Enhanced presynaptic neurotransmitter release in the anterior cingulate cortex of mice with chronic pain

Abstract

The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in learning and memory. Recent studies show that painful stimuli activate the prefrontal cortex and that brain chemistry is altered in this area in patients with chronic pain. Components of the CNS that are involved in pain transmission and modulation, from the spinal cord to the ACC, are very plastic and undergo rapid and long-term changes after injury. Patients suffering from chronic pain often complain of memory and concentration difficulties, but little is known about the neural circuitry underlying these deficits. To address this question, we analyzed synaptic transmission in the ACC from mice with chronic pain induced by hindpaw injection of complete Freund's adjuvant (CFA). In vitro whole-cell patch-clamp recordings revealed a significant enhancement in neurotransmitter release probability in ACC synapses from mice with chronic pain. Trace fear memory, which requires sustained attention and the activity of the ACC, was impaired in CFA-injected mice. Using knock-out mice, we found that calmodulin-stimulated adenylyl cyclases, AC1 and/or AC8, were crucial in mediating the long-lasting enhanced presynaptic transmitter release in the ACC of mice with chronic pain. Our findings provide strong evidence that presynaptic alterations caused by peripheral inflammation contribute to memory impairments after injury.

Figure 1.

Trace fear memory deficit in CFA-injected mice. A, B, Freezing during trace training (A) and testing (B) over 10 trials 3 d after injection of CFA (n = 8) or saline (n = 7) in wild-type mice. The asterisks indicate significantly different from saline-injected mice; in all cases, p < 0.05 is considered significant.

Figure 2.

Potentiation of input–output relationship in CFA-injected mice. A, Representative traces show averages of five EPSCs at 4 and 7 V stimulation intensity in the ACC. B, Plot of input–output curves shows significant enhancement of synaptic transmission in the ACC of CFA-injected mice. Open circles, From saline mice, n = 10; filled circles, from CFA-injected mice, n = 16. C, Representative traces show averages of five EPSCs at 4 and 7 V stimulation intensity in the SSC. D, Plot of input–output curves shows no difference in synaptic transmission in the SSC between saline- and CFA-injected mice. Open circles, From saline control mice, n = 6; filled circles, from CFA-injected mice, n = 8.

Figure 3.

Paired-pulse facilitation in mice with chronic pain. PPF (the ratio of EPSC2/EPSC1) was recorded with intervals of 35, 50, 75, 100, and 150 ms. A, Representative traces of PPF with an interval of 50 ms recorded in the ACC. B, PPF was significantly reduced at each interval in CFA-injected mice. Open circles, From saline control mice, n = 10; filled circles, from CFA-injected mice, n = 17. *p < 0.05; **p < 0.01. C, Representative traces of PPF with intervals of 50 ms recorded in the SSC. D, PPF was not altered in the SSC of CFA-injected mice. Open circles, From saline mice, n = 6; filled circles, from CFA-injected mice, n = 8.

Figure 4.

Miniature EPSCs in ACC neurons. A, Representative mEPSCs recorded in pyramidal neurons at a holding potential of −70 mV from saline-injected (top) and CFA-injected (bottom) mice. B, Top left, Average mEPSC of 50 events from a saline mouse; bottom left, average mEPSC of 53 events from a CFA-injected mouse. Right, Time constant of mEPSC decay (τ) versus the rising time (10–90%) in the recordings from saline-injected (triangle; n = 13) and CFA-injected (circle; n = 10) mice. C, Cumulative frequency (left) and amplitude (right) histogram of the mEPSCs from the cells in A. Solid line, Recording from a saline mouse; dashed line, recording from a CFA-injected mouse. D, mEPSCs frequency (left) and amplitude (right) in neurons from saline-injected (n = 14) and CFA-injected (n = 11) mice. *p < 0.05. E, Current–voltage plots of peak mEPSCs recorded at holding potentials of −70 to +70 mV. Open circle, Recording from saline control mice (n = 6); filled circles, recording from CFA-injected mice (n = 7). Values were obtained by normalizing the mean peak currents at each holding potential to a holding potential of −70 mV. Inset shows the mEPSCs recording at +50 and −70 mV.

Figure 5.

Faster MK-801 blockade of NMDA EPSCs in CFA-injected mice. A, Representative traces show NMDA EPSCs at 0, 2, and 20 min in the presence of MK-801 (35 μm) in saline- and CFA-injected mice. B, Plot of time course of MK-801 blockade of NMDA EPSCs in saline- and CFA-injected mice. Open circles, From saline mice, n = 9; filled circles, from CFA-injected mice, n = 8. C, Individual and statistical results showed the decay time required for the peak amplitude of NMDA EPSC to decrease to 50% of initial value in MK-801. Significantly faster time was found in CFA-injected mice (n = 8) than control mice (n = 9; p < 0.05).

Figure 6.

ACC synaptic transmission in AC1 KO mice. A, Representative traces show averages of five EPSCs at 4 and 7 V stimulation intensity in the ACC. B, Plot of input–output curves shows no difference of synaptic transmission in the ACC between the saline- and CFA-injected AC1 KO mice. Open circles, From saline mice, n = 6; filled circles, from CFA-injected mice, n = 7. C, Representative traces of PPF with interval of 50 ms recorded in the ACC. D, PPFs were no different at each interval between the saline- and CFA-injected AC1 KO mice. Open circles, From saline mice, n = 7; filled circles, from CFA-injected mice, n = 6.

Figure 7.

ACC synaptic transmission in AC1 and AC8 KO mice. A, Representative traces show averages of five EPSCs at 4 and 7 V stimulation intensity in the ACC. B, Plot of input–output curves shows no difference of synaptic transmission in the ACC between the saline- and CFA-injected AC1/AC8 KO mice. Open circles, From saline mice, n = 6; filled circles, from CFA-injected mice, n = 6. C, Representative traces of PPF with interval of 50 ms recorded in the ACC. D, PPFs were no different at each interval between the saline- and CFA-injected AC1/AC8 KO mice. Open circles, From saline mice, n = 9; filled circles, from CFA-injected mice, n = 13.

Figure 8.

CFA and trace fear memory in adenylyl cyclase knock-out mice. A–C, Freezing during trace testing over 10 trials, 3 d after injection of CFA or saline in AC1 KO (A), AC8 KO (B), or AC1/AC8 KO (C) mice. D, Freezing during trace testing over 10 trials after intra-ACC forskolin or vehicle injection in AC1/AC8 KO mice. E, F, Average freezing over the 10 trials of the training and testing sessions. *p < 0.05, significantly different from saline-injected mice. ^#p < 0.05, significantly different from wild-type mice.