Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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Baba, Hiroshi,
Timothy P. Doubell,
Kimberly A. Moore, and
Clifford J. Woolf.
Silent NMDA Receptor-Mediated Synapses Are Developmentally
Regulated in the Dorsal Horn of the Rat Spinal Cord.
J. Neurophysiol. 83: 955-962, 2000.
In vitro whole cell
patch-clamp recording techniques were utilized to study silent
pure-N-methyl-D-aspartate (NMDA)
receptor-mediated synaptic responses in lamina II (substantia
gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify
whether these synapses are present in the adult and contribute to
neuropathic pain, transverse lumbar spinal cord slices were prepared
from neonatal, naive adult and adult sciatic nerve transected rats. In
neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal
intraspinal stimulation (n = 15 of 20 neurons) or
focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent
pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal
stimulation (n = 27), and only one pure-NMDA EPSC
was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were
elicited by focal intraspinal stimulation in only 2 of 45 SG neurons.
Although a large increase in A fiber evoked mixed
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA
receptor-mediated synapses was detected after sciatic nerve injury,
A
fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by
dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are
therefore a transient, developmentally regulated phenomenon, and,
although they may have a role in synaptic refinement in the immature
dorsal horn, they are unlikely to be involved in receptive field
plasticity in the adult.
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INTRODUCTION |
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Neurons in the dorsal horn of the spinal cord show
a marked receptive field plasticity that contributes to pain
hypersensitivity (Woolf and Doubell 1994). Nociceptor
sensory inputs to these neurons can induce a rapid onset, use-dependent
increase in dorsal horn neuron membrane excitability, and central
sensitization. This functional plasticity manifests as an increase in
responsiveness and spatial extent of the cutaneous receptive fields of
dorsal horn neurons (McMahon et al. 1993
; Woolf
1983
), and it appears, from intracellular recordings in vivo,
to be due to the recruitment of previously subthreshold synaptic inputs
(Woolf and King 1990
). Central sensitization, which
lasts for tens of minutes after the initiating C-fiber conditioning
input, results in both hyperalgesia (exaggerated responses to noxious
stimuli) and allodynia (the production of pain by normally innocuous
stimuli) (Koltzenburg et al. 1994
; Torebjork
et al. 1992
). Allodynia represents the novel generation of pain
in response to low-threshold mechanoreceptive A
fibers, which
parallels the recruitment of A
fiber inputs to nociceptive-specific
cells in the superficial and deep dorsal horn during central
sensitization (Simone et al. 1989
; Woolf et al.
1994
). Peripheral nerve injury also results in allodynia, again
due to an abnormal response to A
fiber inputs (Campbell et
al. 1988
), which is caused by a combination both of central sensitization and a structural reorganization of sensory synaptic inputs in the superficial dorsal horn (Woolf and Doubell
1994
). Following peripheral nerve injury, A
fibers sprout
from their normal termination site in the deep dorsal horn into lamina
II where they make synaptic contact with cells that normally receive C-fiber terminals (Koerber et al. 1994
; Woolf et
al. 1992
, 1995
).
Wall first suggested that following deafferentation produced by
peripheral nerve or dorsal root injury, preexisting ineffective, or
silent synapses, in the spinal cord might be unmasked and begin to
activate dorsal horn neurons (Wall 1977). Glutamatergic
pure-N-methyl-D-aspartate (NMDA)
receptor-mediated synapses are one potential type of silent synapse.
At these synapses, transmission would not manifest at the resting
membrane potential of postsynaptic neurons due to the voltage-dependent
Mg2+ block of the NMDA receptor ion channel;
however, following postsynaptic depolarization, these inputs would be
revealed. No direct evidence for the presence of such pure-NMDA
synapses was available until Li and Zhuo (1998)
and
Bardoni et al. (1998)
recently demonstrated the presence
of this type of "silent" synapse in the isolated neonatal spinal
cord. In these neonatal preparations, pure-NMDA receptor-mediated
excitatory postsynaptic currents (EPSCs) could be evoked in superficial
dorsal horn neurons by low-intensity intraspinal and dorsal root entry
zone focal stimulation (Bardoni et al. 1998
; Li
and Zhuo 1998
). Because the silent pure-NMDA receptor-mediated EPSCs were evoked by very low-intensity stimulation, Li and Zhuo suggested that primary afferent A
terminals may be the presynaptic component of these synapses. These silent synapses could be converted to functional ones by depolarization or activation of
5-HT2 receptors (Li and Zhuo
1998
). Although the relative occurrence of silent synaptic
responses decreased over the first 10 postnatal days, Li and
Zhuo (1998)
concluded that silent synapses have a role in
nociception and neural plasticity in the adult spinal cord and
represent a potential therapeutic target for the treatment of
persistent pain.
During development, low-threshold A fibers project to lamina II, but
over the first postnatal month, they progressively retreat to deeper
layers (Fitzgerald et al. 1994
) such that in normal adult rats, no A
fibers are present in lamina I and II
(Robertson and Grant 1985
; Woolf 1987
).
Li and Zhuo (1998)
may have been able to elicit
low-threshold, primary afferent-mediated silent synaptic responses in
superficial dorsal horn neurons only because they recorded from
neonatal rats. Interestingly, after sciatic nerve transection in adult
rats, A fibers sprout from deeper lamina back into lamina II
(Woolf et al. 1992
). Immature synaptic terminals of A
fibers, detected by electron microscopy, are increased 15-fold in
lamina II after sciatic nerve transection (Woolf et al.
1995
). An electrophysiological study has demonstrated that
after sciatic nerve transection, A
fiber stimulation of injured
afferents can elicit EPSCs in most substantia gelatinosa (SG) neurons,
in contrast to A
fiber stimulation in naive rats, which evokes EPSCs
in only a very few SG neurons (Okamoto et al. 1996
).
However, the vast majority of A
fiber-mediated EPSCs revealed by
sciatic nerve transection were found to be polysynaptic in nature
(Okamoto et al. 1996
), suggesting that the newly formed,
morphologically identified, synaptic terminals of sprouting A fibers
are not functional, at least not at the resting membrane potential
(approximately
70 mV). These novel synapses may be then pure-NMDA
receptor-mediated silent synapses, functional only at strongly
depolarized potentials. Accordingly, Okamoto et al.
(1996)
may not have been able to detect monosynaptic EPSCs at a
holding potential of
70 mV.
To investigate whether silent synapses play a role in synaptic plasticity in adult rats, we have examined whether pure-NMDA receptor-mediated synaptic currents can be measured in SG and lamina III neurons of intact and sciatic nerve transected rats.
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METHODS |
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Spinal cord slice preparation
Lumbosacral spinal cords were removed under urethan anesthesia
(1.5-2.0 g/kg ip) from neonatal rats [postnatal day 2-7
(P2-P7)], naive adult rats (10-12 wk old), and adult rats
subjected to a sciatic nerve transection on the left side (10-12 wk
old, 4 wk postinjury). The isolated spinal cord was placed in
preoxygenated ice-cold Krebs solution (2-4°C). After removal of the
dura mater, all ventral and dorsal roots were cut and the pia-arachnoid
membrane removed. In those experiments involving primary afferent
stimulation, a dorsal root (left side, L4) was
preserved to enable primary afferent fiber stimulation as described
previously (Baba et al. 1999; Yoshimura and
Jessell 1989
). The spinal cord was placed in a shallow groove
formed in an agar block, glued to the bottom of the microslicer stage
with cyanoacrylate adhesive, and immersed in ice-cold Krebs solution. A
200- to 300-µm (thin slice with or without dorsal root, Fig.
1) or 600-µm (thick slice with dorsal root) transverse spinal cord slice was then cut on a vibrating microslicer (DTK1500, Dosaka, Kyoto, Japan). For the minimal
stimulation protocol with focal glass electrodes, thin slices were used
to reduce polysynaptic inputs, which prevent analysis of pure-NMDA responses. Thick slices, in which a larger neuronal network is preserved, were used for protocols involving dorsal root stimulation with a suction electrode. The spinal cord slice was then placed on a
nylon mesh in the recording chamber and perfused with Krebs solution
(10 ml/min) saturated with 95% O2-5%
CO2 at 36-37°C. The Krebs solution contained
(in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose. When thin slices were
used, the experiments were performed in the presence of bicuculline (20 µM) and strychnine (5 µM) to block GABAA and
glycine receptors, respectively.
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Patch-clamp recording from lamina II and III neurons
Blind whole cell patch-clamp recordings were made from neurons
located in the SG and lamina III of the spinal dorsal horn (Yoshimura and Nishi 1993). In adult spinal cord slices,
the SG is readily identifiable as a distinct translucent region, and lamina III is a relatively wide band (150-200 µm) ventral to the SG.
When recording from SG neurons, the recording electrode was positioned
in the middle third of the SG. Recently in the adult, we have found
that when the electrode is targeted in this way, a heterogeneous group
of intrinsic stalk and islet cells located in both inner and outer
lamina II can be labeled with neurobiotin (Baba et al.
1999
). When recording from lamina III neurons, the electrodes
were positioned 100-150 µm ventral to the border of the SG. In the
neonatal spinal cord, the SG is clearly identifiable from the shape of
the superficial dorsal horn; however, the borders with the adjacent
dorsal and ventral laminae (lamina I and III) are not as clear as that
in the adult spinal cord. Therefore when recording from neonatal SG
neurons, the recording electrodes were positioned 50-100 µm from the
most dorsal surface of the gray matter, which is known from
histological studies to represent the most ventral region of lamina II
(Fitzgerald 1987
). After establishing the whole cell
configuration, neurons were voltage clamped to a membrane potential of
70 mV. To record NMDA currents, neurons were voltage clamped to +70
mV. The pipette solution contained (in mM) 110 Cs-sulfate, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 TEA, and 5 ATP-Mg salt. The resistance of a typical
borosilicate glass patch pipette (1.5 mm OD, World Precision
Instruments, Sarasota, FL) was 5-10 M
. Membrane currents were
amplified with an Axopatch 200A amplifier (Axon Instruments, Foster
City, CA) in voltage-clamp mode. Signals were filtered at 2 kHz and
digitized at 5 kHz. Data were stored on a personal computer and
analyzed with pCLAMP 6 software (Axon Instruments). Numerical data are
expressed as means ± SD.
Identification of pure-NMDA synapses by minimal stimulation
In thin slices, intraspinal and dorsal root focal stimulations
were performed using the same type of borosilicate glass electrode as
that used for recording. Usually three to six different locations around the recorded cell (100-200 µm from the tip of the recording electrode) or within the dorsal root (3-5 mm from the entry zone) were
stimulated. A minimal stimulation protocol was used to identify pure-NMDA currents (Isaac et al. 1997). Briefly, each
cell was initially held at
70 mV, and intraspinal or dorsal root
focal stimulation was applied at a relatively high intensity (50-200 µA, 0.05 ms). At
70 mV, evoked EPSCs were mainly mediated by
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors. The stimulus intensity was then progressively decreased
until AMPA receptor-mediated EPSCs disappeared (10-50 µA, 0.05 ms). The holding potential was then switched to +70 mV to reveal pure-NMDA EPSCs (slow outward synaptic currents). Pure-NMDA EPSCs were very vulnerable to high-frequency stimulation, so focal stimulation was
applied at a low frequency (0.1-0.2 Hz).
Dorsal root stimulation by suction electrode
In thick slices with an attached dorsal root (16-20 mm), the
dorsal root was stimulated using a suction electrode. The threshold of
stimulation intensity and duration of A (~10 µA, 0.05 ms), A
(~25 µA,0.05 ms), and C fibers (~200 µA, 0.5 ms) for this
suction electrode have been established previously (Baba et al.
1999
). Classification of fibers responsible for synaptic
responses into A
, A
, and C fibers was based solely on EPSC
threshold. Identification of EPSCs as monosynaptic was based on a
constant latency with graded intensity and high-frequency repetitive
stimulation (20 Hz) (Yoshimura and Jessell 1989
).
Polysynaptic EPSCs, in contrast, had variable latencies with such
stimulation protocols.
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RESULTS |
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Pure-NMDA receptor-mediated EPSCs in the neonatal spinal cord
Initially, we set out to confirm the presence of pure-NMDA
receptor-mediated synapses in neonatal SG neurons using intraspinal focal stimulation. This was achieved by decreasing the stimulus intensity until no fast EPSC was apparent at 70 mV (Fig.
1A). When the holding potential was then changed to +70 mV,
a slow APV (100 µM)-sensitive outward synaptic current could be
recorded in 15 of 20 neurons tested (Fig. 1A). The
proportion of pure-NMDA receptor-mediated EPSCs in SG neurons of the
neonatal spinal cord is similar to that reported previously
(Bardoni et al. 1998
; Li and Zhuo 1998
).
The decay time course of most of the pure-NMDA receptor-mediated EPSCs
could be well described by double exponential functions
(n = 11,
1 = 26 ± 20 ms,
2 = 369 ± 240 ms, mean ± SD), but some pure-NMDA
receptor-mediated EPSCs were best fitted by a single exponential
(n = 2,
= 167 ms and 351 ms, Fig.
1A). The remaining EPSCs were not well fitted by exponential
functions (n = 2).
Previous studies (Bardoni et al. 1998; Li and
Zhuo 1998
) did not directly establish the presence of primary
afferent evoked pure-NMDA receptor-mediated EPSCs. Therefore we tested
whether or not focal dorsal root stimulation evoked pure-NMDA
receptor-mediated EPSCs using a neonatal slice with a long (~5 mm)
attached dorsal root. In two of seven neurons examined, we observed
pure-NMDA receptor-mediated EPSCs in response to low intensity (10-50
µA) focal stimulation of the dorsal root (Fig. 1B).
Therefore pure-NMDA receptor-mediated synapses in the neonatal spinal
cord are, at least in part, primary afferent mediated. Both of these
EPSCs were fitted by double exponentials (
1 = 24 ms,
2 = 221 ms, and
1 = 11 ms,
2 = 85 ms, Fig.
1B).
Absence of pure-NMDA receptor-mediated EPSCs in the adult spinal cord
We next tested, using the minimal stimulation protocol in
thin slices, whether pure-NMDA receptor-mediated EPSCs could be detected in any adult spinal cord SG neurons. Unlike the neonatal spinal cord, however, we could find no pure-NMDA receptor-mediated EPSCs in SG neurons of naive adult rats (n = 27). In
all cases, when the stimulus intensity was reduced such that fast EPSCs
at 70 mV were eliminated, no EPSC could then be elicited at +70 mV.
EPSC thresholds at
70 and +70 mV were always identical (Fig. 2A, left). At threshold
stimulation, glutamatergic EPSCs recorded at +70 mV from neurons in
adult rat spinal cord preparations always consisted of an early AMPA
and a later voltage-dependent NMDA component, as revealed by the
appearance of a slow current at +70 mV that was not present at
70 mV.
A slow NMDA current was never present in the absence of an early, fast
AMPA current. An AMPA receptor antagonist,
2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX) (10 µM), blocked the transient nonvoltage dependent
synaptic component, whereas an NMDA receptor antagonist,
2-amino-5-phosphonovaleric acid (APV; 100 µM), blocked the
voltage-dependent slow current component (Fig. 2A,
right).
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Because the presynaptic component of silent pure-NMDA
receptor-mediated synapses may be limited to A afferent terminals
that do not normally terminate in the SG in naive adult rats, we
examined lamina III where there are many synaptic terminals of A
fibers. However, we observed only one pure-NMDA receptor-mediated EPSC in lamina III (n = 22 neurons tested, Fig.
2C). As for the SG neurons, at a holding potential of
+70 mV, the vast majority of the lamina III neurons exhibited EPSCs
with both AMPA and NMDA components at threshold stimulation (Fig.
2B).
At 70 mV, most SG neurons from thick, naive adult rat slices with an
attached dorsal root exhibit either monosynaptic or polysynaptic A
fiber-mediated EPSCs on stimulation of the dorsal root. A small
proportion of the neurons (14 of 57 cells; 25%) have A
fiber-mediated EPSCs. However, the latencies of all these EPSCs were
variable at graded intensity or with 20-Hz repetitive stimulation,
indicating that the A
fiber inputs are likely to be polysynaptic. In
contrast to naive adult rats, SG neurons in slices from sciatic nerve
transected rats exhibited A
fiber-mediated polysynaptic EPSCs in
the majority of cases (15 of 22 cells; 68%), similar to results
reported by Okamoto et al. (1996)
. However, in sciatic
nerve-transected animals, we detected no evidence of A
fiber-mediated EPSCs at
70 mV that had characteristics typically associated with a monosynaptic input (e.g., fixed latency and high-frequency following).
Figure 3B shows the
distribution of the minimum stimulus intensity threshold for eliciting
EPSCs in slices from naive and sciatic nerve-transected rats. In
slices from sciatic nerve-transected rats, the mean threshold
intensity for evoking EPSCs was 20.6 ± 7.3 µA
(n = 22), which was significantly lower than that
in the naive preparation (33.2 ± 15.1 µA, n = 57, P = 0.004, unpaired t-test)
(see also initial observations by Okamoto et al. 1996). Figure 3C shows the distribution of the latencies of
EPSCs evoked at a stimulus intensity of 100 µA, 0.05 ms (supramaximal
for A
fiber and above the A
threshold). Mean EPSC latency at this
intensity in naive (3.1 ± 1.1 ms; n = 53) and
sciatic nerve-transected rats (3.0 ± 1.1 ms;
n = 18) were not significantly different
(P = 0.64, unpaired t-test).
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We then tested whether pure-NMDA receptor-mediated synapses
reappear in the SG after nerve lesions. In thin slices from nerve injured rats, only 2 of 45 cells exhibited pure-NMDA receptor-mediated EPSCs in response to minimal intraspinal focal stimulation (Fig. 3,
A and E). To further extend these
observations, we also tested whether pure-NMDA synapses mediated by the
central terminals of sprouting A fibers could be observed in SG
neurons in thick slices with an attached dorsal root. Although no A
fiber-mediated monosynaptic EPSCs were detected at a holding potential
of
70 mV, it is possible that monosynaptic pure-NMDA EPSCs might be
revealed by changing the holding potential to +70 mV. However, no fast
monosynaptic EPSCs with short latencies (<1.5 ms) were observed at +70
mV (Fig. 3D, top). The shortest EPSC latencies with
supramaximal A
fiber stimulation (100 µA) were always identical at
70 and +70 mV (n = 18, Fig. 3D).
These observations indicate that after peripheral nerve injury,
sprouted A
fiber terminals do not generate pure-NMDA receptor-mediated silent synapses.
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DISCUSSION |
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We have found no evidence for pure-NMDA
receptor-mediated synaptic responses in SG neurons of the adult rat
spinal cord, although we did confirm their presence in the SG of
neonatal spinal cord. Pure-NMDA receptor-mediated responses were also
extremely rare in lamina III of the adult spinal cord, where many A
fibers terminate. Furthermore, chronic peripheral nerve transection did
not result in the reappearance of any NMDA receptor-mediated silent synapses.
Existence of pure-NMDA receptor-mediated synapses
Physiological studies in various parts of the CNS indicate that
during early development there are many glutamatergic synapses at which
transmission is mediated by activation of NMDA receptors alone
(Bardoni et al. 1998; Durand et al. 1996
;
Isaac et al. 1997
; Li and Zhuo 1998
;
Wu et al. 1996
). Either an absence of functional AMPA
receptors, or a low concentration of glutamate (i.e., spillover from
neighboring synapses) sufficient to activate only the higher affinity
NMDA receptors, could account for these observations. Recent
immunohistochemical studies have provided direct evidence that NMDA
receptors are distributed at most glutamatergic synapses in the
neonatal hippocampus. In contrast, AMPA receptors are located at only a
few synapses in the neonate, but at most synapses in the adult
(Petralia et al. 1999
). In the spinal cord, however, morphological data on the developmental distribution of AMPA and NMDA
receptors have yet to be established.
Most in vitro experiments demonstrating the presence of pure-NMDA
receptor-mediated EPSCs have been conducted at subphysiological temperatures. Extrasynaptic spillover of glutamate is regulated by
glutamate transporters in a temperature-dependent manner, such that at
physiological temperatures pure-NMDA receptor-mediated EPSCs are
rarely observed unless glutamate transporters are blocked (Asztely et al. 1997; Kullmann 1999
).
This suggests that it might be easier to record pure-NMDA
receptor-mediated EPSCs at subphysiological temperatures where
extrasynaptic spillover of glutamate is more likely to occur. We have,
however, recorded pure-NMDA receptor-mediated EPSCs at physiological
temperature, suggesting that an absence of AMPA receptors accounts for
pure-NMDA receptor-mediated EPSCs in the dorsal horn of the neonatal
spinal cord.
Selective stimulation of high-threshold, unmyelinated C fibers by the
minimal stimulation protocol is impossible and precludes our ability to
observe pure-NMDA receptor-mediated EPSCs mediated by these afferents
in the adult spinal cord (Fig.
4C). Additionally, differences
in the extent of dendritic arborization between adult and neonatal
spinal dorsal horn neurons may complicate detection of silent synapses.
Adult SG neurons have much larger dendritic arbors than neonatal SG
neurons (Falls and Gobel 1979). Usually, we stimulated
at up to six different sites around the recorded neurons. If pure-NMDA
receptor-mediated synapses are located only at the most distal points
of the dendritic arbor, they may not have been stimulated in adult
slices. However, the most parsimonious interpretation of our results is
that although silent pure-NMDA receptor-mediated synapses do exist in
neonatal spinal cord, they do not exist, or are extremely rare, in the
adult spinal cord (Fig. 4, A and B).
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Synaptogenesis has been observed in the SG following sciatic
nerve transection, and the presynaptic component of these newly formed
synapses has been identified by electron microscopy as the central
terminals of sprouted A fibers (Woolf et al. 1995
). Because newly formed synapses have a relatively high NMDA/AMPA receptor
current ratio (reviewed by Feldman and Knudsen 1998
), we
assumed that some of the newly formed synapses might consist of
postsynaptic NMDA receptors in the absence of AMPA receptors. However,
we obtained no evidence of an increase in the number of pure-NMDA
receptor-mediated synapses in lamina II despite recording from a large
number of neurons from sciatic nerve-transected rats. Furthermore, in
thick slices with an attached dorsal root recorded at +70 mV, we did
not observe any A
fiber-mediated short-latency NMDA
receptor-mediated EPSCs following dorsal root stimulation.
Although sciatic nerve transection substantially increased the
proportion of lamina II cells in the lumbar spinal cord with A fiber
input at
70 mV, these inputs did not fulfill the established criteria
for classification as monosynaptic; constant latency at graded
intensity or high-frequency repetitive stimulation. There are at least
two explanations consistent with this observation. First, the sprouted
A
fibers may not make direct functional synaptic contacts with SG
neurons, but instead may contact other neuronal components such as the
dendrites of lamina I or deep dorsal horn neurons with dorsally
directed dendrites. Alternatively, the sprouted fibers may synapse
directly with lamina II cells, but these fibers may not behave like
classical monosynaptic inputs.
If sprouted A fibers make functional monosynaptic contacts with SG
neurons, one would expect a reduction in EPSC latency after nerve
transection. Although the mean EPSC threshold decreased after sciatic
nerve transection, the mean EPSC latency was not changed significantly.
It is possible that sprouting A
fibers are so thin that the
conduction velocity of these new fibers is insufficient to alter EPSC
latency. The failure of the novel A
fiber input to follow
high-frequency stimulation or display a fixed latency at graded
stimulation might reflect the fact that, like immature axons and
synapses, sprouted fibers may be less reliable in conveying impulses to
postsynaptic cells. This unreliability may result from a propagation
failure of action potentials (or a conduction delay) at branch points
of axon collaterals due to impedance mismatches, as well as
transmission failure at functionally immature synapses due to impaired
calcium entry or vesicle release (Debanne et al. 1997
;
Kopysova and Debanne 1998
; Markram et al. 1997
; Streit et al. 1992
). Such an impairment of
axonal conduction, well-known in the immature nervous system, has also
been shown in regenerating axons (Kocsis et al. 1982
;
Meiri et al. 1981
). Newly sprouted A
fibers with thin
axon terminals, many branch points, and possible immature vesicle
release mechanisms might very well exhibit a variable latency at graded
intensity or with repetitive stimulation, in spite of a morphological
monosynaptic connection. What is clear is that, although there is no
evidence for the appearance of novel NMDA receptor-mediated silent
synapses in lamina II after nerve injury, further work is required to
establish whether the sprouted fibers do indeed make direct functional
contacts with lamina II neurons.
Involvement of pure-NMDA receptor-mediated synapses in synaptic plasticity
Silent NMDA receptor-mediated synapses are likely to
reflect the absence of functional AMPA receptors on the postsynaptic membrane (Gomperts et al. 1998). Conversion to
functional synapses may result either from a modification of
nonfunctional AMPA receptors or insertion of AMPA receptors into the
postsynaptic membrane. Such a conversion of silent to functional
glutamatergic synapses has been reported to contribute to long-term
potentiation in the hippocampus and cortex (Durand et
al. 1996
; Isaac et al. 1997
; Rumpel et
al. 1998
). In the neonatal spinal cord, Li and Zhuo (1998)
reported that the recruitment of silent synapses
contributes to the augmentation of monosynaptic excitatory input to
superficial dorsal horn neurons. They argued, moreover, that silent
synapses might represent an important mechanism for plastic changes in the spinal cord in the adult after inflammation or peripheral nerve
injury. However, our results simply do not support this. We conclude
that the transformation of silent synapses into functional ones is
unlikely to play a major, or perhaps any, role in changing synaptic
efficacy in the adult superficial dorsal horn. Silent synapses may
subserve an important developmental role in the spinal cord, acting in
an activity-dependent fashion to stabilize/eliminate synapses, as they
do in the hippocampus, cortex (Isaac et al. 1997
;
Rumpel et al. 1998
), and optic tectum (Wu et al.
1996
). In the immature spinal cord, there is a transient
projection of A fibers to the superficial laminae of the dorsal horn
(Fitzgerald et al. 1994
), and the distribution of
ionotropic receptors undergoes major developmental modification
(Jakowec et al. 1995
; Kalb and Fox 1997
),
both of which may contribute to transient silent synapses in the dorsal
horn. Although silent synapses may have an important role in the
formation of the immature somatosensory system, receptive field
plasticity in the adult appears to reflect the recruitment of
subliminal, but not silent, inputs (Woolf and King
1990
). Great care must be taken, therefore in using data
obtained from immature spinal cord preparations as a predictor or
surrogate of synaptic mechanisms in the adult. Silent synapses in the
superficial dorsal horn of the spinal cord appear to have a
developmental role only.
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ACKNOWLEDGMENTS |
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We thank Drs. P. A. Goldstein and R. Bardoni for technical advice regarding electrophysiological recordings in the neonatal spinal cord. H. Baba is on leave from The Department of Anesthesia, Niigata University, Niigata, Japan.
This work was supported by Human Frontier Science Program Grant RG73/96 and National Institute of Neurological Disorders and Stroke Grant NS-38253-01. H. Baba was also supported by the Ministry of Education, Science, Sports and Culture of Japan (Niigata University).
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FOOTNOTES |
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Address for reprint requests: H. Baba, Neural Plasticity Research Group, Department of Anesthesia, Massachusetts General Hospital and Harvard Medical School, MGH-East 4th Floor, 149 13th St., Charlestown, MA 02129.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 July 1999; accepted in final form 14 October 1999.
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REFERENCES |
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