Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, New York 11794-5230
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ABSTRACT |
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Arvanian, Victor L. and Lorne M. Mendell. Removal of NMDA Receptor Mg2+ Block Extends the Action of NT-3 on Synaptic Transmission in Neonatal Rat Motoneurons. J. Neurophysiol. 86: 123-129, 2001. NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABAA, GABAB and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1- to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg2+ concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0-2 wk). In low-Mg2+, NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg2+ block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg2+ concentration in fluid bathing the spinal cord.
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INTRODUCTION |
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Studies of the
physiological effects of neurotrophins in neonatal rat spinal cord have
shown that neurotrophin-3 (NT-3), but not nerve growth factor (NGF),
administered to isolated spinal cord acutely increases the amplitude of
the fast monosynaptic AMPA/kainate-mediated excitatory postsynaptic
potential (EPSP) evoked by electrical stimulation of dorsal root (DR)
(Arvanov et al. 2000). This effect is age-specific in
that NT-3 action on the DR-EPSP is restricted to very young (<1 wk
old) animals. It is also synapse-specific in that NT-3 did not affect
the AMPA/kainate-mediated EPSPs produced by activation of the
descending ventrolateral funiculus (VLF) fibers in the same motoneurons
whose AMPA/kainate receptor-mediated EPSPs from the DR were strongly
facilitated. The action of NT-3 on the DR-EPSP was prevented by
K-252a, an inhibitor of receptor tyrosine kinases
(Knusel and Hefti 1992
), suggesting the involvement of
the trk family of receptor tyrosine kinases. Blockade of the N-methyl-D-aspartate (NMDA) receptor by
D-2-amino-5-phosphovaleric acid (D-APV) prior
to NT-3 administration also prevented its action, indicating that
activation of NMDA receptors is required to initiate the NT-3-induced
increase of AMPA/kainate receptor-mediated responses. MK-801
administered inside the motoneuron had an effect similar to
bath-applied APV, suggesting that NMDA receptors in the motoneuron are
crucial for the effect of NT-3 to be expressed.
Because trkC receptors for NT-3 are expressed on Ia afferents
(McMahon et al. 1994) and
motoneurons
(Johnson et al. 1996
) throughout the life span of rats,
we have hypothesized that the age-related changes in NT-3 action are
likely to reflect changes in NMDA receptors. These undergo modification
during the neonatal developmental period (Kalb et al.
1992
) (see DISCUSSION). Specifically, we were drawn
to the possibility that NMDA receptors in motoneurons become more
susceptible to Mg2+ block as the animal matures
and that this might be the cause of the inability of NT-3 to affect
synaptic transmission. To check this hypothesis, we studied the
NMDA-mediated synaptic responses evoked by DR and VLF stimulation in
animals in the first (1 WO) and second (2 WO) postnatal weeks. To
accomplish this, we blocked the action of other known receptors
pharmacologically by treating the isolated spinal cord with a
"cocktail" made up of CNQX (to block AMPA/kainate receptors),
strychnine (to block glycine receptors), bicuculline (to block
GABAA receptors), and CGP35348 (to block GABAB receptors). Studies under these conditions
revealed that the NMDA receptor (NMDAR)-mediated responses were
enhanced by NT-3. Furthermore, changes in NMDAR-mediated responses were
developmentally regulated, and changes in them paralleled the
sensitivity of AMPA/kainate receptor-mediated responses to NT-3 at the
same connections. Finally, elevating the sensitivity of the NMDA
receptor by reducing Mg2+ concentration in the
bath increased the response of both the NMDA receptor and the
associated AMPA/kainate receptors (studied in the absence of the
blockers) to NT-3. Thus these experiments confirmed the relationship
between NMDAR function and sensitivity to NT-3 and this suggests
strategies for extending the effects of NT-3 in modulating synaptic
transmission in the spinal cord.
A preliminary account has been presented in abstract form
(Arvanov and Mendell 2000).
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METHODS |
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These experiments were carried out in spinal cords removed from
neonatal male Sprague-Dawley rats using methods previously described in
detail (Arvanov et al. 2000; Fulton and Walton
1986
; Seebach and Mendell 1996
; Seebach
et al. 1999
). Two age groups were used: 1-5 days old (1WO) and
9-15 days old (2WO). After removal of the spinal cord from the animal,
a section spanning segments from approximately T1
to S3 was placed in a chamber superfused (10 ml/min) with cold (10°C) artificial cerebrospinal fluid (ACSF) containing (in mM) 117 NaCl, 4.7 KCl, 2.5 CaCl2,
2.0 MgSO4, 25 NaHCO3, 1.2 NaH2PO4, and 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 silicone elastomer (Sylgard)-coated surface in
the recording chamber. The L5 dorsal and ventral
roots as well as the cut VLF (dissected from the spinal cord at
T12) (Pinco and LevTov 1994
)
were placed in suction electrodes with silver-silver chloride internal
wires for stimulation. The preparation was then allowed to equilibrate
slowly to 30°C over a period of 1-1.5 h and the experiment was
carried out at 30°C.
Intracellular recordings (microelectrodes 70-110 M filled with 3 M
potassium acetate) were made in lumbar spinal motoneurons in the
L5 segment that were identified by their
antidromic response to VR stimulation. Initially, the synaptic response
to 10 stimuli of 50-µs duration delivered separately to DR and VLF
was averaged (pClamp 8, Axon Instruments). Stimulation rate was 0.05 or
0.1 Hz for experiments in which the fast monosynaptic EPSP was studied (Fig. 4), but only 0.01 Hz when NMDAR-mediated responses were studied
(Fig. 1). Five-minute breaks were
interspersed between groups of 10 stimuli in all experiments. To record
the NMDAR-mediated synaptic responses, the AMPA/kainate receptor
antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 10 µM), the
GABAA receptor antagonist bicuculline (5 µM), the GABAB receptor antagonist CGP 35348 (10 µM)
(Bertrand and Cazalets 1999
; Peshori et al.
1998
) and the glycine receptor antagonist strychnine (5 µM)
were included in the perfusate to isolate NMDAR-mediated responses
pharmacologically. All drugs were added to the perfusion solution. The
neurotrophins NT-3 and NGF were administered at a concentration of 0.2 µg/ml. In some experiments, saline with reduced concentrations of
Mg2+ or increased concentration of
Ca2+ was substituted for the artificial bathing
solution described in the preceding text. In these cases, corresponding
equiosmolar changes in Na+ concentration were
made.
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The peak of the monosynaptic component was detected most clearly at low
stimulus intensity below the levels required to evoke the later
components. A cursor was placed at this peak and used to measure the
amplitude of the EPSP in the single sweep determined to contain the
maximum monosynaptic response (see Figs. 1A1 and 3A). Motoneuron input resistance was estimated by passing
current pulses (100 ms) through the intracellular recording electrode as described previously (Arvanov et al. 2000;
Fulton and Walton 1986
).
The results are presented as means ± SE. Each cell in the present data series was from a different spinal cord. t-tests were used to determine the significance of the differences. Bonferroni's correction was used as required to adjust for multiple comparisons.
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RESULTS |
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NMDAR-mediated synaptic responses in motoneurons
To study the NMDAR-mediated response in isolation, all other
known inputs to the motoneuron were blocked pharmacologically, and
synaptic responses evoked by stimulation of DR and VLF were studied as
a function of stimulus intensity. In the presence of AMPA/kainate-,
GABAA-, GABAB- and
glycine-receptor antagonists, the fast monosynaptic component of
DR-EPSP was blocked. Under these conditions, low-intensity DR
stimulation evoked a slightly slower response whose latency (6.4 ± 0.5 ms compared with 5.5 ± 0.4 ms in the same cells;
n = 37; P > 0.05), threshold (86 ± 8 vs. 68 ± 6 µA; P > 0.05), and sensitivity
to high-frequency stimulation (both followed repetitive stimulation
only at frequencies of 0.05-0.1 Hz or lower) were similar to those of
the initial component observed in the absence of the blockers. This
response was completely blocked by APV, indicating that it was mediated by NMDA receptors. We conclude that this NMDA component was also monosynaptically driven (see also Pinco and Lev-Tov
1994) and refer to it as the NMDA-m response. A further
increase of stimulus intensity (
500 µA) and/or duration (
500
µs) had no effect on maximum amplitude or latency of NMDA-m response
(Fig. 1A1; see also Fig. 3A).
Once the NMDA-m component reached maximum amplitude, a further increase in stimulus intensity of ~20-30 µA evoked a much later APV-sensitive component (150- to 250-ms latency at the peak), referred to as NMDA-l. The amplitude of NMDA-l increased to some intermediate value and then jumped in an all-or-none fashion to a maximum value suggestive of a region of negative resistance in the motoneuron (Fig. 1A; n = 19). With further increase of stimulus intensity, the latency of NMDA-l decreased (see Fig. 3). However, this decrease in latency had no effect on the peak amplitude of the NMDA-m component (see Fig. 3A). NMDA-l could follow stimulus rates of only 0.01-0.02 Hz, considerably lower than those seen for the NMDA-m component (see preceding text). In this study, we did not investigate the properties of this complex presumably polysynaptic NMDA-l component further by subjecting the motoneuron to voltage clamp. Its polysynaptic linkage makes interpretation of changes after neurotrophin administration equivocal, and so the evaluation of the action of these agents is restricted to their effect on NMDA-m response.
Additional experiments (n = 6) were carried out with
the NMDA antagonist MK-801 in the recording electrode (Arvanov
et al. 2000). In these experiments, the antagonist cocktail
(without APV) was introduced into the bathing solution ~0.5 h before
penetration of the motoneuron. In all six cells recorded with the
electrode containing MK-801, we were able to record the first DR-evoked NMDAR-mediated response that disappeared within 10-20 min (Fig. 2), suggesting that the NMDA receptors
that mediate these responses are located in the motoneuron membrane.
Note, in the control recordings performed with electrodes filled
with 3 M potassium acetate without MK-801, the DR-evoked NMDA
receptor-mediated synaptic responses persisted with no decay for
1 h in the presence of the antagonist cocktail (without APV) in ACSF
(n = 37; see Fig.
3).
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VLF stimulation elicited no NMDAR-mediated responses on motoneurons in 1 WO animals in contrast to such responses evoked in the same motoneuron by DR-stimulation (n = 17; Figs. 1 and 4). Similarly, only very small or in many cases no NMDAR-mediated responses (mean = 0.4 ± 0.1 mV, n = 17, Figs. 1 and 4) were observed at DR-synapses on motoneurons in 2 WO animals. VLF stimulation also failed to induce the NMDA response in 2 WO animals (n = 14, Table 1).
Effects of NT-3 on DR-evoked NMDA-receptor mediated responses in motoneuron of 1 WO rat
NT-3 increased the peak amplitude of the NMDA-m component evoked
by DR stimulation in 1 WO neonates (Fig. 3) by 59 ± 10% (Table 1, n = 9, P = 0.0004). It also reduced
the latency of the polysynaptic NMDA-l component by an amount similar
to that observed in response to increasing the intensity of the
afferent volley to include C fibers (see Fig. 3 for comparison of these
2 maneuvers in the same motoneuron). However, as noted
previously with higher intensity volleys, the decrease in latency of
NMDA-l had no effect on the amplitude or time course of the NMDA-m
component. Thus the increase in amplitude of NMDA-m and the decrease in
latency of NMDA-l are considered to be independent actions of NT-3
(Fig. 3). In agreement with findings in ACSF containing no antagonists
(Arvanov et al. 2000), administration of NT-3 to ACSF
containing non-NMDAR antagonists did not affect the resting membrane
potential (
65.4 ± 1 mV in control vs.
66.1 ± 1 mV in
NT-3, n = 9, P = 0.3) or input
resistance (32.5 ± 2.5 M
in control vs. 30.9 ± 2.9 M
in NT-3, n = 5, P = 0.23) of
motoneurons. In contrast with the persistence (>4 h) of the
enhancement of AMPA/kainate receptor-mediated synaptic responses after
NT-3 removal (Arvanov et al. 2000
), the NMDA
receptor-mediated responses recovered to control levels within 20-30
min of washout of the NT-3 (Fig. 3). In the same motoneurons NGF had no
effect on these synaptic responses (Fig. 3, mean = 3.1 ± 1.04 mV in control vs. 3.32 ± 1 in NGF, n = 5, P = 0.98).
Extending NMDA receptor function
As mentioned in the preceding text, pharmacological blockade of
AMPA/kainate, GABAA, GABAB,
and glycine receptors revealed no NMDAR-mediated responses at VLF
connections on motoneurons in 1 WO animals or on DR-synapses in 2 WO
animals (Fig. 1 and 4). Activation of NMDA receptors depends on
extracellular Mg2+ concentration because of the
voltage-dependent Mg2+ block of these receptors
(Ault et al. 1980; Nowak et al. 1984
). We
investigated whether the absence of NMDAR-mediated synaptic responses
as a function of stimulus source (DR, VLF) or age (1 or 2 WO) was due
to a systematically higher sensitivity of motoneuron NMDA receptors
associated with these inputs to Mg2+. Reducing
the level of Mg2+ in the bathing solution to 1 µM increased the peak amplitude of the existing DR-activated NMDA-m
response in 1 WO animals to 6.2 ± 1.2 mV from 3.2 ± 0.4 mV
measured in control solution with 2 mM Mg2+
(Table 1, n = 5). Notably, NMDAR-mediated responses
elicited in 2 WO animals by DR stimulation (n = 6, Fig.
4, Table 1) and VLF stimulation
(n = 9; Fig. 4, Table 1) became evident, increasing significantly from the negligible values in 2 mM
Mg2+. Input resistance of the cells in low
Mg2+ did not change significantly indicating that
the larger NMDAergic EPSP was not due to changes in passive properties
of the motoneurons.
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NT-3 effects are extended in parallel with NMDA receptor function
In solutions containing low Mg2+ (1 µM) the ability of NT-3 to affect synaptic responses was extended to new inputs (VLF) and at both afferent and descending inputs beyond the usual postnatal 1-wk limit. Under these conditions NT-3 enhanced the amplitude of the "uncovered" NMDAR mediated responses [52.7 ± 10% VLF 1WO (n = 4), 62.4 ± 11.7% DR 2WO (n = 6), 65.5 ± 12.4% VLF 2WO (n = 5)], similar to the increase observed for DR 1WO NMDA responses in low 1 µM Mg2+ (72.7 ± 9.1%, n = 5) or standard 2 mM Mg2+ (59.1 ± 10.2%, n = 9) solutions (Fig. 4 and Table 1). NT-3 also significantly facilitated AMPA/kainate receptor-mediated EPSPs in cells displaying novel DR or VLF activated NMDAR-mediated responses (Fig. 4 and Table 1).
We conclude that a major reason for the loss of NT-3 action in normal ASCF is the reduced activity of the NMDA receptor as the animal matures. Lowering extracellular Mg2+ results in the reappearance of NMDA transmission and this extends the action of NT-3 on the AMPA-kainate response.
Effects of increased levels of Ca2+
Reducing extracellular Mg2+ (1 µM Mg/2.5 mM Ca) increases presynaptic release of glutamate in addition to disinhibiting NMDA receptors. Therefore we have carried out control experiments in elevated Ca2+ (2 mM Mg/4 mM Ca) where transmitter release should be enhanced but without removal of the NMDA receptor block. Increasing extracellular Ca2+ to 4 mM uniformly enhanced the mean amplitude of DR-evoked AMPA/kainate-receptor mediated responses comparable to those induced by lowering Mg2+ levels (Table 1). However, unlike lowering Mg2+ concentration, elevating Ca2+ concentration did not cause the appearance of novel DR-NMDAR-mediated responses in 2WO animals, nor the appearance of VLF- NMDAR-mediated responses in 1 or 2 WO animals. However, DR NMDA receptor-mediated responses were somewhat elevated in 1WO rats (Table 1; Fig. 5) as a consequence of the enhanced release of glutamate and perhaps the enhanced driving force on Ca2+ ions permeating through the NMDA receptor.
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The % increase in NMDAR-(DR 1WO) and AMPA/kainate R-mediated EPSPs induced by NT-3 in elevated Ca2+ was similar to that in standard solution, rather than the much larger increases observed in low Mg2+ solutions (Table 1). Thus the increase in the effect of NT-3 in low Mg2+ solutions appears to be related to unblocking of the NMDA receptor rather than enhanced release of glutamate.
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DISCUSSION |
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Pharmacological isolation of NMDA receptor-mediated transmission
using AMPA/kainate, glycine, GABAA and
GABAB receptor antagonists revealed two NMDA
receptor-mediated synaptic responses (NMDA-m and NMDA-l). MK-801 passed
directly into the motoneuron through the recording microelectrode
blocked the NMDA-m response indicating that the NMDA receptors
responsible for this EPSP are located in the motoneuron. The NMDA-m
response is monosynaptic based on the finding that the stimulus
intensity to elicit the maximum NMDA-m response and its ability to
follow repetitive stimulation were similar to those for the
monosynaptic AMPA/kainate-mediated response. The slightly higher
latency for NMDA-m probably reflects the slower kinetics of NMDA
receptors as compared with AMPA/kainate ones (Abdrachmanova et
al. 2000).
In contrast, the NMDA-l component required a higher stimulus intensity
and a lower rate of stimulation corresponding to values of these
parameters necessary to elicit the long latency ventral root reflex
recorded without pharmacological blockade in neonates (Thompson
et al. 1993). This probably reflects the action of A
fibers
(Thompson et al. 1993
) involving NMDA receptors on
interneurons intercalated between dorsal roots and motoneurons although
its abolition by MK-801 administered in the motoneuron indicates that the last order interneuron makes an NMDAergic synapse. These
pharmacologically isolated NMDA receptor-mediated DR-evoked responses
have been described previously (Pinco and Lev-Tov 1993
;
Ziskind-Conhaim 1990
).
Here we show that NT-3, but not NGF, induces a marked facilitation of
NMDA-mediated synaptic responses in motoneurons. We focus on the
potentiation of NMDA-m because of the evidence that this is generated
monosynaptically (see above) simplifying the interpretation of its
mechanism of action. The simplest, most likely interpretation is that
NT-3 directly potentiates the response of the postsynaptic NMDA
receptor to glutamate since it has been shown to enhance the
depolarizing response of motoneurons to bath-applied NMDA
(Arvanov et al. 2000) and because the effect is
eliminated by MK-801 in the motoneuron. However, it might also be
argued that NT-3's effect on the synaptically evoked NMDA response was to reduce presynaptic inhibition of transmission from spindle afferent
fibers onto the motoneuron. This seems unlikely since MK-801 or BAPTA
delivered intracellularly to the motoneuron in the absence of non NMDA
receptor antagonists prevented NT-3 from enhancing AMPAR-mediated
transmission from the same afferents (Arvanov and Mendell
2000
). If NT-3 was reducing presynaptic inhibition of these
afferents, AMPA/kainate receptor-mediated transmission should have been
potentiated under these conditions. Thus we conclude that NT-3's
action on NMDA-m was localized to the motoneuron membrane. Although it
is simplest to conclude that NT-3 enhancement of later NMDAR-mediated components (NMDA-l) was also exerted via an action on postsynaptic NMDA receptors, the interneurons intercalated in this
pathway makes it impossible to localize the site of NT-3 action definitively.
Interestingly, the effect of NT-3 on NMDA receptor-mediated
transmission was transient since the amplitude of NMDA-responses recovered to control values within about 20 min after washout of NT-3
(Fig. 3). In contrast, NT-3-induced enhancement of the monosynaptic
AMPA/kainate receptor-mediated response, requiring motoneuron NMDA
receptors to trigger it, persisted for at least 4 h after washout
(Arvanov et al. 2000). We hypothesize that NT-3 enhances
the response of postsynaptic NMDA receptors in motoneurons (Arvanov et al. 2000
), which in turn triggers a
long-lasting, possibly permanent increase in the response of
AMPA/kainate receptors to synaptically released glutamate. Although
these findings do not speak to the mechanism of NT-3's interaction
with NMDA receptors, neurotrophins have previously been suggested to
affect NMDA receptors at central synapses either by interaction with
the NR1 (Suen et al. 1997
) and NR2B (Lin et al.
1998
) subunits, or at the glycine site (Jarvis et al.
1997
).
Consistent with previous findings from this laboratory (Arvanov
et al. 2000), NT-3 was unable to facilitate monosynaptic
AMPA/kainate receptor-mediated responses at DR-synapses from 2WO
animals and at VLF-synapses on motoneurons. The latter are more mature,
having previously been shown to form at least 1 wk earlier than DR
synapses (Pinco and Lev-Tov 1994
). Each of these
NT-3-insensitive connections also lacked NMDAR-mediated synaptic
responses (Figs. 1 and 3; Table 1). Postnatal decreases in NMDA
receptor responses have been observed in various structures including
the visual cortex (Carmignoto and Vicini 1992
;
Tsumoto et al. 1987
), the lateral geniculate nucleus
(Ramoa and McCormick 1994
), the thalamocortical synapse
in somatosensory barrel cortex (Crair and Malenka 1995
), and the superior colliculus (Hestrin 1992
). At central
glutamatergic synapses in rat the developmental decrease in the NMDAR
component of the postsynaptic responses during postnatal days 12-18 is
accompanied by a corresponding increase in the AMPAR component
(Bellingham et al. 1998
). The developmental reduction in
the NMDAR-mediated current must involve a modification of the NMDA
receptor complex itself since it is observed in excised membrane
patches in response to glutamate application (Carmignoto and
Vicini 1992
; Hestrin 1992
).
The present study demonstrates that the developmental decrease in the
NMDAR-mediated synaptic responses may result from the increased
Mg2+ block of NMDAR in spinal motoneurons during
the second postnatal week. These results are not inconsistent with
previous reports of enhanced NMDA responsiveness in older
neonates (Palecek et al. 1999) since those
determinations were carried out in 0 Mg2+. The
age-related reduction in NMDAR-mediated transmission demonstrated here
is likely to reflect a developmental switch in subunit composition of
the NMDA receptor (Flint et al. 1997
; Monyer et
al. 1994
). Molecular cloning has identified several cDNA
species encoding NMDA receptor subunits in neonatal rat motoneurons
including NMDAR2A (NR2A), NR2B, and NR2C (Abdrachmanova et al.
2000
). These display different sensitivity to
Mg2+ with NR2A channels being more susceptible to
block than NR2C channels (Monyer et al. 1992
). We
speculate that developmental switch between NR2B or NR2C and NR2A
subunits of NMDAR, as occurs in other regions of the CNS (Adams
et al. 1999
; Pollard et al. 1993
; Zhong
et al. 1996
), may determine the increased susceptibility to
Mg2+ block of NMDAR in the postnatal rat spinal cord.
The timing of this developmental switch in the NMDA receptor's
functional properties suggests that it plays an important role in the
development of the segmental reflex by determining the timing of
NT-3's effect on the AMPA/kainate current. The initial postnatal week
is marked by a large increase in motoneuron size. Despite this the
amplitude of the monosynaptic AMPA/kainate receptor-mediated EPSP
remains roughly constant indicating a gain in synaptic strength (Seebach and Mendell 1996). The possibility that
individual spindle afferent fibers make more profuse connections (i.e.,
more boutons and/or more release sites per bouton) on their target
motoneurons as the spinal cord develops cannot be ruled out at present.
Nonetheless, the long-lasting effect of brief NT-3 treatments on the
monosynaptic EPSP via an LTP-like mechanism during this developmental
stage (Arvanov et al. 2000
) suggests that it might play
a role in the maturation process. Indeed, we found that pulses of
exogenous NT-3 delivered periodically during this time enhanced EPSP
size, and trkC-IgG, which reduces endogenous levels of NT-3
(Ashkenazi et al. 1993
), had a tendency to reduce it
(Seebach et al. 1999
). We do not know at present
precisely where the NT-3 would come from in vivo but it is present in
motoneurons (Buck et al. 2000
; Johnson et al.
2000
) and in glia (Dreyfus et al. 1999
) and
could presumably be released by these cells.
It is somewhat surprising that the effects of NT-3 on the amplitude of
the monosynaptic EPSP were restricted to the motoneuron given the fact
that trkC receptors are known to also be expressed on group Ia fibers
(McMahon et al. 1994). NT-3 is known to have a
presynaptic physiological effect at the neuromuscular junction (Yang et al. 2001
) indicating a physiological action on
motoneurons, although different from the one proposed in the present
studies. It has further been suggested that neurotrophin trk receptors have access to several intracellular signaling systems through which
they can elicit different effects including survival, growth and
synaptic function (Kaplan and Cooper 2001
). NT-3 has
been demonstrated to encourage growth of group Ia fibers after damage (Mendell et al. 1999
; Ramer et al. 2000
)
indicating that the role for NT-3 on afferent fibers may be quite
different from that on motoneurons.
Recent experiments (Kerr et al. 1999) have demonstrated
an increase in c-fos staining in dorsal horn neurons in response to intrathecal NT-3, very similar to that observed in response to intrathecal BDNF, both presumably due to activation of NMDA
receptors in neurons of the dorsal horn. In these same experiments no
effect of NT-3 was found on the late component of the ventral root
reflex. These experiments were carried out in 12-14-day-old rat pups
where the motoneuron NMDA receptors are largely inactive, and we
speculate that this eliminated the ability of the motoneurons to
respond to exogenous NT-3.
The present results suggest that re-establishing functional NMDA
receptors might be required for neurons to regain sensitivity to NT-3.
Presumably, lowering Mg2+ is not a practical way
to accomplish this in vivo. However, if other approaches could be
found, this might turn out to be a useful role for NT-3 in promoting
recovery of the damaged spinal cord that complements its ability to
promote regeneration of descending fibers (McTigue et al.
1998; Schnell et al. 1994
).
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ACKNOWLEDGMENTS |
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We thank the Statistical Consulting Unit in the Department of Applied Mathematics and Statistics at SUNY-Stony Brook for help with the statistical procedures. NT-3 was provided by Regeneron Pharmaceuticals Inc.
Support was provided by National Institute of Neurological Disorders and Stroke (NINDS) 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-39420 to L. M. Mendell.
Victor L. Arvanian published formerly as Viktor L. Arvanov.
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FOOTNOTES |
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Address for reprint requests: L. M. Mendell, Dept. of Neurobiology and Behavior, SUNY at Stony Brook, Life Sciences Building, Rm. 550, Stony Brook, NY 11794-5230 (E-mail: lorne.mendell{at}sunysb.edu).
Received 1 February 2001; accepted in final form 28 March 2001.
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REFERENCES |
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