NT-3 Evokes an LTP-Like Facilitation of AMPA/Kainate Receptor-Mediated Synaptic Transmission in the Neonatal Rat Spinal Cord

Viktor L. Arvanov, Bradley S. Seebach, and Lorne M. Mendell

Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794-5230


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Arvanov, Viktor L., Bradley S. Seebach, and Lorne M. Mendell. NT-3 Evokes an LTP-Like Facilitation of AMPA/Kainate Receptor-Mediated Synaptic Transmission in the Neonatal Rat Spinal Cord. J. Neurophysiol. 84: 752-758, 2000. Neurotrophin-3 (NT-3) is a neurotrophic factor required for survival of muscle spindle afferents during prenatal development. It also acts postsynaptically to enhance the monosynaptic excitatory postsynaptic potential (EPSP) produced by these fibers in motoneurons when applied over a period of weeks to the axotomized muscle nerve in adult cats. Similar increases in the amplitude of the monosynaptic EPSP in motoneurons are observed after periodic systemic treatment of neonatal rats with NT-3. Here we show an acute action of NT-3 in enhancing the alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptor-mediated fast monosynaptic EPSP elicited in motoneurons by dorsal root (DR) stimulation in the in vitro hemisected neonatal rat spinal cord. The receptor tyrosine kinase inhibitor K252a blocks this action of NT-3 as does the calcium chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) injected into the motoneuron. The effect of NT-3 resembles long-term potentiation (LTP) in that transient bath application of NT-3 to the isolated spinal cord produces a long-lasting increase in the amplitude of the monosynaptic EPSP. An additional similarity is that activation of N-methyl-D-aspartate (NMDA) receptors is required to initiate this increase but not to maintain it. The NMDA receptor blocker MK-801, introduced into the motoneuron through the recording microelectrode, blocks the effect of NT-3, indicating that NMDA receptors in the motoneuron membrane are crucial. The effect of NT-3 on motoneuron NMDA receptors is demonstrated by its enhancement of the depolarizing response of the motoneuron to bath-applied NMDA in the presence of tetrodotoxin (TTX). The potentiating effects of NT-3 do not persist beyond the first postnatal week. In addition, EPSPs with similar properties evoked in the same motoneurons by stimulation of descending fibers in the ventrolateral funiculus (VLF) are not modifiable by NT-3 even in the initial postnatal week. Thus, NT-3 produces synapse-specific and age-dependent LTP-like enhancement of AMPA/kainate receptor-mediated synaptic transmission in the spinal cord, and this action requires the availability of functional NMDA receptors in the motoneuron.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Neurotrophin-3 (NT-3) is well established as a trophic factor for group Ia axons supplying muscle spindles (Chen and Frank 1999). Prenatally it acts as a "survival" factor for group I spindle afferents in rats (Zhou et al. 1998), although this effect is transient and restricted to early developmental stages (Horton et al. 1998). In the adult cat, chronic application of NT-3 to the proximal cut end of a muscle nerve reverses the decline in conduction velocity and central synaptic drive of axotomized group Ia fibers (Mendell et al. 1999; Munson et al. 1997). Additionally, in the first postnatal week when the strength of synaptic inputs from dorsal root (DR) to motoneurons is increasing (Seebach and Mendell 1996), systemic treatment of rats with NT-3 results in larger than normal fast excitatory postsynaptic potentials (EPSPs) evoked by dorsal root stimulation (DR-EPSPs) (Seebach et al. 1999). Furthermore, there is some indication that endogenous NT-3 acts tonically over this period to promote development of this synaptic connection (Seebach et al. 1999). Since the blood brain barrier is not well developed in these young rats (Tonra and Mendell 1997), and in view of the acute effects of NT-3 at central synapses (Kang and Schuman 1995; Stoop and Poo 1996), a direct action of NT-3 on the spindle afferent connection to motoneurons is a possible explanation for these findings during early postnatal development. This has prompted an investigation of the acute action of NT-3 on EPSPs produced in neonatal rat motoneurons.

DR-EPSPs in neonatal rat motoneurons are mediated by activation of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. An early, fast, likely monosynaptic component is mediated by non-NMDA alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and blocked by the AMPA/kainate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Pinco and Lev-Tov 1993). The NMDA receptor that is selectively inhibited by the competitive antagonist 2-amino-5-phosphonovaleric acid (APV) (Davies et al. 1981) and the noncompetitive antagonist MK-801 (Davies et al. 1988) contributes mainly to generating a slower component of EPSP that is likely at least partly polysynaptic. Activation of NMDA receptors depends on membrane potential because of the Mg2+ block (Ault et al. 1980; Nowak et al. 1984) that is reduced with depolarization. These components exhibit changes during perinatal development (Jiang et al. 1990; Pinco and Lev Tov 1993; Ziskind-Conhaim 1990) with transmission being restricted initially to NMDA receptor (NMDAR)-mediated slow EPSPs. In the postnatal period, fast EPSPs mediated by AMPA/kainate receptors become evident and within 1-2 wk after birth the NMDAR-mediated component disappears. Another AMPA/kainate receptor-mediated synaptic projection onto mammalian spinal motoneurons from axons of the ventrolateral funiculus (VLF) becomes functional about 1 wk earlier (Pinco and Lev-Tov 1994).

Here we compared the effect of NT-3 on intracellularly recorded EPSPs evoked by electrical stimulation of DR and VLF in antidromically identified motoneurons in the hemisected spinal cord from animals of two age groups, "postnatal" 1- to 7- and 8- to 15-day old rats. We provide evidence that during the first postnatal week a brief exposure to NT-3 acutely increases the amplitude of the "fast" AMPA/kainate receptor-mediated DR-evoked fast EPSP, a potentiation that persists well beyond removal of NT-3. Motoneuron NMDA receptors are required to initiate the NT-3-induced EPSP increase but not to maintain it. This action is synapse specific in that it does not affect fast AMPA/kainate receptor-mediated EPSPs produced by VLF axons. It is also age specific in that it cannot be elicited in either pathway after the first week. We demonstrate that this action of NT-3 resembles LTP and speculate that this effect may play an important role in the development of the Ia/motoneuron connection in the neonatal rat.

Portions of these results have been reported previously in abstract form (Arvanov et al. 1999a; Mendell and Arvanov 1999).


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INTRODUCTION
METHODS
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Intracellular recordings were obtained from antidromically identified lumbar spinal motoneurons in the L5 segment in 121 spinal cord preparations isolated from neonatal male Sprague-Dawley rats 1-15 days old. The general experimental methods have been described previously (Fulton and Walton 1986; Seebach and Mendell 1996; Seebach et al. 1999). Rats were anesthetized in ether, or in the case of 1- and 2-day-old rats, by placing them on ice, and killed by decapitation. After laminectomy from the ventral surface, the lumbar region of the spinal cord was removed. It was hemisected in a dissection chamber superfused with cold (10°C) artificial cerebrospinal fluid (ACSF) containing as follows (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 2.0 MgSO4, 25 NaHCO3, 1.2 NaH2PO4, 11 dextrose, aerated with 95% O2-5% CO2 (pH 7.4). These Mg2+ and Ca2+ concentrations were chosen to eliminate the discharge of motoneurons in response to stimulation of the entire L5 dorsal root. The medial surface of the left hemicord was pinned to a Sylgard-coated surface in a recording chamber that was continuously perfused at 10 ml/min with aerated ACSF at 30°C. The VLF was dissected free of the spinal cord at T12 by the use of sharpened tungsten microneedles, as previously described (Pinco and Lev-Tov 1994). Suction stimulating electrodes with silver-silver chloride internal wires were attached to the L5 ventral root, to peeled, cut VLF axon bundles, and to the cut L5 dorsal root (Fig. 1). To minimize generation of action potentials evoked by the VLF, it was necessary to use VLF axon bundles of <= 100 µm in diameter.



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Fig. 1. Diagram of electrode placement.

Intracellular recordings (microelectrodes 70-110 MOmega were filled with 3 M potassium acetate) were made from motoneurons identified by antidromic response to ventral root stimulation. Electrical stimuli of 60-µs duration were delivered separately to the DR and VLF to synaptically activate the motoneuron. The intensity (80-200 µA) was chosen to be just supramaximal for evoking the maximum monosynaptic fast EPSP, the location of whose peak was determined as described previously (Seebach and Mendell 1996). The responses to 10 stimuli were averaged (pClamp 8, Axon Instruments). A stimulation rate of 0.05-0.1 Hz was chosen to prevent frequency-dependent changes in successive responses either in controls or after NT-3 administration. This protocol was delivered every 5 min in control periods and during/after NT-3 or drug administration. Each spinal cord was used for only a single NT-3 application. Motoneuron input resistance and rheobase were estimated by passing current pulses (100 ms) through the intracellular recording electrode as described previously (Fulton and Walton 1986; Seebach and Mendell 1996).

Unless otherwise stated, drugs were added to the perfusion solution. NT-3 was administered at a concentration of 0.2 µg/ml, except as noted. The change in the peak amplitude of the monosynaptic fast EPSP after NT-3 or other treatments was calculated using the following formula: % change in EPSP = [(100 × EPSP2/EPSP1) - 100]. The results are presented as mean ± SE. Unless otherwise mentioned, a paired two-sided t-test was used to evaluate the statistical significance of the observed changes; in these cases the response of the same cell was compared before and after drug application. Because multiple such tests were carried out, it is necessary to use a more conservative value of P for judging statistical significance. Using the Bonferroni correction, we established a value of P = 0.004 for study-wide statistical significance at the 0.05 level.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Administration of NT-3 (0.2 µg/ml) to spinal cords removed from rats during the first postnatal week resulted in facilitation of the monosynaptic fast component of DR-EPSPs beginning 10-15 min after application and lasting at least 3 h after washout of NT-3 (Fig. 2, A and C; also see Fig. 5C). This fast component is mediated by CNQX-sensitive AMPA/kainate receptors (Fig. 4A) (Thomas et al. 1998). Figures 2A and 5A also suggest that NT-3 leads to potentiation of a late polysynaptic response. This could not be evaluated quantitatively because of the contribution of the falling phase of the monosynaptic EPSP to the later components of the response. NT-3 produced no significant effect on the CNQX-sensitive VLF-EPSPs (-8.5% ± 3.7, n = 18; P = 0.16) recorded in the same motoneurons indicating that the DR-evoked changes were not due to generalized changes in motoneuron properties (Fig. 2, B and C). The mean maximum facilitation of the fast component of DR-EPSPs averaged over all motoneurons studied was 48.2 ± 9.5% (n = 29, P < 0.00007). Although dose-response effects were not investigated systematically, in five preparations NT-3 produced a similar potentiation at a lower concentration (0.1 µg/ml, Fig. 1A), while at 0.01 µg/ml, it was without effect (n = 4). Treatment with NT-3 produced no significant action on the resting membrane potential (-68.7 ± 1 mV in control versus -69.6 ± 1 mV in NT-3, n = 29, P = 0.17) or input resistance (31 ± 2.5 MOmega in control versus 27 ± 3 MOmega in NT-3, n = 29, P = 0.12) of the motoneurons. Treatment with other neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5)] at similar doses did not elicit these synaptic effects (not illustrated).



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Fig. 2. Neurotrophin-3 (NT-3) produces a synapse-selective long-lasting enhancement of the monosynaptic fast excitatory synaptic transmission in motoneurons. A and B: representative traces of the recorded intracellularly synaptic potentials elicited by stimulation of dorsal root (DR) and ventrolateral funiculus (VLF) in the same motoneuron, before and 10 min after acute administration of NT-3, respectively. All records are 10-sweep computer averages. C: the time course of NT-3-induced changes in amplitude of the fast monosynaptic EPSP is illustrated for the cell held for about 4 h. Each point represents the amplitude of the fast monosynaptic EPSP in response to a single stimulus (DR or VLF) relative to the average peak amplitude of that component before NT-3 administration.



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Fig. 3. NT-3-induced facilitation of the dorsal root-excitatory postsynaptic potential (DR-EPSP) depends on Trk family receptor tyrosine kinases, intracellular Ca2+, and motoneuron resting membrane potential. All data from rats in first postnatal week. A: effect of NT-3 (0.2 µg/ml) on the DR-EPSPs is abolished by prior treatment with K-252a (0.2 µM, 15 min). B: effect of NT-3 (0.2 µg/ml) on the DR-EPSPs is abolished by intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) allowed to diffuse in from the recording microelectrode filled with 2 M K-acetate plus 0.2 M BAPTA (tetrapotassium salt, pH = 7). Depression of afterhyperpolarization of antidromically activated action potential was used as an index that BAPTA had reached an effective concentration in the cell. C: % change in peak amplitude of monosynaptic fast EPSP plotted versus membrane potential at EPSP peak. Abscissa denotes membrane potential at monosynaptic EPSP peak [resting membrane potential (RMP) + EPSP amplitude] in the absence of NT-3, i.e., the potential at which the effect of NT-3 would be exerted. Further details in text.



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Fig. 4. Postsynaptic NMDA-receptor activation is necessary for the induction of synaptic facilitation by NT-3. A: NMDA receptor antagonist d-APV (40 µM, 15 min) added to artificial cerebrospinal fluid (ACSF) and B: NMDA receptor channel blocker MK-801 (500 µM, 30-40 min) included in the recording electrode both prevent NT-3-induced facilitation of the DR-EPSPs. Note both d-APV and MK-801 markedly decreased the late, slow component of DR-EPSPs, but not that of VLF-EPSPs in the same motoneurons (while recorded with MK-801 electrode, the late component of DR-EPSPs was markedly decreased and stabilized at the new baseline within about 30 min of recording). The fast component was blocked by further addition of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM).



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Fig. 5. NMDA-receptor blockade did not significantly diminish the peak amplitude of fast DR-EPSP at preparation whose amplitude was already enhanced by prior treatment with NT-3. A and B: representative traces of the DR-EPSP and VLF-EPSP in the same motoneuron, (1) before, (2) 10 min after acute administration of NT-3, and (3) 20 min after administration of 40 µM APV, respectively. Note that APV selectively inhibits slow component of the EPSP, while fast EPSP amplitude is blocked only minimally by APV, indicating that incremental EPSP after NT-3 is largely not NMDA-mediated. C: the time course of NT-3-induced changes in fast EPSP amplitude and the lack of effect of APV is illustrated for the same cell. See Fig. 2C for further details.

The facilitatory action of NT-3 on the DR-EPSP was prevented by pretreatment with K-252a (200 nM) (P = 0.32; n = 6; Fig. 3, A and C; Fig. 6), an inhibitor of receptor tyrosine kinases (Knusel and Hefti 1992), suggesting the involvement of the trk family of receptor tyrosine kinases. Administration of K-252a alone did not significantly alter the fast component of DR-EPSPs (0.80 ± 0.98%; n = 6; P = 0.74). The NT-3 effects probably involve increased intracellular calcium since allowing bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) to diffuse into the motoneuron from the recording microelectrode (Lancaster and Nicoll 1987) prevented NT-3 from potentiating the fast EPSP (P = 0.31; n = 5; Figs. 3B and 6).



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Fig. 6. Pooled data to demonstrate the effects of bath-applied 0.2 µg/ml NT-3 and 10 µM NMDA on peak amplitude of monosynaptic fast EPSPs at DR synapses on motoneurons. NT-3 enhanced the DR-EPSPs during the first, but not the second postnatal week. NT-3-induced enhancement of DR-EPSP was abolished by prior treatment with K-252a (see Fig. 3A), intracellular Ca2+ chelator BAPTA (see Fig. 3B), and NMDA receptor blockers (extracellularly applied APV or intracellular MK-801; see Fig. 4). All cells included in the chart had membrane potential at the peak of the monosynaptic fast EPSP between -52 and -63 mV (see Fig. 3C). Asterisk indicates significance at the 0.004 level as required by the Bonferroni correction. See text for further details.

Interestingly, the facilitatory action of NT-3 on DR-EPSPs was greater in the motoneurons with resting membrane potential of -60 to -66 mV than in more hyperpolarized ones (not illustrated). This effect was not correlated with EPSP amplitude itself (not illustrated). However, as displayed in Fig. 3C, we observed that the effect of NT-3 was greater in cells in which the membrane potential at the peak of the monosynaptic EPSP was more positive. We tested the significance of this difference by dividing the data into two groups according to the median value of membrane potential at EPSP amplitude peak (i.e., median split along the abscissa). The potentiating effect of NT-3 was significantly greater in motoneurons where membrane potential at EPSP peak was more positive than -63 mV (86.3 ± 11.8%; n = 15) than in those in which it was more negative (9.2 ± 3.9%; n = 14) (P < 0.00004; 2-sided t-test with unequal variance; df = 27). Since the NMDA receptor is known to function in a voltage-dependent manner so that it is more active at a more depolarized membrane potential (see INTRODUCTION), this finding suggested a possible role for the NMDA receptor in mediating the NT-3 induced effect.

NT-3 did not produce a significant action on DR-EPSPs in motoneurons taken from animals older than 1 wk even when tested on motoneurons with membrane potential at the peak of the monosynaptic fast EPSP between -52 and -63 mV (8-15-day-old rats; P = 0.37, n = 8; Fig. 6). This is consistent with a role for the NMDA receptor in the NT-3 induced effect since the contribution of NMDA receptors to the DR-EPSPs decreases during the first postnatal week (see INTRODUCTION; Jiang et al. 1990; Pinco and Lev-Tov 1993; Ziskind-Conhaim 1990).

The effect of the NMDA-receptor antagonist d-APV on NT-3 induced potentiation of the DR EPSP was tested on motoneurons with membrane potential at the peak of the fast EPSP in the range of -52 to -63 mV (i.e., normally sensitive to NT-3) in animals younger than 1 wk. Addition of 40 µM d-APV alone to the bath produced a small decrease in the peak amplitude of the fast DR-EPSP averaging about 10%, but markedly shortened its decay, indicating a greater effect on later components of the response (Ziskind-Conhaim 1990; Fig. 4A). APV had no effect on the VLF-EPSP in the same cell (Fig. 4A). Addition of NT-3 to d-APV-treated preparations resulted in no change in the fast component of the DR-EPSP (P = 0.44; n = 6; Figs. 4A and 6), indicating a role for NMDA receptors in the development of NT-3 induced potentiation of the EPSP. Addition of CNQX to the bath eliminated most of the surviving response, indicating that it was mediated by AMPA/kainate receptors. The small remaining depolarizing response was not present in experiments where bicuculline and strychnine were also added (not illustrated; see also Pinco and Lev Tov 1993), indicating that it was a depolarizing inhibitory response.

To examine the possibility that motoneuron NMDA receptors are necessary for the NT-3-induced effect, we applied the NMDA antagonist MK-801 intracellularly by allowing it to leak from the recording microelectrode. This restricted blockade of NMDA receptors to those in the recorded motoneuron (Arvanov et al. 1999b; Berretta and Jones 1996). In control experiments, motoneuron depolarization produced by bath-applied NMDA but not that produced by AMPA was blocked by this procedure (not illustrated). Within 20-45 min of impalement, the amplitude of the DR-evoked slow EPSP diminished substantially with only a small effect on the fast DR-EPSP and almost no action on VLF-EPSPs in the same motoneuron (Fig. 4B), i.e., similar to the effect of d-APV. Under these conditions (membrane potential of -53 to -63 mV at monosynaptic EPSP peak), NT-3 had no significant effect on the fast DR EPSP in animals younger than 1 wk (P = 0.95; n = 7; Figs. 4B and 6). This confirmed that postsynaptic NMDA receptors play a crucial role in this action of NT-3. Consistent with the blockade of NT-3's action by NMDA receptor antagonists, NMDA itself mimicked the action of NT-3 by enhancing fast DR-EPSPs (P = 0.02; n = 8; Fig. 6). Although this effect was not significant on applying the strict Bonferroni criterion, the potentiating effect of NMDA was observed in all eight cells tested (mean increase = 36 ± 12%).

The potentiation of the monosynaptic EPSP induced by NT-3 persisted after APV was added to the bath (Fig. 5C). A small reduction in the amplitude of the monosynaptic EPSP was detected in all five cells tested (-7.8 ± 1.9%; P = 0.02; n = 5) (Fig. 5), but this decrease was much smaller than the increase produced by NT-3 in the absence of APV. Thus, NMDA receptors are required for initiation of the long-lasting NT-3 effect on peak amplitude of fast DR-EPSPs, but not for its maintenance. In all of these respects, the effect of NT-3 resembles long-term potentiation (LTP) studied at other synapses (for review see Malenka and Nicoll 1999).

To examine whether NT-3 produces a direct action on motoneuron NMDA receptors, we studied the effect of NT-3 on NMDA-induced depolarization of the motoneuron in the presence of bath-applied tetrodotoxin (TTX) to block impulse activity in spinal neurons presynaptic to the motoneuron. Administration of 20 µM NMDA for 1 min produced a moderate, reproducible depolarization of 5-10 mV that was blocked by d-APV. NT-3 enhanced NMDA-induced depolarization in all three motoneurons tested by an average of 86 ± 17% (n = 3, P < 0.05; paired t-test, Fig. 7A). There was no sign of synaptic noise (Fig. 7A) that would have suggested increased presynaptic release of glutamate, as was observed in the absence of TTX (not illustrated). Also, application of NT-3 never depolarized the motoneuron, indicating that its action on the motoneuron is one of modulation rather than activation.



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Fig. 7. A: NT-3 enhances direct NMDA-evoked depolarization response of the motoneuron. Typical responses of motoneuron to bath-applied 20 µM NMDA before (control) and after NT-3 administration (0.2 µg/ml, 10 min) and after d-APV administration (40 µM, 15 min). After identification of the motoneuron using antidromic stimulation, tetrodotoxin (TTX, 0.5 µM, the concentration required for complete blockade of the spontaneous and electrically evoked EPSPs) was included in the perfusate. A 30 min inter-application interval was needed to avoid desensitization and to maintain a stable baseline. B: scheme to explain the results obtained in these experiments. Motoneuron NMDA receptors are activated by NMDA (open arrow) or are modulated by NT-3 [either directly via glycine site or indirectly via second messengers (closed arrows)] to increase AMPA receptor sensitivity or number (closed short arrow).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We provide direct physiological evidence that brief administration of NT-3 rapidly induces a synapse-selective and age-specific enhancement of fast synaptic transmission in the neonatal rat spinal cord. The data support a mechanism whereby NT-3-induced synaptic facilitation of the fast AMPA/kainate receptor-mediated DR-evoked synaptic responses results from modulation of motoneuron NMDA receptors by NT-3. The requirement for NMDA receptors, the persistence of the increase in AMPA/kainate receptor-mediated EPSP amplitude despite subsequent blockade of the NMDA receptor, and the long-lasting increase induced by a brief pulse of NT-3 all point to a mechanism similar to that responsible for LTP in area CA1 of the hippocampus (Malenka 1991).

NT-3 has also been found to elicit long-lasting changes in synaptic transmission in the adult hippocampus that resemble LTP (Kang and Schuman 1995), although the evidence there indicates that the NT-3-evoked LTP is at least partially independent of the stimulus-evoked LTP at CA1 synapses. First, unlike stimulus-evoked LTP, NT-3 could produce LTP in the presence of APV, indicating an effect independent of the NMDA receptor (Kang and Schuman 1995). Second, antibodies to NT-3 did not have any effects on the stimulus-evoked LTP (Chen et al. 1999). Our present findings indicate that NT-3-induced long-lasting enhancement of synaptic transmission in the spinal motoneuron resembles stimulus-evoked LTP at the Schaffer-collateral CA1 synapses in that both processes require NMDA receptors.

LTP induced by high-frequency dorsal root stimulation has also been reported in motoneurons of neonatal rats (Lozier and Kendig 1995). As in the hippocampus, the properties of LTP induced by stimulation and by NT-3 differ considerably. Stimulus-induced LTP in motoneurons is confined to a very late component of the synaptic response elicited by stimulation of C fibers. Unlike NT-3, repetitive stimulation did not potentiate the short-latency, CNQX-sensitive component of the EPSP. Furthermore, potentiation of the late response by high-frequency stimulation occurred in the presence of APV, unlike its complete blockade of NT-3-induced potentiation.

Neurotrophins have previously been suggested to affect central synapses via NMDA receptors either indirectly by interaction at the level of intracellular signaling cascades (Suen et al. 1997) or directly at the glycine site of the NMDA receptor (Jarvis et al. 1997) (Fig. 7B). Although we found that NT-3 enhances the initial AMPA/kainate response evoked by stimulation of the dorsal root (Figs. 2 and 6), the requirement for NMDA receptors in motoneurons to obtain this effect (Fig. 4) and the ability of NT-3 to enhance the response of motoneuron NMDA receptors (Fig. 7A) together indicate that the direct action of NT-3 in this system is also on NMDA receptors. Also in agreement with the present scheme (Fig. 7B) are reports that activation of NMDA receptors can increase the response of AMPA receptors (Benke et al. 1998). Our results suggest that these two mechanisms can operate in series, i.e., neurotrophins modulate NMDA receptors which in turn increase the response of AMPA/kainate receptors to synaptically released glutamate (Fig. 7B).

The finding that K-252a eliminated the response to NT-3 suggests that it was mediated by the high-affinity trkC receptor. A possible role for the p75 receptor was not considered directly but the finding that no other neurotrophin elicited this effect suggests that the p75 receptor is not a crucial component of the response since it is responsive to all neurotrophins.

Although details of the cellular mechanisms mediating these actions remain obscure for the present, the NT-3 effects probably involve signaling cascades related to intracellular calcium because chelating Ca2+ in the motoneuron by BAPTA loaded into the electrode prevented NT-3 potentiation of the monosynaptic fast EPSP (Fig. 3B). Ca2+ entering via NMDA receptors initiates the signaling cascade that results in enhanced AMPA/kainate responses. For example, in the hippocampus, Ca2+ influx through the NMDA-type ion channel can activate CaM-kinase II (Strack and Colbran 1998), which in turn can phosphorylate and regulate AMPA/kainate-type GluR ion channels (McGlade-McCulloh et al. 1993; Raymond et al. 1993; Stricker et al. 1999). An alternate cellular mechanism for interaction of NMDA and AMPA receptors can involve cytoplasmic C termini of AMPA-receptor subunits and the glutamate receptor interacting protein (GRIP) (Li et al. 1999). Postsynaptic NMDA receptors have recently been shown to play a role in the accumulation of postsynaptic AMPA receptors, and the increase in the magnitude of the EPSP they evoke as a consequence of LTP (Shi et al. 1999). Despite the evidence for a requirement for postsynaptic NMDA receptors in the changes reported here, at the present time we cannot discard the possibility that activating NMDA receptors in the postsynaptic cell retrogradely causes the presynaptic terminal to increase release of transmitter (Malgaroli et al. 1992; Robert 1998).

The effects of NT-3 were highly selective. NT-3 was unable to influence the AMPA/kainate-mediated EPSPs produced by activation of the descending fibers in the ventrolateral funiculus in the very same motoneurons whose AMPA/kainate-mediated EPSPs from the dorsal root were strongly facilitated (Fig. 2). Also, NT-3-facilitated DR-EPSPs only in very young animals (<1 wk, Fig. 6). One possibility is that trkC receptors are located in dorsal root afferents, but not in the descending fibers, and these receptors in the dorsal roots decrease in number during development and cannot mediate the effect of NT-3 beyond the initial postnatal week. However, high expression levels of trkC receptors have been found in lumbar dorsal roots in 3-mo-old rats (Bergman et al. 1999) and in the descending pathways as well (King et al. 1999). Moreover, in adult cats it is known that NT-3 applied directly to axotomized afferent fibers can reverse the decline in conduction velocity and EPSP amplitude associated with such damage (Mendell et al. 1999), indicating that trkC receptors associated with spindle afferents remain functional, at least in adult cats. This suggests that the changes are in NMDA receptors and there is considerable evidence that these undergo substantial changes on their subunit composition (Flint et al. 1997; Monyer et al. 1994) and a decline in their sensitivity (Carmignoto and Vicini 1992; Crair and Malenka 1995; Feldman and Knudsen 1998; Hestrin 1992; Tsumoto et al. 1987) in the first two postnatal weeks. Thus, as things stand at present, it seems more likely that the synapse-specific and age-specific effects noted here are due to differences in NMDA receptor properties rather than trkC receptor expression. We speculate that NMDA receptor properties must be highly specialized according to the nature of the presynaptic input (DR versus VLF in neonates).

To understand the role of NT-3 in normal function, it is important to know where it is expressed. Its expression in adult muscle is well established, particularly in association with spindles (Copray and Brouwer 1997), which suggests that its role may be to maintain the monosynaptic reflex pathway as well as other spindle projections. There is increasing evidence that NT-3 is also expressed in cells of the spinal cord, specifically in astrocytes of the white matter and in motoneurons as well as smaller cells in the gray matter (Scarisbrick et al. 1999; Schober et al. 1999). The persistence of the change induced by NT-3 during the initial postnatal week raises the possibility that this mechanism participates in the strengthening of DR connections to motoneurons known to be occurring in neonatal rats at that time (Seebach and Mendell 1996). Together, these recent findings raise the possibility that NT-3 is available in the vicinity of these synapses and can exert actions on the monosynaptic reflex connection. However, whether it is released in sufficient amounts and whether it exerts physiological effects during development and/or in the adult is not yet known.


    ACKNOWLEDGMENTS

We thank Dr. Paul Adams for valuable comments on a draft of the manuscript and the Statistical Consulting Unit in the Department of Applied Mathematics and Statistics at the State University of New York at Stony Brook for help with the statistical procedures. Neurotrophins were received courtesy of Regeneron Pharmaceuticals Inc.

Support was provided by National Institute of Neurological Disorders and Stroke Grant NS-16696 to L. M. Mendell (Javits Neuroscience Award) and by the Christopher Reeve Paralysis Foundation. Additional support was provided by NINDS Grants RO1 NS-32264 and PO1 NS-14899 to L. M. Mendell.

Present address of B. S. Seebach: Dept. of Biology and Microbiology, University of Wisconsin at La Crosse, 1725 State St., La Crosse, WI 54601.


    FOOTNOTES

Address for reprint requests: L. M. Mendell, State University of New York at Stony Brook, Life Sciences Building, Room 550, Stony Brook, NY 11794-5230 (E-mail: lorne.mendell{at}sunysb.edu).

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 29 December 1999; accepted in final form 25 April 2000.


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ABSTRACT
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