Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2679_Библиотеки_им_академика_М_И_Перельмана
.pdf
146 Chronic Pain: New Molecular Insights into Pain and Treatment
https://t.me/medicina_free
ACC LTD is input specific
To test whether LTD in the ACC is input-specific, two-pathway experiments were performed. An ACC slice was divided by a cut to separate two
populations of axons (Figure 2). A paired-pulse facilitation (PPF) protocol was used to examine the presynaptic relationship between the two
pathways (Figures 2(b) and (c)). As shown in Figure 2(d), whereas synaptic responses were significantly depressed in the stimulated pathway,
synaptic responses in the second, independent pathway, were not significantly affected. These results suggest that LTD in the ACC is
input-specific.
Voltage-dependent synaptic plasticity
It has been reported that LTD is dependent on the level of depolarization
of the postsynaptic neurons [10,11]. To examine which voltage is optimal
for induction of LTD, we used different holding potentials (−70, −30, −15,
and +30 mV) during presynaptic stimulation. Holding potentials at −70,
−30, and −15 mV could not induce LTD (Figure 3). By contrast, a holding
potential at +30 mV induced long-lasting potentiation of responses
(Figure 3). These results suggest that neurons that were only slightly
depolarized (−45 mV) during presynaptic stimulation were in a suitable
condition for the induction of LTD.
Requirement of NMDA receptor activation and
postsynaptic calcium
In many brain regions — including the hippocampus, visual cortex, somatosensory cortex, and peripheral cortex — the induction of LTD was
dependent on the synaptic activation of NMDA receptors [2,12,13]. First,
NMDA receptor EPSCs were isolated pharmacologically, and these
NMDA receptor EPSCs were completely blocked by bath application of a
selective NMDA receptor antagonist, 50 µM AP5 (Figure 4). To determine if NMDA receptor activation is required for LTD induction within
Cortical Depression 147
(a) (b) (c)
(d)
https://t.me/medicina_free
(e)
Figure 2. Two-pathway experiments show that LTD is input-specific. (a) Diagram of
anACC slice showing the placement of stimulating electrodes in two divided pathways
(S1, S2). EPSCs are recorded in ACC neurons in whole-cell patch-clamp. (b) Paired pulse
to one pathway shows PPF, while cross-facilitation is not observed between pathways.
(c)Statistical summary of paired-pulse ratio between two pathways (n = 6). PPF is calculated as the ratio of the second EPSC amplitude to the first EPSC amplitude. *P < 0.05
compared with across. (d) Summary of results for two pathway experiments (n = 6).
Stimulated pathway (S1) only shows LTD. Sample traces show averaged EPSCs during
baseline responses and 25 min after the pairing procedure (bar). (e) Statistical summary of
EPSCs between two pathways (n = 6). **P < 0.01 compared with baseline responses
(adapted from Toyoda et al. [15]).
the ACC, AP5 (50 µM) was applied and LTD induced by pairing training
was completely blocked (Figure 4).
It has been clearly shown that influx of Ca2+ into the postsynaptic
cell and the resulting rise in intracellular Ca2+ concentration are
148 Chronic Pain: New Molecular Insights into Pain and Treatment
https://t.me/medicina_free
Figure 3. Effects of voltage-dependent thresholds on induction of LTD. (a,b) Different
holding potentials (HP) at −70 and −15 mV fail to induce LTD (−70 mV, 103.9 ± 8.2%
of baseline responses, n = 6; −15 mV, 106.9 ± 6.3% of baseline responses, n = 7). The
insets show averaged EPSCs during baseline responses and 25 min after the pairing procedure (bar). (c) Synaptic potentiation is induced by holding potentials at +30 mV in
pyramidal ACC neurons (140.6 ± 12.2% of baseline responses, n = 10). The insets show
averaged EPSCs during baseline responses and 25 min after the pairing procedure (bar).
(d) Summary results for the effects of different holding potentials (−70, −45, −30, −15,
and +30 mV). Only a slightly depolarized (−45 mV) holding potential can induce LTD.
*P < 0.05 and ***P < 0.001 compared with baseline responses (adapted from Toyoda
etal. [15]).
(a) (b)
(c) (d)
https://t.me/medicina_free
(e) (f)
Cortical Depression 149
Figure 4. LTD induced by postsynaptic NMDA receptor activation. (a,b) LTD is completely blocked by bath applied AP5 (50 µm, n = 8) and BAPTA (11 mm, n = 9) in the
intracellular solution. Insets show averaged EPSCs recorded during baseline responses and
25 min after the pairing procedure (bar). (c) LTD is completely blocked by NVP-AAM077
(0.4 µm, n = 6, 103.8 ± 5.4% of baseline responses). (d) In the presence of NVP-AAM077,
a holding potential at −15 mV during presynaptic stimulation induces LTD (79.3 ± 4.8%,
n = 6, P < 0.05 compared with baseline responses). Insets show averaged EPSCs recorded
during baseline responses and 25 min after the pairing procedure (bar). (e) LTD is completely blocked by Ro25-6981 (0.3 µM, n = 7, 104.9 ± 4.9% of baseline responses) or
ifenprodil (3 µM, n = 9, 97.0 ± 6.7% of baseline responses). (e,f) In the presence of Ro256981, a holding potential at −15 mV during presynaptic stimulation does not induce LTD
(96.7 ± 4.6%, n = 7, P > 0.05 compared with baseline responses), but a holding potential
at −30 mV in the presence of Ro25-6981 induces LTD (84.8 ± 7.3%, n = 6, P < 0.05 compared with baseline responses). Insets show averaged EPSCs recorded during baseline
responses and 25 min after the pairing procedure (bar) (adapted from Toyoda et al. [15]).
150 Chronic Pain: New Molecular Insights into Pain and Treatment
https://t.me/medicina_free
necessary for the induction of both LTP and LTD. Whether the synapse
undergoes LTP or LTD is dependent on the intracellular Ca2+ concentration. For example, large Ca2+ influx leads to LTP and slight Ca2+ influx
leads to LTD. In the induction of homosynaptic LTD, postsynaptic
increases in Ca2+ are shown to be mediated by either NMDA receptors or
voltage-dependent Ca2+ channels. There are many reports which say that
Ca2+ influx through voltage-dependent Ca2+ channels is important for
LTD induction. To determine if postsynaptic Ca2+ signaling pathways are
involved, a pipette solution containing 11 mM BAPTA was used and the
induction of LTD was also completely abolished (Figure 4). These findings indicate that this form of LTD is NMDA receptor-dependent and a
postsynaptic Ca2+ increase is required for the induction of LTD in the
ACC neurons.
NR2A- vs. NR2B-containing NMDA receptors are
required for the induction of LT D
It has been reported that NR2B (GluN2B)-containing receptors, but not
NR2A(GluN2A)-containing receptors, contribute to LTD in the hippocampus [14]. However, little is known about whether GluN2A (NR2A)and/or NR2B-containing NMDA receptors contribute to the induction of
LTD in the ACC. First, the contribution of the NR2A subunit to NMDA
receptor-mediated EPSCs in ACC neurons was examined. A selective
pharmacological antagonist for NR2A was used to examine synaptically
induced NMDA receptor-mediated EPSCs. The averaged decay time constant and rising time (10–90%) of NR2A-mediated currents were 95 ± 8
and 13 ± 1 ms, respectively (Figure 4). The effect of NVP-AAM077 on
PPF was also examined, which is thought to be a presynaptic process. In
the presence of NVP-AAM077, PPF was unchanged, suggesting that this
drug did not affect neurotransmitter release [15].
Blocking the NR2A subunit with NVP-AAM077 (0.4 µM) abolished
LTD (Figure 4). To test whether LTD could be induced in the presence of
NR2A blockade, a different form of stimulus (holding potential at −15
mV during presynaptic stimulation) was used. Interestingly, this form of
Cortical Depression 151
https://t.me/medicina_free
stimulation in the presence of NVP-AAM077 was able to induce LTD,
indicating that NR2A is not absolutely required for ACC LTD.
The contribution of the NR2B subunit to NMDA receptor-mediated
EPSCs was also investigated in ACC neurons. A selective antagonist for
the NR2B subunit was used to examine synaptically induced NMDA
receptor-mediated EPSCs. Bath application of a selective NR2B subunit
antagonist, Ro25-6981 (0.3 µM), depressed the total NMDA receptormediated currents by 19% of control, indicating that the NMDA receptor
EPSCs in ACC neurons are mostly mediated by NR2A-containing receptors. As with NVP-AAM077, application of Ro25-6981 had no effect on
AMPA receptor-mediated EPSCs. The average decay time constant and
rising time (10–90%) of NR2B-mediated currents were 129 ± 10 and 19 ±
2 ms, respectively. The rising time (10–90%) and decay time constant of
NR2B-mediated EPSCs were significantly longer than those of NR2Amediated currents in ACC pyramidal neurons. Furthermore, Ro25-6981
did not affect PPF. By contrast, a blockade of the NR2B subunit with
Ro25-6981 or ifenprodil abolished the induction of LTD. These results
show that the NR2B subunit also contributes to the induction of LTD in
the ACC. To test whether LTD could be induced with a different form of
stimulus (holding potential at −30 and −15 mV during presynaptic stimulation) in the presence of Ro25-6981, LTD was repeated in the presence of
NR2B blockade. Unlike NVP-AAM077, this induction protocol at a holding potential of −15 mV failed to induce LTD in the presence of Ro256981 (Figure 4). However, a holding potential at −30 mV in the presence
of Ro25-6981 induced LTD (Figure 4). Taken together, these results indicate that both NR2A and NR2B subunits are required for the formation of
LTD in the ACC and the abolishment of LTD by these compounds could
be due to the reduction in calcium influx via NMDA R receptors.
NMDA receptor-independent form of LTD
In addition to NMDA receptor-dependent LTD, there is NMDA receptorindependent LTD in the ACC as well. Using field recording of EPSP in
adult ACC slices, repetitive stimulation at a low frequency induced LTD.
152 Chronic Pain: New Molecular Insights into Pain and Treatment
(a) (b) (c)
(d)
https://t.me/medicina_free
Both voltage-gated calcium channels and mGluRs contribute to ACC LTD
[16]. Nimodipine (10 µM) completely blocked the induction of LTD
(Figure 5), although basal synaptic responses were not significantly
affected. By contrast, 50 µM AP-5 did not affect field LTD (Figure 5).
MCPG (500 µM), a metabotropic glutamatergic receptor antagonist, also
blocked LTD. These results suggest that both L-type calcium channels and
(e)
(g)
Figure 5. Pharmacological aspects of ACC LTD. (a) Controls show LTD after 1 Hz, 15
min low-frequency stimulation (78 ± 2%; n = 9/9). (b) NMDA receptor antagonist, AP5
(50 μM) partially blocked LTD (88 ± 2%; n = 7/ 7). (c) L-VGCC blocker, nimodipine
(10μM) blocked LTD (95 ± 3%; n = 6/6). (d) Groups I and II mGluR antagonist, MCPG
(500 μM) also blocked LTD (93 ± 3%; n = 6/ 6). (e) mGluR5 antagonist, MPEP (10 μM)
had no effect on ACC LTD (77 ± 2%; n = 4/4). (f) mGluR1 antagonist, LY367385
(100μM) blocked LTD (96 ± 2%; n = 5/5). (g) Summarized results of the averaged fEPSP
slope of the last 10 min of each experiment (F
Bonferroni post hoc; p < 0.001 for control versus nimodipine, MCPG and LY367385)
(adapted from Kang et al. [16]).
(f)
= 10.64, p < 0.01; one-way ANOVA with
(5,31)
Cortical Depression 153
https://t.me/medicina_free
mGluRs are critical for the induction of LTD in the ACC. LTD was not
affected in the presence of picrotoxin (100 µM), a GABAA receptor
antagonist, indicating that inhibitory influences are not required for LTD.
Postsynaptic expression of LTD
Two major possible mechanisms may contribute to LTD: the reduction of
the release of glutamate from presynaptic terminals and decreases in postsynaptic AMPA receptor-mediated responses [11,17–20]. To test if the
interaction between the GluR2/3 C-terminal and its specific PDZ binding
partners is important for the induction of LTD, several synthetic peptides
that disrupt the interaction between AMPA subunits and PDZ-containing
proteins were used. Peptides were applied through the patch recording
electrode into postsynaptic neurons. First, a control peptide (Pep2-SVKE),
in which the PDZ interaction motif is inhibited by substituting the last
amino acid (isoleucine) with glutamate was used. Postsynaptic application
of Pep2-SVKE (100 µM) did not affect basal synaptic transmission in
ACC slices (Figure 6). In the presence of Pep2-SVKE, LTD was not
affected (Figure 6). Next, a Pep2-SVKI peptide, which interferes with
interactions between GluR2 and GRIP (glutamate receptor interaction
protein), ABP (AMPA receptor binding protein), and PICK1, was used. In
the presence of Pep2-SVKI (100 µM), basal synaptic responses were also
unchanged. However, LTD was completely blocked by the presence of
Pep2-SVKI. These results provide direct evidence that postsynaptic interaction between AMPA GluR2 receptor and PDZ protein is critical for the
induction of cingulate LTD.
Another Pep2-AVKI peptide, which disrupts binding of GluR2 to
PICK1 (protein interaction with C kinase), was also used. Postsynaptic
application of Pep2-AVKI (100 µM) had no effect on basal synaptic transmission, but LTD was blocked. These results strongly suggest that both
GluR2/3-PICK1 and GluR2/3-GRIP1/ABP interactions are likely contributing to cingulate LTD. Cingulate LTD requires activation of postsynaptic
NMDA receptors. To exclude the possible inhibition of NMDA receptors
by these peptides, we measured the effects of postsynaptic injection of the
peptides on NMDA receptor-mediated EPSCs. We found that these peptides had no effect on NMDA receptor-mediated EPSCs, indicating that
154 Chronic Pain: New Molecular Insights into Pain and Treatment
(a) (b)
(c)
https://t.me/medicina_free
Figure 6. Pep2-SVKI blocks the induction of LTD. (a, c) Pep2-SVKE (100 µM) and
Pep2-SVKI (100 µM) do not affect baseline response. The insets show averages of six
EPSCs at the time points of 5 (1) and 35 min (2) during the recording. The dashed line
indicates the mean basal synaptic response. (b) Pep2-SVKE (100 µM, n = 8) has no effect
on the induction of LTD. (d) In the presence of Pep2-SVKI (100 µM) in the intracellular
solution, LTD is blocked (n = 10). (b and d) Traces show averages of six EPSCs at baseline
responses (1) and 30 min (2) after the paired training (bar). The dashed line indicates the
mean basal synaptic response (adapted from Toyoda et al [27]).
(d)
the blocking effects are not simply due to the inhibition of NMDA receptor functions in the cingulate neurons.
To examine if GluR2 subunit contributes to the expression of LTD,
Pep2-SVKI was injected into neurons 5 min after the induction of LTD.
No significant effect was found on the expression of LTD during the 25
min treatment with Pep2-SVKI. This result suggests that GluR2 receptormediated possible trafficking events are completed within 5–10 min after
LTD induction.
Cortical Depression 155
(a) (b) (c)
https://t.me/medicina_free
Figure 7. Synaptic depression in GluR2 KO mice. (a) LTD is induced in ACC neurons
in wild-type mice (n = 7 slices/6 mice). (b) LTD is absent in ACC neurons in GluR2 KO
mice (GluR2
mice (GluR3
−/−
) (n = 9 slices/6 mice). (c) LTD is absent in ACC neurons in GluR3 KO
−/−
) (n = 9 slices/6 mice) (adapted from Toyoda et al. [27]).
Genetic studies of LTD
Although the use of peptide inhibitors offers possible insights for the
involvement of AMPA receptor subtypes in LTD, it is difficult to rule out
possible non-selective interactions. To study the subunit-specific function
of GluR2 and GluR3 in cingulate LTD, LTD was recorded from ACC
neurons of GluR2 or GluR3 KO mice (GluR2
LTD in GluR2
−/−
mice was completely abolished (Figure 7), while normal
LTD was induced in wild-type controls (Figure 7). These results suggest
that the GluR2 contributes to the induction of cingulate LTD. Additional
experiments found that GluR2 deletion did not affect basal excitatory
synaptic transmission, PPF, and NMDA receptor-mediated responses.
The possible role of GluR3 in LTD was also examined. LTD in ACC
slices from GluR3
−/−
and wild-type mice was comparable in both groups,
suggesting that synaptic depression was not affected by the deletion of
GluR3 subunit.
−/−
or GluR3
−/−
). As expected,
LTD in other pain-related cortical areas
The IC is another cortical area that is critical for pain perception and emotion. LTD has been reported in the IC using multiple electrode field
recording system or whole-cell patch-clamp recording [21–23]. In field
Соседние файлы в папке Библиотека им академика М.И. Перельмана