptors . Therefore, we investigated the
mGlu5-induced potentiation of NMDA receptors at resting potential in the
presence of 0.7 mM Mg2+, by recording currents and
calcium transients (Figure 7 ). Whole-cell current recordings
revealed a residual NMDA-induced current of 1.80 + 0.30 pA/pF
density, which was potentiated to 2.40 + 0.40 pA/pF by DHPG
co-application (Figure 7A ). The full current potential
relationships are displayed in Figure 7B .
In a second set of experiments, we measured calcium fluctuations, still
in presence of physiological concentrations of Mg2+and following successive application of DHPG and AP5 (Suppl
Video 3 ). GCaMP6s fluorescence fluctuations highlighted basal
spontaneous Ca2+ transients, of various shape, size,
and kinetics, as illustrated by a projection of all spontaneous
Ca2+ transients recorded during 2 min and 15 sec
(Figure 7C ). The variety of these Ca2+ events
certainly relies on the nature of the receptors and channels involved in
their triggering. To focus on synaptic events, we selected small and
non-propagating calcium transients. The majority of these events were
blocked by AP5 (50 µM) at the end of the experiment, revealing
NMDA-dependent events (Figure 7D ). We could a posteriorfocused in these regions of interest to measure the influence of mGlu5
activation on NMDA function (Figure 7E ). DHPG increased the
number of basal calcium transients (Figure 7D ) and their area
under curve (AUC indeed significantly increased from 0.102 +0.001 to 0.154 + 0.002 following DHPG application, Figure
7F ). More importantly, DHPG changed the shape of the NMDA-dependent
events inducing a more sustained calcium inflow over time
(Figure 7E ). Taken together, our data show that the mGlu5
receptor potentiates NMDA receptor activity at resting membrane
potential, increasing currents and particularly calcium influx, thus
expanding the functional importance of NMDA receptors to resting
neurons.