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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2679_Библиотеки_им_академика_М_И_Перельмана
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166 Chronic Pain: New Molecular Insights into Pain and Treatment
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Experimentally induced LTP and LTD
In a rodent model of neuropathic pain, synaptic responses in the ACC are
potentiated at the time that allodynia develops, assessed 1–2 weeks after
nerve injury (see Chapter 6). At this time, the late component of LTP can
no longer be induced by theta-burst stimulation, indicating that maximal
potentiation has already occurred. These findings suggest that neuropathic
pain is linked to the mechanisms that underlie the expression of LTP in the
ACC. Pain-related long-term changes in synaptic transmission are not
limited to potentiation. LTD induced by repetitive stimulation of ACC
neurons is also strikingly affected after peripheral tissue injury. For example, in rodents with digit or tail tip amputation, the induction of LTD in
the ACC, measured 45 min and 2 weeks after amputation, is suppressed
[10,14]. Furthermore, in a mouse model of bone cancer pain, LTD in the
ACC is also impaired (see Chapter 6). Based on these observations, we
propose that chronic pain is associated with a saturated late component of
LTP, and with a suppression of LTD, in the ACC.
Presynaptic glutamate release
In addition to postsynaptic changes, the release of glutamate is also
enhanced in the ACC in animal models of chronic pain [15–17] (Figure3).
One to two weeks after peripheral nerve ligation (or 3–5 days after CFA
injection to induce inflammation of the hind paw in adult mice), PPF is
reduced in the ACC, indicating presynaptic enhancement of excitatory
synaptic transmission. Furthermore, an increase in the frequency of
AMPA receptor-mediated mEPSCs occurs in ACC neurons after peripheral nerve injury or inflammation in mice [18].
Postsynaptic AMPA receptor changes
AMPA receptor-dependent EPSCs, recorded from pyramidal neurons of
layers II/III and V, and evoked by focal electrical stimulation within the
ACC, are increased following peripheral nerve ligation in mice [18–21]
(Figure 3). Similar changes are found in ACC neurons in a murine model
Cortical Sensitization and Reorganization 167
(a) (b) (c)
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Figure 3. Increased synaptic transmission in the ACC following peripheral nerve ligation. (a) Diagram illustrates the nerve injury (CPN) and experimental design. (b) Synaptic
input–output curves in slices from control (n = 6 neurons) and nerve-ligated mice (n = 7
neurons). *P < 0.05 and **P < 0.01 compared with those of control group. Open circles:
neurons from control mice; solid circles: neurons from mice with nerve ligation. (c)
Representative traces with an interval of 50 ms recorded in the layer II/III of the ACC.
Paired pulse facilitation (PPF: 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); solid circles: neurons from mice with nerve ligation (n =
19 neurons). *P < 0.05, **P < 0.01, and ***P < 0.001 (adapted from Xu et al. [18]).
of inflammation, induced by CFA. These changes are associated with an
alteration in the rectification of AMPA receptor-mediated synaptic transmission and the development of sensitivity to an inhibitor of CP-AMPA
receptors. Consistent with this, the population of membrane-bound
GluA1-containing AMPA receptors is increased, whereas GluA2/3containing AMPA receptors are not significantly affected. The selective
contribution of GluA1 to the increase in EPSCs is further supported by
genetic studies. Deletion of GluA1, but GluA2, significantly reduces
peripheral injury-triggered c-fos activation in the ACC, as well as in the
spinal cord dorsal horn. Behavioral responses to the peripheral injury are
also reduced [22]. Recent electron microscopy data further supports the
conclusion that postsynaptic GluA1-containing receptors are increased
after peripheral injury (Figure 5).
168 Chronic Pain: New Molecular Insights into Pain and Treatment
(a) (b)
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Figure 4. Altered phosphorylation of GluR1 and rectification index of AMPA receptormediated 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 (adapted from Xu et al. [18]).
Postsynaptic NMDA receptors
In addition to the enhancement of AMPA receptor EPSCs, peripheral
nerve injury leads to increased GluN2B-containing NMDA receptormediated responses [23] ( see Chapter 8). Furthermore, in models of
persistent inflammation, the expression of GluN2B subunits in the ACC is
upregulated, thereby increasing the GluN2B component in NMDARmediated responses. Administration of GluN2B receptor-selective antagonists — either systemically or directly to the ACC — inhibits pain
hypersensitivity associated with peripheral inflammation. Interestingly,
recent studies of the IC and PFC have shown that upregulation of
GluN2B-containing NMDA receptors after nerve injury also occurs in
these structures. This may therefore constitute a generalized cortical
response to peripheral injury that facilitates the induction of NMDA
receptor-dependent LTP. Such metaplasticity could contribute to the sustained enhancement of synaptic transmission that is associated with the
chronic pain state.
Figure 5. Postsynaptic accumulation of GluA1 in the ACC after nerve injury. (a, b) SDS-
(a) (b)
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digested freeze-fracture replica labeling EM samples showing increased GluA1 particles
in the synaptic region on ACC layer V neurons in mice with nerve injury compared with
mice with sham surgery. Bar equals 200 nm. (C) Cumulative histograms showing the distribution of postsynaptic GluA1 density. (d) Averaged density of the postsynaptic GluA1.
**P< 0.01 (adapted from Chen et al. [19]).
Cortical Sensitization and Reorganization 169
Calcium-stimulated AC1
Calcium-stimulated AC1 is critical for cortical excitation triggered by
injury [7,24–26]. For example, in AC1
sitization was found in inflammatory pain and neuropathic pain models.
Moreover, following nerve injury, the AC1
reduction in PPF of AMPA receptor-mediated EPSCs as shown by the
wild-type mice after the nerve injury [18]. While frequency and amplitude
of mEPSCs in ACC neurons were consistently increased in wild-type
−/−
mice, reduced chronic pain sen-
−/−
mice did not demonstrate a
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mice following nerve injury, no increase in mEPSC frequency or amplitude was observed in AC1
−/−
mice [18]. These results indicate that both
presynaptic and postsynaptic enhancement of excitatory synaptic transmission in the ACC is dependent on AC1 in neuropathic pain. Biochemical
studies found that injury-induced increases of the phosphorylation levels
of GluR1 induced by nerve injury were blocked in the ACC of AC1
−/−
mice
compared to wild-type mice, indicating that AC1 is involved in the phosphorylation of GluR1 receptors in the ACC during neuropathic pain
(Figure 4). Similar results are also found in animal models of chronic
visceral pain [27].
Upregulation of AC1 in chronic
visceral pain
In addition to being an activity-dependent signaling protein, essential for
producing the second messenger cAMP, AC1 is also activity-dependent
for regulation in cortical neurons. Liu et al. (2019) reported that AC1 significantly increased in the ACC in an animal model of IBS and persisted
for at least a few weeks. Furthermore, inhibiting AC1 activity by NB001
significantly reduced the upregulation of AC1 proteins in the ACC, suggesting that AC1 activity itself is critical for AC1 protein upregulation
[28]. This finding indicates that AC1 may form a positive regulation in the
cortex during chronic visceral pain. Figure 9 is a proposed model for AC1
positive feedback control in disease condition.
Upregulation of PKMζζ
An atypical PKC isoform (either PKMζ or PKCι/λ) is critical for maintaining enhanced synaptic responses in the ACC in rodent models of neuropathic pain [29] (Figure 6). Peripheral nerve injury causes activation of
PKMζ in the ACC, and locally applied ZIP erases synaptic potentiation
caused by nerve injury (Figure 7). In addition, microinjection of ZIP into
the ACC blocks mechanical allodynia in mice [29]. These observations
suggest that chronic pain activates signaling cascades that are similar to
Cortical Sensitization and Reorganization 171
(a) (b)
(c)
(e) (f)
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(d)
Figure 6. Expression and phosphorylation of PKMζ in the ACC during neuropathic
pain. (a) Mechanical allodynia was tested in the sham and nerve injury groups from wildtype (WT) and AC1
PKMζ and p-PKMζ in the ACC obtained between 3 days and 2 weeks after nerve injury
(the ACC of mice from the sham and nerve injury groups was used for western blot analysis 90 min after the allodynia test). (c) Level of PKMζ in the ACC of mice from the sham
and nerve injury groups. Level of PKMζ increased significantly 3 days after nerve injury
compared with PKMζ in the sham group. (d) Level of p-PKMζ increased significantly
3days after nerve injury. This effect lasted over 2 weeks after nerve injury compared with
p-PKMζ in the sham group. (e) Peripheral nerve injury did not increase the levels of
PKMζ and p-PKMζ in the ACC of AC1
(gray) and p-PKMζ (blue) levels in the ACC of WT mice (adapted from Li et al. [29]).
−/−
animals 3 days after nerve injury. *P < 0.05. (b) Western blots for
−/−
mice. (f) Forskolin incubation increased PKMζ
those activated by experimental stimuli used to induce NMDARdependent LTP at ACC synapses.
Altered intrinsic properties after injury
In addition to changes in synaptic transmission, long-term changes in firing patterns and intrinsic electrical properties have been noted in ACC
neurons in chronic pain states [30,31]. There are at least three major types
of pyramidal cells in the ACC, classified according to their action potential firing pattern: regular spiking, intrinsic bursting, and intermediate
cells. The population distribution and the single action potential properties
of these three groups are not affected 1–2 weeks after nerve injury in
172 Chronic Pain: New Molecular Insights into Pain and Treatment
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Figure 7. Inhibition of PKMζ selectively decreased the amplitude of eEPSCs in ACC
neurons of mice with neuropathic pain by reducing the number of active AMPA receptors.
(a, b) Samples showed the effect of ZIP (5 mM) on the amplitude of eEPSCs in the ACC
neurons of animals from the nerve injury [black circles in (a)] and sham group [black
circles in (b)]. The gray open circles represent the change of membrane resistance during
recording. Black traces in the upper part of (a) and (b) indicate the averaged response at
baseline, and blue traces indicate the average of 2 min responses collected 10 min after ZIP
application. (c) Pooled data of effects of ZIP on the eEPSCs recorded from the ACC of
mice in the sham (open) and nerve injury (solid) groups 10 min after ZIP application.
*P<0.05 (adapted from Li et al. [29]).
mice. However, intermediate cells from animals with neuropathic pain
showed higher initial firing frequencies. Furthermore, it has been shown
that the temporal precision of action potential firing in the ACC is reduced
1–3 days after injection of CFA, or 1–2 weeks after nerve injury, as
reflected in increased jitter [31] (Figure 8). These findings suggest that
chronic pain does not cause dramatic changes in the frequency of the ACC
neuronal firing pattern but that the temporal precision of information coding in the ACC is reduced for long periods of time.
Activation by allodynic stimulation
Recent studies have consistently indicated that the Erk signaling cascade
plays an important role in activity-dependent plasticity and may contribute to the molecular mechanisms underlying learning, memory, and
Cortical Sensitization and Reorganization 173
(A) (B)
(C)
(D)
(G)
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(a)
(b)
(E) (F)
(H) (I)
(a)
(b)
(a)
(b)
(c)
Figure 8. Inflammation of mice decreased the temporal precision of AP firing of neurons
in the ACC of mice in vitro. (A) CFA significantly decreased the paw withdrawal threshold
of mice on 1 and 3 d after injection. (B(a)) The traces showed 15 responses under the
steady-state current stimulations recorded in the ACC from control mice. (B(b)) The raster
plot shows the APs recorded in (A(a)). (C(a)) The traces showed 15 responses under the
steady-state current stimulations recorded in the ACC from CFA-injected mice (CFA-1D).
(C(b)) The raster plot shows the APs recorded in (B(a)). (D) Significant differences
weredetected in the jitter of the first AP and the slope of jitter between saline (open black
circle) and CFA-1D (filled red circle), CFA-3D group (open red circle); *P < 0.05. (E)
The jitter of AP firing from saline-injected mice (black bar) was significantly different from
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Figure 8. (Continued) CFA-injected mice (red bar for CFA-1D and green bar for
CFA-3D; two-way ANOVA; *P < 0.05). (F(a)) Represents 15 traces elicited by simulated
EPSCs (τ
τ
: 1/10 ms). (F(c)) Represents the simulated EPSCs (τ
decay
function I(t) = λ[exp(−t/τ
:0.3/3 ms). (F(b)) Represents 15 traces elicited by simulated EPSCs (τ
rise/τdecay
: 1/10 ms) generated by
rise/τdecay
) − exp(−t/τ
decay
)] (see Materials and Methods). (G) The sum-
rise
rise
marized data show the jitter of AP firing evoked by simulated EPSCs with different rise
and decay times (rise/decay: 0.3/3 or 1/10 ms). The jitter of latency from CFA-injected
mice was higher than the control with the stimulations of simulated EPSCs with rise/
decay: 1/10. *P < 0.05. (H) CPN ligation significantly increased the slope of jitter change
tested 1 week after surgery; t-test, P < 0.05. (I) Summarized data present the jitter change
induced by CPN ligation (adapted from Li et al. [31]).
/
Figure 9. Positive feedback control of intracellular AC1 in case of chronic visceral pain.
Peripheral injury activates significant increases in AC1 protein synthesis through NMDA
receptor-dependent mechanism. NMDA NR2B containing receptor is required for the
induction. In addition to the increase in AC1 protein, NMDA NR2B subtype is also
increased. In this positive feedback loop, the activity of AC1 is required.
persistent pain. Electrophysiological experiments have demonstrated
that the activity of Erk contributes to ACC LTP and that such inhibitors
are relatively selective and do not affect basic synaptic transmission.
Interestingly, rapid increases in P-Erk expression were found in some of
the layer II neurons in the bilateral ACC at 15 min after tissue injury.
Cortical Sensitization and Reorganization 175
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Theactivated Erk level in the ACC was reduced at 45 min and declined
further at 90 min after formalin injection. There was no obvious Erk
activation in the deep-layer neurons in the ACC. Thus, the P-Erk expression pattern in the region is different from that of immediate early genes,
such as c-Fos, which is widely expressed in ACC neurons located in all
layers after formalin injection. It has previously been demonstrated that
neurons in the layer II/III receive ascending noxious inputs from the
thalamus and communicate with other cortical areas. Thus, the Erk activation in ACC neurons may play a role in Erk-dependent neuronal plasticity in the ACC during the induction and/or development of
inflammatory pain. More importantly, 2weeks after the injury, peripheral gentle non-noxious stimuli can cause wide-spread activation of Erk
in ACC neurons, indicating that injury can cause long-term changes in
neuronal excitation [12].
Loss of inhibition
Besides changes in excitatory synapses, it has previously been reported
that there is a striking loss of connections between excitatory and inhibitory neurons in the ACC after nerve injury [32]. No significant changes in
synaptic efficacy in the remaining connected pairs were found. These
changes were reflected at the network level by a decrease in the mEPSC
and mIPSC frequencies. Additionally, nerve injury resulted in a potentiation of the intrinsic excitability of pyramidal neurons, whereas the cellular
properties of interneurons were unchanged. These changes at the cortical
network level might therefore contribute to the neuropathic pain condition, via disinhibition of the anterior cingulate cortex.
Possible roles of microglia
While the role of microglia in chronic pain-related spinal plasticity has
been researched, microglia seem to be inactive in responding to peripheral injury in cortical areas, such as the ACC and IC [33,34]. Direct
whole-cell recordings from microglia in the ACC found that microglia
are not responsive to excitatory and inhibitory transmitters [35] while
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