Silent NMDA Receptor-Mediated Synapses Are Developmentally Regulated in the Dorsal Horn of the Rat Spinal Cord

Hiroshi Baba, Timothy P. Doubell, Kimberly A. Moore, and Clifford J. Woolf

Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Abeta fiber evoked mixed alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta 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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Abeta fibers, which parallels the recruitment of Abeta 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 Abeta 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, Abeta 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 Abeta 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 Abeta 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 Abeta 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, Abeta fiber stimulation of injured afferents can elicit EPSCs in most substantia gelatinosa (SG) neurons, in contrast to Abeta fiber stimulation in naive rats, which evokes EPSCs in only a very few SG neurons (Okamoto et al. 1996). However, the vast majority of Abeta 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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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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Fig. 1. Silent pure-N-methyl-D-aspartate (NMDA) excitatory postsynaptic currents (EPSCs) recorded from thin slices from neonatal rats. Examples of pure-NMDA receptor-mediated EPSCs evoked by intraspinal (A) and dorsal root (B) focal stimulation. Threshold intensities for eliciting fast EPSCs at -70 mV were 24 µA (A) and 26 µA (B). The stimulus intensity was decreased to subthreshold level (20 µA, A; 24 µA, B) such that alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs disappeared at -70 mV. NMDA receptor-mediated silent EPSCs were revealed by depolarizing the cell to +70 mV and reapplying the same "subthreshold" stimulus. The response at +70 mV was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV; 100 µM). The example shown in A was well fitted by a single exponential function (tau  = 167 ms, chi 2 = 0.039), whereas the example shown in B was best fitted by a double exponential (tau 1 = 11 ms, tau 2 = 85 ms, chi 2 = 0.312). Arrows indicate the sites of focal stimulation. All currents are averages of 5 traces.

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 MOmega . 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 alpha -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 Abeta (~10 µA, 0.05 ms), Adelta (~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 Abeta , Adelta , 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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, tau 1 = 26 ± 20 ms, tau 2 = 369 ± 240 ms, mean ± SD), but some pure-NMDA receptor-mediated EPSCs were best fitted by a single exponential (n = 2, tau  = 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 (tau 1 = 24 ms, tau 2 = 221 ms, and tau 1 = 11 ms, tau 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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Fig. 2. Absence of silent synapses in the naive adult spinal cord. A: glutamatergic EPSCs recorded from a substantia gelatinosa (SG) neuron. Left: initially, at -70 mV, a fast EPSC was evoked by a threshold intensity stimulus of 25 µA. The intensity was then slightly reduced to 23 µA, so that no fast EPSC was evoked at -70 mV. Unlike in the neonatal spinal cord, when the holding potential was changed to +70 mV, no EPSC was observed. The threshold intensity at both +70 and -70 mV was identical (25 µA). At -70 mV, EPSCs evoked by threshold intensity stimulation (25 µA) consisted of an early, transient component. However, at +70 mV, threshold intensity stimulation evoked an EPSC consisting of both an early component and a late, sustained component. Right: at +70 mV, the early component was blocked by the AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX; 10 µM). Simultaneous application of NBQX (10 µM) and a NMDA receptor antagonist, APV (100 µM) blocked both components. B: glutamatergic EPSCs recorded from a lamina III neuron. Left: threshold intensity at -70 and +70 mV was identical (16 µA). As for SG neurons, the threshold intensity evoked EPSC at +70 mV consisted of both early and late components. Right: the late component was blocked by APV (100 µM). Simultaneous application of APV (100 µM) and NBQX (10 µM) completely abolished the EPSC recorded at +70 mV. C: pure-NMDA EPSC recorded from a lamina III neuron. The stimulus intensity was decreased until no EPSC was evoked at -70 mV. Only NMDA receptor-mediated EPSCs were revealed when the holding potential was changed to +70 mV and the subthreshold stimulus was repeated. The response at +70 mV was completely blocked by the NMDA receptor antagonist, APV (100 µM). The example shown in C was well fitted with a double exponential function (tau 1 = 15 ms, tau 2 = 180 ms, chi 2 = 0.04). All currents are averages of 5 traces. All records shown were obtained from thin slices.

Because the presynaptic component of silent pure-NMDA receptor-mediated synapses may be limited to Abeta 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 Abeta 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 Adelta fiber-mediated EPSCs on stimulation of the dorsal root. A small proportion of the neurons (14 of 57 cells; 25%) have Abeta fiber-mediated EPSCs. However, the latencies of all these EPSCs were variable at graded intensity or with 20-Hz repetitive stimulation, indicating that the Abeta fiber inputs are likely to be polysynaptic. In contrast to naive adult rats, SG neurons in slices from sciatic nerve transected rats exhibited Abeta 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 Abeta 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 Abeta fiber and above the Adelta 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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Fig. 3. Synaptic responses recorded from SG neurons from sciatic nerve-transected (SNT) rats. A: pure-NMDA EPSCs recorded from a SG neuron in a thin slice. The stimulus intensity was decreased until no EPSC was evoked at -70 mV. Pure-NMDA receptor-mediated EPSCs were revealed when the holding potential was changed to +70 mV and the subthreshold stimulus was repeated. The response at +70 mV was completely blocked by an NMDA receptor antagonist, APV (100 µM), whereas an AMPA receptor antagonist, NBQX (10 µM), had no effect. The example shown in A was best fitted with a single exponential function (tau  = 138 ms, chi 2 = 0.084). All currents are average of 5 traces. B: distribution of the minimum stimulus threshold intensities necessary for eliciting EPSCs in SG neurons from spinal cord slices prepared from naive and SNT rats. The mean stimulus threshold intensity required to evoke EPSCs was 33.1 ± 15.1 µA (n = 57) for naive and 20.6 ± 7.3 µA (n = 22) for SNT rats (P = 0.004; unpaired t-test). C: distribution of the latencies of EPSCs evoked by supramaximal Abeta fibers stimulation (100 µA, 0.05 ms). Mean latency of EPSCs in naive and sciatic nerve transected rats were not significantly different (3.1 ± 1.1 ms; n = 53 for naive and 3.0 ± 1.1 ms; n = 18 for SNT, P = 0.64, unpaired t-test). There was no significant difference in the length of attached dorsal roots between slices from naive and SNT rats (18.6 ± 0.7 mm; n = 12 for naive and 17.6 ± 2.1 mm; n = 4 for SNT, P = 0.16, unpaired t-test). D: Abeta fiber-evoked polysynaptic EPSCs recorded from a SG neuron in a spinal cord slice from a sciatic nerve transected rat. At a holding potential of -70 mV, when the stimulus intensity was increased (10-100 µA), the EPSC latency decreased (bottom). The shortest latency was 3.5 ms. When the holding potential was changed to +70 mV (top), the latency also decreased as the stimulation intensity increased (10-100 µA). Note that the shortest latencies at -70 and +70 mV are the same. The length of dorsal root was 17.5 mm. Data shown in B-D were obtained from thick slices with attached dorsal roots. E: the percentage of silent pure-NMDA synapses in SG neurons of neonatal, naive adult, and adult SNT rats and in lamina III neurons of naive adult rats.

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 Abeta fibers could be observed in SG neurons in thick slices with an attached dorsal root. Although no Abeta 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 Abeta 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 Abeta fiber terminals do not generate pure-NMDA receptor-mediated silent synapses.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Abeta 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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Fig. 4. Schematic diagram of glutamatergic transmission in neonatal and adult spinal cord. A: in neonatal spinal cord, pure-NMDA glutamatergic synapses exist on many dorsal horn neurons. Pure-NMDA synapses are revealed at strongly depolarized membrane potentials (+70 mV) by minimal stimulation during which only one, or a few, fibers are activated. B: in the adult spinal cord, in contrast, most synapses possess both NMDA and AMPA receptors postsynaptically and, as such, pure-NMDA synapses are very rare. The EPSCs evoked by threshold intensity stimulation consist of both AMPA and NMDA components even with minimal stimulation. C: it is not possible to electrically stimulate only high-threshold, unmyelinated C fibers. The stimulus intensity sufficient to activate high-threshold fibers will simultaneously activate nearby low-threshold fibers. Therefore if the terminals of these high-threshold fibers are the sole presynaptic component of pure-NMDA synapses, threshold responses will always be recorded as consisting of both AMPA and NMDA components.

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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta 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 Abeta fibers are so thin that the conduction velocity of these new fibers is insufficient to alter EPSC latency. The failure of the novel Abeta 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 Abeta 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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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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