Loss of M1 Receptor Dependent Cholinergic Excitation Contributes to mPFC Deactivation in Neuropathic Pain

Abstract

In chronic pain, the medial prefrontal cortex (mPFC) is deactivated and mPFC-dependent tasks such as attention and working memory are impaired. We investigated the mechanisms of mPFC deactivation in the rat spared nerve injury (SNI) model of neuropathic pain. Patch-clamp recordings in acute slices showed that, 1 week after the nerve injury, cholinergic modulation of layer 5 (L5) pyramidal neurons was severely impaired. In cells from sham-operated animals, focal application of acetylcholine induced a left shift of the input/output curve and persistent firing. Both of these effects were almost completely abolished in cells from SNI-operated rats. The cause of this impairment was an ∼60% reduction of an M1-coupled, pirenzepine-sensitive depolarizing current, which appeared to be, at least in part, the consequence of M1 receptor internalization. Although no changes were detected in total M1 protein or transcript, both the fraction of the M1 receptor in the synaptic plasma membrane and the biotinylated M1 protein associated with the total plasma membrane were decreased in L5 mPFC of SNI rats. The loss of excitatory cholinergic modulation may play a critical role in mPFC deactivation in neuropathic pain and underlie the mPFC-specific cognitive deficits that are comorbid with neuropathic pain.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) undergoes major reorganization in chronic pain. Deactivation of mPFC output is causally correlated with both the cognitive and the sensory component of neuropathic pain. Here, we show that cholinergic excitation of commissural layer 5 mPFC pyramidal neurons is abolished in neuropathic pain rats due to a severe reduction of a muscarinic depolarizing current and M1 receptor internalization. Therefore, in neuropathic pain rats, the acetylcholine (ACh)-dependent increase in neuronal excitability is reduced dramatically and the ACh-induced persisting firing, which is critical for working memory, is abolished. We propose that the blunted cholinergic excitability contributes to the functional mPFC deactivation that is causal for the pain phenotype and represents a cellular mechanism for the attention and memory impairments comorbid with chronic pain.

Keywords: SNI; internalization; muscarinic; pyramidal cell; working memory.

Figure 1.

Tactile threshold is markedly reduced in the injured paw of SNI rats 1 week after surgery. Before surgery (week 0), there was no significant difference in the tactile threshold of the right and left hindpaws of sham (14.47 ± 3.5 g and 12.2 ± 3.0 g, respectively, n = 19) or SNI animals (15.8 ± 4.8 g and 14.97 ± 3.8 g, respectively, n = 14). One week after surgery (week 1), tactile thresholds in sham animals were 12.7 ± 2.6 g on the right paw contralateral to the surgery site and 8.6 ± 1.4 g on the left paw. In SNI animals, no significant change in threshold was detected in the right hindpaw (10.4 ± 2.8 g), whereas a significant drop was observed in the left hindpaw ipsilateral to the surgery site (0.4 ± 0.1 g, p = 0.002).

Figure 2.

Location and firing properties of the recorded mPFC L5 pyramidal neurons. A, All current-clamp (and a subset of voltage-clamp) recorded cells were filled with biocytin; the positions were then mapped onto an image of the mPFC (Paxinos and Watson, 1998) and plotted as a function of distance from pia (x-axis) and distance from apex (y-axis). Cell bodies of sham neurons were located at a distance of 726.4 ± 22 μm from the pia and 2227 ± 61.6 μm from the apex (n = 31). SNI neurons were located 685.6 ± 22 μm from pia and 2315.7 ± 57 μm from apex (n = 22), in the prelimbic region of the rat mPFC. B, Left, Current-clamp recordings in slices from sham and SNI rats. Middle, Cells from both sham and SNI animals exhibited accommodation, as evidenced by interspike interval ratios (first interspike interval divided by the average interval) of 0.2 ± 0.02 and 0.29 ± 0.07 (n = 10, 9, respectively) and a time-dependent reduction in instantaneous firing frequency in response to a current injection of 150 pA (13.21 ± 1.51 Hz at 1 s, and 11.53 ± 1.12 Hz at 2 s, paired t test p = 0.01; n = 19; right).

Figure 3.

ACh increases pyramidal cell excitability in sham, but not SNI, animals. A, Voltage responses recorded from L5 pyramidal neurons in acute slices in the presence of blockers of fast synaptic transmission (100 μm picrotoxin, 20 μm DNQX, 50 μm APV); cells were stimulated with 2 s current injections of either 50 or 100 pA in control conditions (black) and after a 1 mm, 100 ms focal application of ACh (black; arrow indicates the time of ACh application) in a slice from a sham animal. At rest, both neurons were held at −70 mV. B, In slices from SNI animals, the same stimulation protocol failed to increase cellular excitability in response to a 50 pA current injection and actually decreased excitability in response to a 100 pA current injection (gray) compared with control conditions (black). C, In sham rats, ACh application induced a leftward shift in the I/O curve. Fitting the data points with Boltzmann equations showed that ACh shifted the midpoint from 99.5 ± 8.1 pA to 45 ± 6.3 pA (n = 10). D, ACh failed to shift the I/O curve of pyramidal neurons from SNI animals (gray) (I_1/2 = 104.9 ± 7.8 pA, in control and 120.9 ± 14.2 pA in ACh, in SNI cells, n = 9).

Figure 4.

ACh-mediated persistent firing is abolished in SNI animals. A, Whole-cell, current-clamp traces of an ACh-dependent sADP response immediately after a 100 pA, 2-s-long current injection (inset) in a cell from a sham (black) and a 150 pA, 2-s-long current injection from a SNI (gray) rat. B, Average of the maximum peak sADP voltage response in sham and SNI animals (10.55 ± 0.8 mV and 5.5 ± 1.4 mV, n = 7 and 9, respectively, p = 0.01). C, Top trace, Current-clamp response of a mPFC pyramidal neuron to a 150 pA, 2 s current injection in control conditions. Bottom trace, Voltage response of an mPFC pyramidal neuron to 1 mm, 100 ms focal application of ACh (large arrow), followed by a 150 pA, 2 s current injection. Note the persistence of action potentials (small arrows) after the end of the current step. D, Left, Time, relative to the end of the current step (time 0, dashed line), of the last recorded action potential after focal application of ACh plotted for both sham and SNI animals. Note that, in 55% of the tested pyramidal neurons from sham animals, persistent activity was observed for several seconds after the end of the current step. Right, Average time, in seconds, of the last recorded action potential relative to step repolarization after focal application of ACh in sham and SNI animals (2.7 ± 1.1 s and −0.015 ± 0.16 s, respectively, n = 11, 9 p = 0.036).

Figure 5.

ACh induced depolarization is blunted in SNI animals. A, Voltage responses of L5 pyramidal neurons to 100-ms-long focal applications of ACh (1 mm, at the soma) in slices from sham and SNI animals. The bath temperature was ∼31°C and the resting potential −70 mV, in both cells. The resting membrane potential was kept at −70 mV in all cells (if necessary, a small holding current was injected). In some cells, the ACh-dependent depolarization was preceded by a hyperpolarization (bottom, gray). B, Left, When present, the average hyperpolarizing responses to ACh (1 mm) were significantly different in sham animals (black, −0.42 ± 0.19 mV, 11 cells) and SNI animals (gray, −2.28 ± 0.34 mV, 9 cells, p = 0.017). Right, Average depolarizing voltage responses to ACh (1 mm) were significantly different between sham (black, 7.47 ± 0.95 mV, n = 11) and SNI animals (gray, 3.50 ± 0.78 mV, n = 9, p = 0.005). C, Membrane resistance in SNI cells was larger compared with sham (231.0 ± 19 MΩ and 176.08 ± 14.0 MΩ, respectively, n = 9 and 10, respectively, p = 0.03). D, Responses evoked by focal application of 100 μm ACh were 3.48 ± 0.7 mV after 100 ms applications (10 cell from naive animals) and 2.3 ± 0.5 mV in response to 50 ms applications (7 cells from naive animals). The gray dotted bar shows, for comparison, the depolarizing response observed in sham animals using 1 mm ACh. Note that no hyperpolarizing component was detectable with the reduction in ACh concentration and duration. E, Focally applied ACh (100 μm) caused an increase in the firing frequency (in response to a 100 pA current injection it was 6.1 ± 0.7 Hz in control versus 10.1 ± 0.9 Hz in 100 μm ACh, p = 0.02; 4 cells from naive animals).

Figure 6.

ACh-evoked currents in L5 pyramidal neurons are markedly reduced in mPFC of SNI rats. A, Whole-cell, current traces in sham (left) and SNI (right) pyramidal neurons voltage clamped at −70 mV after bath application of 1 mm ACh (bar). The bath solution contained 100 μm picrotoxin to block fast, inhibitory synaptic currents. B, Average peak current responses evoked by bath application of 1 mm ACh in sham (−89.5 ± 9.6 pA, n = 10) and SNI (−36.5 ± 5.2 pA, n = 10, p = 0.0003) animals. When individual peak current responses are normalized to the capacitance of the cell, the difference between sham and SNI responses remains significant (0.70 ± 0.09 pA/pF and 0.25 ± 0.03, n = 10 and 10, respectively, p = 0.0014). C, Current traces in sham (left) and SNI (right) neurons voltage clamped at −70 mV after a 1 mm, 100 ms picospritzer application of ACh (arrow). Bath and focal application solution contained blockers of fast excitatory (50 μm APV and 20 μm DNQX) and inhibitory synaptic currents (100 μm picrotoxin). D, Average peak current responses evoked by 1 mm ACh in sham (−32.57 ± 1.98 pA, n = 27) and SNI (−18.81 ± 2.69 pA, n = 18, p = 0.0015) animals.

Figure 7.

Depolarizing cholinergic current in mPFC neurons is mediated by the M1 receptor. A, Traces represent peak current responses to focal application of 1 mm ACh (arrow) in control conditions (black) and in the presence of 2 μm bath-applied atropine (gray) in a mPFC neuron from sham (left) and SNI (right) animals. Voltage was −70 mV. B, Atropine blocked the ACh-activated current (the block of the peak current was 96.32 ± 2.17% in sham and 89.2 ± 8.6% in SNI animals (n = 6 for each group). C, Current responses to 1 mm focally applied ACh (arrow) in control conditions (black) and after bath application of either 2 μm pirenzepine (gray) or 10 μm pirenzepine (gray). D, Summary of the effect of either 2 or 10 μm pirenzepine on the ACh-elicited current. Ten micromolar pirenzepine blocked 93.54 ± 5.62% (p = 0.002; 5, cells from sham animals) of the current. Similarly, 2 μm pirenzepine blocked 82.03 ± 13.21% (p = 0.01; n = 3, naive animals) of the peak control current. E, Depolarizing current evoked by ACh (1 mm) was not affected by bath application of the M2 blocker methoctramine (1 μm; the current was 96 ± 0.02% of that in control; n = 5 cells from naive animals).

Figure 8.

Level of M1 mRNA and total protein is unaffected in L5 mPFC of SNI rats. A, qRT-PCR was performed on cDNA from tissue isolated from L5 of the mPFC contralateral to the surgery site to measure the expression of several transcripts potentially involved in the reduction of cholinergic modulation in SNI animals. No significant decrease was observed for any of the transcripts investigated. B, Representative blots showing the total expression of M1 and the AMPAR subunit GluA2 protein in L5 mPFC of sham and SNI animals. C, Total amount of protein in mPFC L5 of SNI rats normalized to actin and compared with expression level in sham (dotted line); similar to the PCR data, no difference was found in M1 expression in SNI relative to sham.

Figure 9.

Surface expression of M1 muscarinic receptor is reduced in L5 mPFC of SNI animals. Representative blots (A) showing the total expression (Homg) and fraction associated with SPM of the indicated proteins in L5 mPFC. B, Bar chart showing the significant decrease of M1 receptor in the SPM fraction (81.2 ± 5.3% of sham level; n = 7 and 7, 3 animals per sample, p = 0.017). C, Slice biotinylation experiment showing the total (Input) and surface expressed (Surface) fraction of the indicated proteins. The cytosolic protein actin was evaluated as a control. D, Quantification of the data in C showing a significant reduction in the levels of surface-expressed M1 receptor in SNI rats (83.5 ± 2.1% of sham level; 4 samples in each condition, 3 animals per sample, p = 0.029).