Metabotropic glutamate receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca²⁺ wave-dependent activation of SK and TRPC channels - PubMed (original) (raw)

Comparative Study

Metabotropic glutamate receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca²⁺ wave-dependent activation of SK and TRPC channels

Lynda El-Hassar et al. J Physiol. 2011.

Abstract

Group I metabotropic glutamate receptors (mGluRs) play an essential role in cognitive function. Their activation results in a wide array of cellular and molecular responses that are mediated by multiple signalling cascades. In this study, we focused on Group I mGluR activation of IP3R-mediated intracellular Ca2+ waves and their role in activating Ca2+-dependent ion channels in CA1 pyramidal neurons. Using whole-cell patch-clamp recordings and high-speed Ca2+ fluorescence imaging in acute hippocampal brain slices, we show that synaptic and pharmacological stimulation of mGluRs triggers intracellular Ca2+ waves and a biphasic electrical response composed of a transient Ca2+-dependent SK channel-mediated hyperpolarization and a TRPC-mediated sustained depolarization. The generation and magnitude of the SK channel-mediated hyperpolarization depended solely on the rise in intracellular Ca2+ concentration ([Ca2+]i), whereas the TRPC channel-mediated depolarization required both a small rise in [Ca2+]i and mGluR activation. Furthermore, the TRPC-mediated current was suppressed by forskolin-induced rises in cAMP. We also show that SK- and TRPC-mediated currents robustly modulate pyramidal neuron excitability by decreasing and increasing their firing frequency, respectively. These findings provide additional evidence that mGluR-mediated synaptic transmission makes an important contribution to regulating the output of hippocampal neurons through intracellular Ca2+ wave activation of SK and TRPC channels. cAMP provides an additional level of regulation by modulating TRPC-mediated sustained depolarization that we propose to be important for stabilizing periods of sustained firing.

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Figures

Figure 1

Figure 1. Synaptically evoked intracellular Ca2+ waves and an associated biphasic membrane potential change in CA1 pyramidal neurons

A, left panel, fluorescence image of a fura-2FF-filled neuron. Middle and right panels, two representations of the same imaging data elicited by synaptic stimulation (50 pulses at 100 Hz; see electrical response in right panel). The middle panel is a pseudo-linescan showing that intracellular Ca2+ waves propagate through hot and cold spots of release. The right panel shows coloured waveforms corresponding to Ca2+ rises occurring at the colour-coded boxes (regions of interest, ROIs) over the cell in the left panel. Note the difference in magnitude and timing of the VGCC-mediated Ca2+ rise occurring during the synaptically elicited action potentials and the delayed intracellular Ca2+ wave. B, Group I mGluR blockers selectively blocked intracellular Ca2+ waves, but not VGCC-mediated rises in [Ca2+]i. Rises in [Ca2+]i were first initiated by activation of VGCCs with current injection-evoked spikes (2 ms, 2 nA, 10 spikes at 100 Hz) followed by synaptic stimulation (30 pulses at 100 Hz). Middle panel, mGluR antagonists, MPEP (10 μ

m

) and LY367385 (100 μ

m

), blocked synaptically elicited internal Ca2+ release. Right panel, the evoked electrical waveforms recorded during the Ca2+ responses shown in the middle panel. Note the suppression of the membrane depolarization following bath application of the mGluR antagonists. C, synaptically elicited intracellular Ca2+ waves correlate with a biphasic membrane potential change. Synaptic stimulation, subthreshold for eliciting a rise in [Ca2+]i (100 pulses at 100 Hz), failed to elicit a biphasic membrane potential change. 30 s after ‘priming’ the cell with a train of current injection-evoked spikes (2 ms, 2 nA current injection; 100 spikes at 100 Hz) and consequent VGCC-mediated Ca2+ influx (not shown), the previously subthreshold synaptic stimulation elicited internal Ca2+ release and a hyperpolarization and depolarization. Note that the fast EPSPs evoked by electrical stimulation were not affected by priming. In this example, mAChRs and GABABRs were blocked (1 μ

m

atropine and 1 μ

m

CGP55845, respectively). D, summary data showing priming-induced facilitation of synaptically elicited internal Ca2+ release and associated membrane potential changes (normalized to pre-priming response averages; n = 7; *P < 0.01, **P < 0.001; ANOVA).

Figure 2

Figure 2. mGluR agonists elicit a Ca2+ wave-dependent hyperpolarization and depolarization

A, left, overlay of a DIC image and fluorescence neuron image showing the position of the pressure application pipette near the primary apical dendrite of the recorded cell. Right, a DHPG puff (400 μ

m

, 50 ms) onto the primary apical dendrite triggered a bidirectionally propagating Ca2+ wave and associated hyperpolarization and depolarization of a CA1 pyramidal neuron. B, upper panel, in a different neuron, an ACPD puff (400 μ

m

, 50 ms) elicited a Ca2+ wave (not shown) and a transient hyperpolarization and a sustained depolarization. Lower panel, when the neuron was held at a membrane potential slightly subthreshold for spiking (∼−53 mV), ACPD elicited a transient hyperpolarization and a sustained train of action potentials. C, in voltage clamp, an ACPD puff elicited an outward current and inward current (see Results for summary data).

Figure 3

Figure 3. The hyperpolarization and depolarization are due to SK channels and CAN channels, respectively

A, left, the reversal potential (_E_rev) of the hyperpolarizing potential was determined to be ∼−85 mV by applying DHPG puffs (400 μ

m

, 50 ms) at different holding potentials in current clamp and measuring membrane potential changes. Intracellular Ca2+ waves were similar at all holding potentials (waves are colour-coded by holding potential). Right panel, summary graph showing the reversal potential for all cells tested (n = 5; each cell is represented by a different colour). B, consistent with a mechanism involving SK channels, apamin (100 n

m

) blocked the hyperpolarization. This treatment unveiled the isolated depolarizing potential and revealed its delayed onset. C, left and middle panels, the mGluR-mediated, Ca2+-dependent depolarizing current was isolated in voltage clamp and its _E_rev was determined to be ∼12 mV. DHPG puffs (400 μ

m

, 50 ms) were delivered to the primary apical dendrite in the presence of voltage-gated K+ channel and Na+ channel blockers, and GABABR blockers (see Results). Right, summary I–V graph for all cells tested (n = 5; each cell is represented by a different colour) shows data consistent with activation of CAN channels.

Figure 4

Figure 4. The sustained depolarization requires both group I mGluR receptor activation and a rise in [Ca2+]i

A, fluorescence image of a neuron filled with fluo-4 (100 μ

m

) and NPE-caged IP3 (97 μ

m

). Coloured boxes indicate the regions of interest ROIs in apical dendrites corresponding to the optical traces showing internal Ca2+ release on the right. UV flashes directed at the proximal primary apical dendrite (20 μm diameter, 400 ms duration; represented by yellow circle) elicited internal Ca2+ release and a hyperpolarizing potential, but not a depolarization. B, a caffeine puff (50 m

m

, 50 ms) onto the proximal apical dendrite elicited intracellular Ca2+ waves and a transient hyperpolarization, but no depolarization. C, the sustained depolarization depended on mGluR activation and a rise in [Ca2+]. Depleting Ca2+ stores with CPA (50 μ

m

) prevented mGluR-mediated internal Ca2+ release and membrane potential changes. In the absence of internal Ca2+ release, pairing mGluR activation with VGCC-mediated Ca2+ influx during spikes evoked by current injection (2 ms, 2 nA current injections; 10–50 spikes at 100 Hz) elicited a similar sustained depolarization. Under these conditions, spiking elicited a VGCC-mediated hyperpolarization that was not affected by agonist application (data not shown). D, summary data showing rescue of membrane potential changes when internal stores are depleted (n = 5, **P < 0.001, ANOVA).

Figure 5

Figure 5. Pharmacological characterization of the sustained depolarization—non-specific blockers of _I_CAN/_I_TRPC suppressed the sustained depolarization

A, internal Ca2+ stores were first depleted with CPA (50 μ

m

). VGCC-mediated rises in [Ca2+]i were elicited with current-injected spikes and paired with puffs of DHPG. Addition of both flufenamate (FFA; 100 μ

m

) and SKF96365 (30 μ

m

) suppressed both the depolarization and the hyperpolarization (n = 5; P < 0.01, t test). B, addition of FFA (100 μ

m

) alone blocked the depolarization (n = 3). C, addition of SKF96365 (30–100 μ

m

) alone had no effect on either the depolarization or the hyperpolarization under these conditions (n = 5).

Figure 6

Figure 6. TRPC1, TRPC4 and TRPC5 antibodies block the mGluR-mediated and intracellular Ca2+ wave-dependent depolarization

Antibodies to TRPC were loaded into patch recording pipettes (1:100 dilution). In some cases antibodies were heat inactivated. Responses recorded ∼5 min after breaking into the cell were compared to responses recorded ∼20 min after breaking in. A, an example of data collected from a CA1 pyramidal neuron loaded with anti-TRPC1 and an example of a neuron loaded with heat-inactivated anti-TRPC1. Anti-TRPC1 selectively blocked the sustained depolarization. B, examples of neurons loaded with anti-TRPC3, anti-TRPC4 or anti-TRPC5. TRPC3 did not affect the depolarization (n = 3, P > 0.1, t test); TRPC4 (n = 5), like TRPC1 (n = 5) and TRPC5 (n = 5; data not shown), suppressed the mGluR/IP3R evoked-depolarization (P < 0.01 for each antibody, t test). C, summary data for anti-TRPCs, and the controls, IgG (n = 7) or inactivated anti-TRPC1 (n = 5) or TRPC5 (n = 5).

Figure 7

Figure 7. Rises in intracellular cAMP differentially suppressed the mGluR-mediated depolarization and hyperpolarization

A, DHPG application paired with current injection-evoked action potentials elicited robust rises in [Ca2+]i and associated membrane potential changes. Bath application of forskolin (5–10 μ

m

) totally blocked the TRPC channel-mediated depolarization. Forskolin also partially suppressed the SK channel-mediated AHP, but not the fast Ca2+-dependent AHP. Forskolin also suppressed internal Ca2+ release in this cell. B, summary data showing suppression of the depolarization (n = 9; *P < 0.01, t test).

Figure 8

Figure 8. TRPC1, 4 and 5 antibodies suppress mGluR-mediated increases in spike frequency

A, mGluR activation of intracellular Ca2+ waves modulates the firing pattern of CA1 pyramidal neurons. Puffing DHPG (50 ms) onto the apical dendrite of a spiking pyramidal neuron held at ∼−45 mV in current clamp suppressed and then increased the firing frequency of this representative pyramidal neuron. B, an example of a neuron loaded with IgG. mGluR regulation of firing frequency was not affected by IgG (or inactivated anti-TRPCs; not shown). C, an example of a neuron loaded with anti-TRPC4. Addition of anti-TRPC1, 4 or 5 (1:100 dilution) to the patch pipette suppressed the increase in firing frequency 20 min after breaking into the cell. D, summary data showing that anti-TRPC1, 4 and 5 suppress mGluR-mediated increases in firing frequency (cumulatively P < 0.01, _t_ test). TRPC3 (_n_ = 3) did not alter mGluR-mediated regulation of firing (_P_ > 0.1, t test). Inactivated anti-TRPCs (n = 4) and IgG (n = 7) did not have a significant effect on mGluR-mediated increases in firing rate (P > 0.1, t test). Caffeine puffs (50 m

m

; n = 5), which activate an RyR-mediated _I_SK but not an _I_TRPC, did not elicit an increase in firing frequency (P > 0.1, t test).

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