Presynaptic and postsynaptic amplifications of neuropathic pain in the anterior cingulate cortex

Abstract

Neuropathic pain is caused by a primary lesion or dysfunction in the nervous system. Investigations have mainly focused on the spinal mechanisms of neuropathic pain, and less is known about cortical changes in neuropathic pain. Here, we report that peripheral nerve injury triggered long-term changes in excitatory synaptic transmission in layer II/III neurons within the anterior cingulate cortex (ACC). Both the presynaptic release probability of glutamate and postsynaptic glutamate AMPA receptor-mediated responses were enhanced after injury using the mouse peripheral nerve injury model. Western blot showed upregulated phosphorylation of GluR1 in the ACC after nerve injury. Finally, we found that both presynaptic and postsynaptic changes after nerve injury were absent in genetic mice lacking calcium-stimulated adenylyl cyclase 1 (AC1). Our studies therefore provide direct integrative evidence for both long-term presynaptic and postsynaptic changes in cortical synapses after nerve injury, and that AC1 is critical for such long-term changes. AC1 thus may serve as a potential therapeutic target for treating neuropathic pain.

Figure 1.

Increased synaptic transmission in layer II/III in the ACC after peripheral nerve ligation. A, Synaptic input–output curves in slices from control (n = 6 neurons) and nerve-ligated (n = 7 neurons) mice. *p < 0.05 and **p < 0.01 compared with those of control group. Open circles, Neurons from control mice; filled circles, neurons from mice with nerve ligation. B, Representative traces with an interval of 50 ms recorded in layer II/III of the ACC. Paired-pulse ratio (the ratio of EPSC2/EPSC1) was recorded at intervals of 35, 50, 75, 100, and 150 ms from control and nerve-ligated mice. Open circles, Neurons from control mice (n = 17 neurons); filled circles, neurons from mice with nerve ligation (n = 19 neurons). *p < 0.05; **p < 0.01; ***p < 0.001. C, PPF in layer V of the ACC from control and nerve-ligated mice. Open circles, Neurons from control mice (n = 9 neurons); filled circles, neurons from mice with nerve ligation (n = 15 neurons). D, PPF in motor cortex neurons from control and nerve-ligated mice. Open circles, Neurons from control mice (n = 5 neurons); filled circles, neurons from mice with nerve ligation (n = 5 neurons).

Figure 2.

mEPSCs recorded in the ACC in mice after peripheral nerve ligation. A, Representative mEPSCs recorded in the ACC neuron in slices from control mice (left) and mice with nerve ligation (right) at a holding potential of −70 mV. B, Cumulative interevent interval (left) and amplitude (right) histograms of mEPSCs recorded in slices from control mice (open circles; n = 9 neurons) and mice with nerve ligation (filled circles; n = 13 neurons). C, Summary plots of mEPSC data. Averaged values of mEPSC parameters: mean peak frequency (left) and amplitude (right). *p < 0.05; **p < 0.01.

Figure 3.

Faster MK-801 blockade of NMDA receptor-mediated EPSCs in mice with nerve ligation. A, Representative traces show NMDA receptor-mediated EPSCs at 0, 5, and 20 min in the presence of MK-801 (35 μm) in control and nerve-ligated mice. B, Plot of time course of MK-801 blockade of NMDA receptor-mediated EPSCs in control mice and mice with nerve ligation. Open circles, From control mice (n = 7 neurons); filled circles, from mice with nerve ligation (n = 8 neurons). C, Individual and statistical data showed the decay time required for the peak amplitude of NMDA receptor-mediated EPSC to decrease to 50% of initial value in the presence of MK-801. Significantly faster time was observed in mice with nerve ligation (n = 8 neurons) compared with control mice (n = 7 neurons). *p < 0.05.

Figure 4.

Altered phosphorylation of GluR1 and rectification index of AMPA receptor-mediated current in the ACC after nerve injury. A, Representative expression of GluR1 and phosphorylation of GluR1 and GluR2/3 by Western blot in the ACC from control and nerve-ligated mice. B, Pooled data showing that phosphorylation of GluR1 was upregulated in mice with nerve ligation. C, Representative traces of evoked AMPA receptor-mediated postsynaptic currents at −65, −5, and +35 mV holding potentials were recorded from one ACC neuron in control and nerve-ligated groups, respectively. Pooled data of rectification of AMPA receptor-mediated peak current show significant differences (*p < 0.05). Open bars, Neurons from control mice (n = 12 neurons); filled bars, neurons from mice with nerve ligation (n = 18 neurons). Rectification index = (amplitude at −65 mV holding potential)/(amplitude at +35 mV holding potential). D, Mean I–V curve of AMPA EPSCs in ACC neurons from control and nerve-ligated mice. *p < 0.05; **p < 0.01.

Figure 5.

The redistribution of AMPA receptor GluR1 subunits in ACC neurons after nerve injury. A, The subcellular distribution of GluR1 subunits in the membrane and cytosolic fraction of ACC neurons. The abundance of GluR1 subunits increased in the membrane fraction, whereas it decreased correspondingly in the cytosolic fraction after nerve injury. B, The subcellular distribution of GluR2/3 subunits was not affected in ACC neurons by nerve injury. The membrane and cytosolic fractions were prepared 1 week after nerve ligation. *p < 0.05; n = 4 mice for each group. C, Bilateral microinjection of CNQX (1 mm) in the ACC reduced the mechanical allodynia (n = 6 mice). D, Microinjection of CNQX did not affect the 50% paw-withdrawal threshold in control mice (n = 3 mice).

Figure 6.

Paired-pulse facilitation in ACC neurons in AC1^−/− mice with nerve ligation. A, Reduced mechanical allodynia in AC1^−/− mice with nerve ligation (n = 4 mice). *p < 0.05. B, The reduction in PPF of AMPA receptor-mediated EPSCs as shown by the wild-type mice after nerve injury was abolished in AC1^−/− mice after nerve injury. **p < 0.01. Insets, Representative traces of PPF with an interval of 50 ms recorded in the ACC. C, mEPSCs recorded in ACC neurons from AC1^−/− mice with nerve ligation. Shown are representative mEPSCs recorded in the ACC neuron in slices from AC1^−/− control mice (left) and mice with nerve injury (right) at a holding potential of −70 mV. D, Summary plots of mEPSC data. Average values of mEPSC parameters are shown. Shown are mean peak frequency (top) and amplitude (bottom) in AC1^−/− control mice (n = 5 neurons) and AC1^−/− mice with nerve ligation (n = 11 neurons; p > 0.05).

Figure 7.

Expression of GluR1 and rectification index of AMPA receptor-mediated currents in ACC neurons from AC1^−/− mice with nerve ligation. A, Representative Western blot of GluR1 and GluR2/3 in the ACC from wild-type and AC1^−/− mice with nerve ligation. B, Representative Western blot and corresponding quantitation of the phosphorylation of GluR1 at Ser845 in the ACC from wild-type mice (n = 6), wild-type mice with nerve ligation (n = 6), AC1^−/− control mice (n = 4), and AC1^−/− mice with nerve ligation (n = 4). The phosphorylation levels of GluR1 were significantly increased in the ACC after nerve injury (**p < 0.01; n = 6) in wild-type mice. The increase of the phosphorylation levels of GluR1 induced by nerve injury was blocked in the ACC of AC1^−/− mice compared with wild-type mice (^##p < 0.01; n = 4). C, Representative traces and pooled data of rectification index of AMPA receptor-mediated current in the ACC from AC1^−/− control and AC1^−/− mice with nerve ligation.