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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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96 Chronic Pain: New Molecular Insights into Pain and Treatment
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of spinal cord slices were recorded by using whole-cell patch-clamp
recording techniques and tested for the possible existence of silent glutamatergic synapses (Figure 3). Fast monosynaptic, excitatory postsynaptic
currents (EPSCs) were induced when cells were held at −70 mV. In order
to detect silent synapses, the intensity of stimulation was decreased so that
no fast EPSC was detected at −70 mV. The holding potential was then
changed to +40 mV to detect NMDA receptor-mediated EPSCs. In about
(d) (e)
Figure 3. Silent glutamatergic synapses in the lumbar spinal cord. (a) Examples of
responses (the average of three continuous traces) at -70 or +40 mV holding potential.
(b)Time course of the experiment shown in A. (c) Responses at + 40 mV were reversibly
inhibited by 50 µM AP-5. Upward arrow indicates the time of stimulation. Downward
arrow indicates the peak currents measured. (d) Developmentally related distribution of
silent synapses in the spinal cord. The percentage of silent synapses in the superficial
dorsal horn of the lumbar spinal cord at P2-17. The number of cells tested is indicated
above the bars. (e) Distribution of labeled spinal neurons (adapted from Li and Zhuo [2]).
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59% of the dorsal horn neurons tested, synaptic responses were found at
+40 mV but not at −70 mV. Synaptic responses at +40 mV were abolished
by the selective NMDA receptor antagonist AP5 (50 µM). The sensitivity
to AP5 and the voltage dependence of channel activation indicates that
synaptic responses measured at +40 mV were NMDA receptor-mediated.
The intensity of stimulation used in these experiments was low, and it is
likely that only low-threshold afferent fibers were activated. However, it
does not rule out the possibility that silent synapses may exist in other
neurons that receive high-threshold afferent inputs in the spinal cord.
Silent synaptic transmission and silent
glutamatergic synapses
It is important to point out that silent synapses should not be confused
with potential “silent synaptic transmission.” The term “silent synapses”
refers to a condition where the postsynaptic cell is clamped at –70 mV and
NMDA receptors are abundantly located. In an unclamped cell, these
NMDA receptors may contribute to sensory synaptic transmission, for
example, in the case of high-intensity sensory fiber activity induced by
tissue injury. These results consistently suggest that different types of
glutamatergic synapses exist in spinal sensory connections between primary afferent fibers and dorsal horn neurons.
Recruitment of silent synapses
Sensory transmission in the spinal cord receives descending modulation
from supraspinal structures including the RVM. 5-HT-containing neurons
in the RVM send descending projection fibers to targets in the spinal
cord, like the superficial dorsal horn. Activation of these descending
pathways can facilitate, or inhibit, spinal nociceptive transmission.
Previous studies of this descending modulation focused on behavioral,
pharmacological, and electrophysiological recordings of spike firing
from dorsal horn neurons. It is important to show that these modulatory
effects are due to changes in spinal sensory synaptic transmission and not
to the modulation of pre-motor spinal interneurons or spinal local inhibitory synapses.
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Consistent with the biphasic modulatory effect of 5-HT on spinal
nociceptive transmission and behavioral reflexes, 5-HT produced biphasic
modulation of excitatory synaptic responses in spinal slices [2,14,15].
5-HT at high doses inhibited AMPA receptor-mediated EPSCs, while a
low dose of 5-HT — or a selective 5-HT2 receptor agonist — induced
facilitation of fast EPSCs in the lumbar spinal cord (Figure 4). 5-HT at
low doses could facilitate fast EPSCs in the presence of an NMDA receptor antagonist AP-5 (50 µM), indicating that the facilitatory effect is
NMDA receptor-independent. Furthermore, the facilitatory effect induced
by 5-HT at low doses persisted during the washout of 5-HT. While the
activation of 5-HT receptors is important for the induction of facilitation,
continuous activation of these receptors is not necessary for the expression
of facilitation. Application of methysergide after the serotonergic receptor
Figure 4. Biphasic modulation induced by 5-HT. (a–b) Examples of 5-HT experiments
at two doses. Upward arrows indicate the time of stimulation. (c) The effect of 5-HT on
amplitudes of EPSCs in experiments shown in a (squares) and b (triangles). (d) Summary
data of 5-HT at four different doses (n = 8 for each dose). (e) Different effects of 5-HT
(8-OH-DPAT) and 5-HT2 (DOI) receptor agonists. The effects were blocked by their receptor antagonists NAN-190 (5-HT1A) and methysergide, respectively. *P < 0.05 (adapted
from Li and Zhuo [2]).
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1A
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agonist DOI failed to reverse the facilitatory effect. One synaptic mechanism for the 5-HT-produced facilitation is due to the recruitment of silent
glutamatergic synapses. Application of 5-HT (5 µM) caused typically fast
EPSCs to appear at synapses initially lacking AMPA/KA receptor-mediated responses (Figure 5).
5-HT may affect spinal sensory transmission by acting on presynaptic
or postsynaptic receptors. Postsynaptic application of G protein inhibitors,
introduced through the recording pipette, abolished the effect of 5-HT on
synaptic transmission, suggesting that postsynaptic 5-HT receptors
are critical for this effect. In support of this notion, postsynaptic
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Figure 5. Transformation of silent synapses into functional synapses by 5-HT or DOI.
(a) The average of 15 continuous responses (collected at 0.05 Hz) before and 15 min after
the end of 5-HT application. (b) Summary data of 5-HT. (c) Responses before and 15
min after DOI application using dorsal root nerve stimulation. (d) Summary of response
ratios before and after 5-HT or DOI application. Symbols for a given cell are connected;
symbols and errors show response ratio (= the number of stimuli with responses/total
number of stimuli × 100) and the calculated 95% confidence intervals (adapted from
Liand Zhuo [2]).
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Ca2+-dependent processes were shown to be required for 5-HT-induced
facilitation. In experiments with BAPTA (1,2-bis-(o-aminophenoxy)
ethane-N,N,N’,N’-tetracetic acid) in the pipette solution, the facilitatory
effect of 5-HT was abolished, demonstrating the requirement for an
increase in postsynaptic Ca2+. Additional evidence arguing against a
mechanism of 5-HT-induced synaptic facilitation involving modulation of
presynaptic glutamate release comes from the observation that while
5-HT application clearly caused AMPA receptor-mediated EPSCs, NMDA
receptor-mediated EPSCs were significantly decreased by 5-HT in the
same neurons. This result suggests that postsynaptic enhancement of
AMPA receptor-mediated currents by 5-HT is selective.
Intracellular mechanisms for 5-HT-induced
facilitation
In central glutamatergic synapses, PKC plays an important role in longlasting synaptic enhancement. Application of the PKC activator phorbol
12,13-dibutyrate (PDBu) produced long-lasting enhancement of AMPA
receptor-mediated responses in hippocampal neurons, and inhibition of
PKC prevented the induction of long-term potentiation induced by tetanic
stimulation. In dorsal horn neurons of the spinal cord, the excitatory
effect of PKC on spinal nociceptive transmission has been reported.
Activation of PKC enhances sensory synaptic transmission in the dorsal
horn, sensitizing the responses of ascending projection neurons to noxious
peripheral stimuli. Mice lacking PKCγ show reduced pain behaviors following nerve injury. Because PKC acts downstream of 5-HT2 receptors
and these receptors are critical for the facilitatory effect of low doses of
5-HT, we examined whether postsynaptic PKC might mediate the facilitatory effect of 5-HT in spinal dorsal horn neurons. When the PKC peptide
inhibitor PKCI was present in the patch pipette, application of 5-HT failed
to cause any synaptic facilitation. Consistently, bath application of the
PKC activator PDBu caused an enhancement of postsynaptic responses in
spinal dorsal horn neurons. The effect of PDBu persisted during the washout of the drug and was independent of NMDA receptors. In a similar
manner as 5-HT, the facilitatory effect induced by PKC involved the
recruitment of silent glutamatergic synapses.
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AMPA receptor and the expression of facilitation
One possible mechanism for the recruitment of silent synapses is through
the interaction of glutamate AMPA receptors and proteins containing
postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domains
[16,17]. GluR2/3 is widely expressed in sensory neurons in the superficial
dorsal horn of the spinal cord. Unlike GluR1 which is mainly expressed
in spinal interneurons, GluR2/3 is mainly expressed in non-local inhibitory neurons. Glutamate receptor-interacting protein (GRIP), a protein
with 7 PDZ domains that binds specifically to the C-terminus of GluR2/3,
is also expressed in spinal dorsal horn neurons. In many dorsal horn
neurons, GluR2/3 and GRIP coexist. Long-term overexpression of the
C-terminus of GluR2 in hippocampal neurons reduces the number of
synaptic AMPA receptor clusters, suggesting that the interaction between
GluR2/3 and PDZ proteins is involved in the postsynaptic targeting of
AMPA receptors [14]. To examine the functional significance of GluR2/3–
PDZ interactions in sensory synaptic transmission, a synthetic peptide
was made corresponding to the last 10 amino acids of GluR2 (“GluR2SVKI”: NVYGIESVKI) which disrupts the binding of GluR2 to GRIP. As
expected, the GluR2-SVKI peptide blocked the facilitatory effect of 5-HT.
The effect of GluR2-SVKI on synaptic facilitation is rather selective, as
the baseline level of evoked EPSCs and currents evoked by glutamate
application did not change over time in these neurons (Figure 6).
Experiments with different control peptides consistently indicate that the
interaction between the C-terminus of GluR2/3 and GRIP/ABP (or called
GRIP1 and GRIP2) is important for 5-HT-induced facilitation (Figures
4–7). Furthermore, synaptic facilitation induced by PDBu is also blocked
by GluR2-SVKI, suggesting that synaptic facilitation mediated by PKC
activation is similar to that produced by 5-HT in its dependence on
GluR2/3 C-terminal interactions (Figure 7).
Coactivation of cAMP signaling pathways in
facilitation of adult sensory synapses
cAMP signaling pathways are implicated in the function of spinal dorsal
horn neurons. Activation of several receptors for sensory transmitters such
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Figure 6. AMPA receptor–PDZ interactions. (a) 5 µM 5-HT activates silent synapses in
dorsal horn neurons. Representative traces show that this activation was prevented when
50 µM GluR2-SVKI was present in the patch pipet. Traces are shown at time points before
5-HT exposure (Pre), at the end of a 10-min bath application of 5 µM 5-HT, and 30 min
after return to control bath solution (washout). Representative traces (b) and pooled data
(c) show that silent synapses in dorsal horn neurons were activated by 0.5 µM PDBu in the
presence of 50 µM GluR2-SVKE (n = 5) or no peptide (n = 5; data were not different
between these two control conditions and were pooled; open squares). However, PDBu
failed to activate silent synapses in most neurons loaded with 50 µM GluR2-SVKI (n = 6;
see text; closed squares). Traces are shown at time points before PDBu exposure (Pre), at
the end of a 10-min application of 0.5 µM PDBu, and 30 min after return to control bath
solution (washout) (adapted from Li et al. [14]).
(d)
as glutamate and calcitonin gene-related peptide (CGRP) is reported to
raise cAMP levels. In a recent study, application of forskolin did not significantly affect the synaptic responses induced by dorsal root stimulation
in slices of adult mice. However, co-application of 5-HT and forskolin
produced long-lasting facilitation of synaptic responses [18] (Figure 8).
Possible contributors to the increase in cAMP levels are calcium-sensitive
ACs. The facilitatory effect induced by 5-HT and forskolin was
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(e) (f)
Figure 7. Postsynaptic PKC activation is necessary and sufficient for synaptic facilitation by 5-HT. (a) 5 µM 5-HT induces a persistent potentiation of EPSC amplitude in representative traces from a dorsal horn neuron. The three traces illustrate EPSCs before drug
application (control), at the end of a 10-min bath application of 5 µM 5-HT, and 30 min
after return to control bath solution (washout). (b, c) Loading postsynaptic neurons with
the PKC inhibitor PKCI (20 µM) abolished the facilitatory effect of both 5-HT (5 µM; b)
and the PKC activator PDBu (0.5 µM; c) on EPSCs in experiments conducted as in (a).
(d–e) Application through the patch pipet of 50 µM GluR2-SVKI (e) but not 50 µM
GluR2-SVKE (d) prevented facilitation of dorsal horn synaptic transmission by bath application of 0.5 µM PDBu. (f) Pooled data show that 50 µM GluR2-SVKI (n = 6) and 20 µM
PKCI (n = 5), but not 50 µM GluR2-SVKE (n = 7), blocked the potentiation of EPSCs by
0.5 µM PDBu (adapted from Li et al. [14]).
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104 Chronic Pain: New Molecular Insights into Pain and Treatment
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Figure 8. Synergistic recruitment of AMPA/KA receptor-mediated responses at pure
NMDA synapses. (a–c) Examples of EPSPs showing synaptic responses before, during,
and after co-application of 5 µM 5-HT and 10 µM forskolin in the presence of 100 µM
AP5. The effect of 5-HT and forskolin on the EPSP slopes in the experiment shown in A.
Spike responses to stimulation of the dorsal root nerves at the subthreshold intensity were
observed during the washout (indicated by arrows). In a separate experiment, 20 µM
CNQX completely blocked both EPSPs and action potentials induced by 5 µM 5-HT and
10 µM forskolin in the presence of AP5. (d) Summary data of 5 µM 5-HT and 10 µM
forskolin. Data contain two sets of experiments: pure NMDA synapses (n = 3) and mixed
AMPA/KA and NMDA synapses (n = 6). In both cases, significant enhancement was
observed and data were pooled together (adapted from Wang and Zhuo [18]).
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Figure 9. Model for 5-HT-induced facilitation in the spinal cord. The model pathway
explains how activation of 5-HT receptor may recruit silent synapses in spinal dorsal horn
neurons. 5-HT released from descending projection fibers activates postsynaptic PKC
through G protein receptors. PKC activation and subsequent AMPA receptor and GRIP
interactions cause the recruitment of AMPA receptors to the synapse (see Li et al. [14] for
details).
completely blocked in mice lacking AC1 or AC8, demonstrating the
importance of calcium-sensitive ACs. These results show that in adult
sensory synapses, cAMP signaling pathways determine whether the activation of 5-HT receptors causes facilitatory, or inhibitory, effects on synaptic responses. This finding provides a possible explanation for the
regulation of two different signaling pathways under physiological or
pathological conditions. Postsynaptic increases in cAMP levels by sensory transmitters may favor 5-HT-induced facilitation (Figure 9). The
interaction between cAMP and 5-HT may provide an associative heterosynaptic form of central plasticity in the spinal dorsal horn which may
allow sensory inputs from the periphery to act synergistically with central
modulatory influences descending from the brainstem RVM.
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