Inhibition of mitochondrial complex II induces a long-term potentiation of NMDA-mediated synaptic excitation in the striatum requiring endogenous dopamine

Abstract

Abnormal involuntary movements and cognitive impairment represent the classical clinical symptoms of Huntington's disease (HD). This genetic disorder involves degeneration of striatal spiny neurons, but not striatal large cholinergic interneurons, and corresponds to a marked decrease in the activity of mitochondrial complex II [succinate dehydrogenase (SD)] in the brains of HD patients. Here we have examined the possibility that SD inhibitors exert their toxic action by increasing glutamatergic transmission. We report that SD inhibitors such as 3-nitroproprionic acid (3-NP), but not an inhibitor of mitochondrial complex I, produce a long-term potentiation of the NMDA-mediated synaptic excitation (3-NP-LTP) in striatal spiny neurons. In contrast, these inhibitors had no effect on excitatory synaptic transmission in striatal cholinergic interneurons and pyramidal cortical neurons. 3-NP-LTP involves increased intracellular calcium and activation of the mitogen-activated protein kinase extracellular signal-regulated kinase and is critically dependent on endogenous dopamine acting via D2 receptors, whereas it is negatively regulated by D1 receptors. Thus 3-NP-LTP might play a key role in the regional and cell type-specific neuronal death observed in HD.

Fig. 1.

Inhibition of mitochondrial complex II but not complex I activity induces LTP in striatal spiny neurons. In spiny neurons, 3-NP enhanced the amplitude of NMDA-mediated corticostriatal EPSPs (in 0 mm Mg plus CNQX), whereas AMPA-mediated potentials (in 1.2 mm Mg plus APV) were unaffected.Traces on the right are an average of four single EPSPs (a). The effect of 3-NP was mimicked by MMA (b). Conversely, rotenone failed to affect either component of excitatory synaptic transmission (c). Calibration in a also applies to b and c. In all the experiments, the resting membrane potential of the recorded cells (dotted lines) was constant and ranged between −84 and −86 mV.

Fig. 2.

Striatal cholinergic interneurons and cortical pyramidal neurons do not show 3-NP-LTP. In cholinergic interneurons, 3-NP did not enhance the amplitude of either NMDA- or AMPA-mediated corticostriatal EPSPs (a). Resting membrane potential = −60 mV. Similar lack of effects was observed in cortical pyramidal neurons (b). Resting membrane potential = −72 mV.

Fig. 3.

3-NP-LTP is caused by enhanced NMDA receptor-mediated synaptic transmission and requires intracellular calcium elevation. Cortically evoked EPSPs after the induction of 3-NP-LTP in spiny neurons were fully suppressed by the NMDA receptor antagonist APV (a). Intracellular injection of the calcium chelator BAPTA fully prevented 3-NP-LTP. Conversely, this form of synaptic plasticity was prevented neither by 10 μm nifedipine nor by 100 nm pirenzepine (b).

Fig. 4.

3-NP-LTP is expressed when striatal neurons are in the up state. In the presence of physiological concentrations of external magnesium, 3-NP enhanced EPSPs recorded from neurons depolarized to −55 mV by intracellular injection of positive current (“up” state), but not EPSPs recorded at resting membrane potentials (−85 mV; “down” state) (a). Note that the data represented ina were obtained by shifting the membrane potential of each single neuron from the up to the down state and vice versa throughout the duration of 10 electrophysiological experiments. This experimental protocol was followed to test whether the induction of 3-NP-LTP on the up state EPSP also influenced the AMPA component of the EPSP as detected in the down state. The NMDA receptor antagonist APV fully prevented the formation of 3-NP-LTP observed in the up state (b). Traces in the bottom part represent examples of EPSPs recorded from two different spiny neurons in the absence (a) and presence (b) of APV at different membrane potential levels.

Fig. 5.

SD inhibitors enhance inward currents induced by bath application of NMDA in striatal spiny neurons but not in cholinergic interneurons. In spiny neurons, bath application of 3-NP significantly enhanced the inward current evoked by application of NMDA but not of AMPA (a). A single experiment obtained from a striatal spiny neuron voltage clamped at −80 mV is shown (b). The effect of 3-NP was mimicked by MMA (c). Application of 3-NP failed to affect NMDA- and AMPA-mediated currents in striatal cholinergic interneurons (d).

Fig. 6.

3-NP enhances basal and NMDA-induced intracellular calcium concentration in striatal spiny neurons. The histogram shows the mean percentage of increase ± SEM of intracellular calcium (expressed as ratio values) in response to NMDA, 3-NP alone, and NMDA in the presence of 3-NP (a). Thetraces in the top panel show the membrane depolarization induced by NMDA (30 μm) in the control condition (left) and after 20 min of 3-NP treatment (right). The bottom panel shows the simultaneous measurements of intracellular calcium levels in the same neuron (b). Resting membrane potential = −84 mV. *p < 0.05; **p < 0.01; ***p < 0.001 (Student's ttest for paired observations).

Fig. 7.

Endogenous DA controls the formation of 3-NP-LTP through the activation of D2 DA receptors. 3-NP-LTP was abolished in slices obtained from DA-depleted striata, whereas it was normally expressed in tissue obtained from intact contralateral striata (a). Quinpirole, a D2-like DA receptor agonist, but not SKF 38393, a D1-like DA receptor agonist, restored 3-NP-LTP in DA-denervated neurons (b). 3-NP-LTP was blocked by l-sulpiride, a D2-like DA receptor antagonist, but not by SCH 23390, a D1-like DA receptor antagonist (c). 3-NP-LTP was also prevented by forskolin, an adenylyl cyclase activator, and by SKF 38393, a D1-like DA receptor agonist (d). Intracellular application of the PKA inhibitor H89 partially prevented the inhibitory effects ofl-sulpiride and forskolin on 3-NP-LTP formation (e). 3-NP-LTP was absent in striatal neurons recorded from mice lacking D2 receptors but not in WT animals. Intracellular injection of H89 partially restored 3-NP-LTP in mice lacking D2 (f). Note that in this latter graph data obtained from WT mice were significantly different from both D2 knock-out (KO) mice (p < 0.01) and D2 KO mice plus H89 (p < 0.001). Moreover, a statistical significance (p < 0.001) was detected between D2 KO mice and D2 KO mice plus intracellular H89.

Fig. 8.

3-NP-LTP induction is blocked by the ERK cascade inhibitor PD98059. Corticostriatal slices were treated with 100 μm 3-NP, with 3-NP plus MEK inhibitor PD 98059 (PD; 10 μm), or 3-NP together with 1 μml-sulpiride as indicated in Materials and Methods. After protein extraction, ERK activation was analyzed by immunoblotting with antibodies specific for phosphorylated, activated ERKs 1 and 2. Equal levels of ERK were confirmed by immunoblotting with anti-ERK1/2 (a). Inhibition of MAP kinase activity by 10 μm PD 98059 prevented 3-NP-induced 3-NP-LTP (b).

Fig. 9.

Hypothetical model to account for the receptor and post-receptor mechanisms underlying 3-NP-LTP in striatal spiny neurons. D2 and D1 receptors exert opposing actions on adenylyl cyclase (AC) activity. The D2 receptor-mediated reduction of cAMP levels leads to the inhibition of protein kinase A (PKA) activity. This suppresses PKA-dependent inhibition of Raf-1 and consequently activates the MEK/ERK cascade. MEK activity is also stimulated by increased intracellular calcium levels ([Ca²⁺]_i). The increase in [Ca²⁺]_i is secondary to NMDA receptor stimulation, impairment of mitochondrial buffering properties caused by SD inhibition, and D2 receptor-mediated stimulation of phospholipase C (PLC). The final result of this cascade of biochemical events is the ERK-dependent induction of nuclear events leading to protein synthesis and altered NMDA receptor-channel complex function.