When are class I metabotropic glutamate receptors necessary for long-term potentiation?

Abstract

The involvement of metabotropic glutamate receptors (mGluRs) in hippocampal long-term potentiation (LTP) is a matter of controversial debate. Using [Ca2+]i measurements by confocal laser scanning microscopy and field recordings of EPSPs (fEPSPs) in the hippocampal CA1-region, we found that the efficacy of the broad-spectrum mGluR-antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and of (S)-4-carboxy-phenylglycine (4-CPG), a selective antagonist at class I mGluRs, in LTP is contingent on the tetanization strength and the resulting [Ca2+]i response. As indicated by experiments in which we blocked voltage-dependent calcium channels (VDCCs) and intracellular Ca2+ stores (ICSs), the functional significance of class I mGluRs in LTP is confined to certain types of potentiation, which are induced by weak tetanization protocols and require the release of Ca2+ from ICSs for induction. During strong tetanic stimulation, this Ca2+ source is functionally bypassed by activating VDCCs.

Fig. 1.

MCPG (400 μm; n= 7) (A) and 4-CPG (100 μm;n = 7) (B) did not influence LTP induced by a strong tetanization (3 × 100 Hz, 500 msec, 2 min interval between trains). The potentiation persisted for at least 240 min. The tetanus was applied at the time point 0. Horizontal bars under the time scale indicate the time of drug application. Analog traces represent typical recordings of single experiments taken 10 min before tetanization (1) and 120 min after tetanization (2). ○, Drug-treated groups; •, controls. Calibration: 2 mV, 3 msec.

Fig. 2.

LTP induced by a weak tetanization protocol was susceptible to the action of MCPG (A) or 4-CPG (B–D). The tetanization consisted of either four × two pulses (200 msec interval between pulse pairs) (A, B) or a single 100 Hz train, 400 msec duration (C). In the experiments depicted inD, two independent pathways in the same slice were used to allow a direct comparison of the effects of 4-CPG on LTP generated by induction protocols of different strength. The groups treated with mGluR antagonists are indicated by open symbols, and their respective controls are indicated by closed symbols. A, Application of 400 μmMCPG led to a significant reduction of LTP, starting at 50 min post-tetanus (n = 7, as compared with controls,n = 9; p < 0.05).B, 4-CPG (50 μm) caused a significant blockade of LTP from 65 min after tetanus (4-CPG groups:n = 6; controls: n = 8;p < 0.05). C, Similarly, an LTP induced by the weak 100 Hz tetanization decayed faster after application of 4-CPG (50 μm). D, 4-CPG (50 μm) significantly impaired a decremental LTP that was induced by a weak tetanization (single 100 Hz train, 400 msec duration;circles) of the first pathway, but had no significant effect on a robust potentiation (3 × 100 Hz, 500 msec duration, 2 min interval between trains; squares) generated 10 min afterward by strong tetanization of the second pathway. Note that inD the first sampling time after tetanus was 5 min, but it was 1 min in A–C. Analog traces represent typical recordings of single experiments taken 10 min before tetanization (1) and 60 min after tetanization (2). Calibration 2 mV, 3 msec.

Fig. 3.

The effectiveness of 4-CPG on LTP was contingent on the strength of tetanization. The fEPSP slope potentiation (normalized to controls 90 min after tetanization) was mostly impaired using tetanization protocols of four × two pulses (82.4 ± 2.5%, n = 7; p < 0.05;left column) and 100 Hz, 400 msec duration (83.4 ± 5.7%, n = 5, p < 0.05). The effect decreased if six instead of four paired pulses were applied in the paired-pulse protocol (89.5 ± 2.9%, n = 5; p < 0.05). Enhancement of the tetanus strength, by adding two more pulses (8 × 2) (data not shown) or by increasing the number of pulses (8 × 4) or by applying a strong tetanization of 3 × 100 Hz (500 msec, 2 min interval between trains) abolished the effect of 4-CPG on LTP (103.9 ± 10.6%,n = 5, and 100.4 ± 2.3%,n = 6, respectively). Coapplication of 4-CPG (100 μm) and nimodipine (10 μm) resulted in a clear decrease of potentiation to 67.8 ± 6.9% (n = 7). This effect could be mimicked by coapplication of thapsigargin (TG) and nimodipine (n = 7).

Fig. 4.

Ca²⁺ imaging of the rise of [Ca²⁺]_i in the dendritic tree of CA1 neurons (filled with the Ca²⁺-sensitive dye Calcium Green-1) on stimulation with different tetanization protocols and bath application of the mGluR class I antagonist 4-CPG (50 μm) and the L-type VDCC antagonist nimodipine (10 μm). A, Averaged Ca²⁺ response curves (transients) of seven neurons to a single set of tetanization paradigms. The Ca²⁺ transients of four dendritic regions were averaged. The tetanization protocols corresponding to the curves are indicated by an arrow. An increasing duration of the 100 Hz stimulation led initially to an increment of the peak amplitude, but after reaching a maximum amplitude the high Ca²⁺ level is maintained, followed by a slower decay (1 sec train). Application of the standard weak tetanization paradigm of four bursts of two pulses at 100 Hz (200 msec interburst interval) triggered a [Ca²⁺]_i rise that resembled the [Ca²⁺]_i responses obtained with the common 100 Hz protocols but was superimposed by steep pinnacles that were synchronized with the interburst interval of 200 msec (5 Hz) (shaded area). B, C, Representative images of the Ca²⁺ response of one neuron. The Ca²⁺ response to a weak (200 msec) (B) and a strong tetanization (1 sec) (C) is illustrated. The examples were taken at the onset of tetanization (top images), at the maximum of [Ca²⁺]_i elevation (middle), and during the decay phase of the response (bottom). Note the clear difference in the Ca²⁺ level during the decay of response at 933 msec (bottom traces). D, As indicated by the averaged areas of the fluorescence intensity changes (Area_F/F0), the rise of [Ca²⁺]_i was closely correlated to both the duration of tetanic 100 Hz stimulation and the pulse width that was used (correlation coefficients of 0.99; data not shown of pulse width 0.2 msec). The area below theF/F₀ curves was calculated asArea_F/F0 = ∫_B(F/F₀ − 1) dt;B = [0 sec, 8 sec]. E, F, After a tetanization of four × two pulses, bath application of 4-CPG (50 μm) led to a significant reduction (p < 0.05) of Area_F/F0 to 67% (F), which was caused predominantly by a slower rise and earlier decay of [Ca²⁺]_i. Note that the effect of 4-CPG was reversible (wash outline in E). G, Only the coapplication of 4-CPG and nimodipine caused a significant reduction ofArea_F/F0 (78%;p < 0.05) during strong tetanization (1 sec, 100 Hz). The application of 4-CPG alone had no effect.

Fig. 5.

Coapplication of mGluR class I antagonists and the L-type calcium channel antagonist nimodipine impaired LTP evoked by a strong tetanization protocol. A, MCPG (400 μm) affected the potentiation when nimodipine (10 μm) was coapplied. The reduction became significant 150 min after tetanization (p < 0.05;n = 7). The control application of nimodipine alone (n = 8) had no effect on LTP. B, Similarly, coapplication of 4-CPG (100 μm) and nimodipine resulted in a significant impairment of LTP starting 80 min after tetanization (p < 0.05;n = 7), whereas nimodipine by itself was not effective (n = 8). C, Application of thapsigargin (6 μm) had the same effect as mGluR class I antagonists if coapplied with nimodipine (○, p < 0.05; n = 7). The application of nimodipine (•,n = 7) and thapsigargin (▪, n= 6) alone had no influence on LTP. See Figure 2 for further explanation.

Fig. 6.

Scheme of the role of the three main Ca²⁺ sources during LTP induction in dependence of the tetanization strength. The final [Ca²⁺]_i necessary for induction of LTP is determined by two factors, the NMDARs and an additional source provided by either VDCCs or the Ca²⁺ release from ICSs after activation of class I mGluRs. Top scheme, A₁, During a weak, single tetanization the rise of [Ca²⁺]_i is fed by the activation of NMDARs and class I mGluRs, whereas the contribution of L-type VDCCs is negligible under these conditions. Thus, application of class I antagonists causes an impairment of LTP (A₂).Bottom scheme, B₁, A strong tetanization paradigm leads to a sustained depolarization enabling the Ca²⁺ entry via NMDARs and VDCCs, as well as to the release of Ca²⁺ from ICSs.B₂, The additional Ca²⁺ that is provided by the release from ICSs via liberation of IP₃on class I mGluR activation is not required for LTP induction.B₃, Blockade of VDCCs is counterbalanced by the class I mGluR-triggered Ca²⁺ release from ICSs.B₄, Concomitant inhibition of class I mGluRs and VDCCs results in a decremental potentiation.