Department of Neurobiology and Behaviour, State University of New York at Stony Brook, Stony Brook, New York 11794-5230
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ABSTRACT |
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Seebach, Bradley S., Viktor Arvanov, and Lorne M. Mendell. Effects of BDNF and NT-3 on development of Ia/motoneuron functional connectivity in neonatal rats. The effects of neurotrophin administration and neurotrophin removal via administration of tyrosine kinase (trk) immunoadhesins (trk receptor extracellular domains fused with IgG heavy chain) on the development of segmental reflexes were studied in neonatal rats. Brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), trkB-IgG, and trkC-IgG were delivered via subcutaneous injection on days 0, 2, 4, and 6 of postnatal life. Electrophysiological analysis of EPSPs recorded intracellularly in L5 motoneurons in response to stimulation of dorsal root L5 was carried out on postnatal day 8 in the in vitro hemisected spinal cord. Treatment with BDNF resulted in smaller monosynaptic EPSPs with longer latency than those in controls. EPSP amplitude became significantly larger when BDNF was sequestered with trkB-IgG, suggesting that BDNF has a tonic action on the development of this synapse in neonates. Treatment with NT-3 resulted in larger EPSPs, but the decrease noted after administration of trkC-IgG was not significant. Neurotrophins had little effect on the response to high-frequency dorsal root stimulation or on motoneuron properties. Polysynaptic components were exaggerated in BDNF-treated rats and reduced after NT-3 compared with controls. As in control neonates the largest monosynaptic EPSPs in NT-3 and trkB-IgG-treated preparations were observed in motoneurons with relatively large values of rheobase, probably those that are growing the most rapidly. We conclude that supplementary NT-3 and BDNF administered to neonates can influence developing Ia/motoneuron synapses in the spinal cord but with opposite net effects.
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INTRODUCTION |
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The role of neurotrophins during development of
the nervous system has been well established. It is now clear that
these naturally occurring substances play important roles in the
survival and development of sensory and motor neurons (Lindsay
1996). Individual neurotrophins [nerve growth factor (NGF),
brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and
neurotrophin-4/5 (NT-4/5)] exhibit selectivity for different classes
of neurons based on the expression of their corresponding high-affinity
tyrosine kinase (trk) receptor (NGF:trkA; BDNF and NT-4/5: trkB; NT-3:
trkC) (see reviews in Lewin and Barde 1996
;
Mendell 1995
) although the selectivity is not absolute
(Barbacid 1994
) (see DISCUSSION). These trk
receptors are expressed differentially on developing rat sensory and
motoneurons (Mu et al. 1993
; Yan et al.
1993
).
Recently, it has become clear in the rat that the function of
neurotrophins extends beyond the time of birth. NGF plays an important
role in the postnatal differentiation and function of nociceptors (see
Lewin and Mendell 1993 for review). Administration of
exogenous BDNF can improve survival of axotomized motoneurons in
neonatal rats (Vejsada et al. 1995
), and can affect
properties of intact motoneurons in the adult (Gonzalez and
Collins 1997
). NT-4/5 plays an important role in the
determination of the conduction velocity of motor axons, and NT-3
influences the conduction velocity of Ia (spindle) afferent fibers and,
to a lesser extent, motor axons in adult rats (Munson et al.
1997b
). Monosynaptic EPSPs evoked in motoneurons by spindle
afferent fibers normally decline in amplitude when the afferent fibers
are axotomized (reviewed in Titmus and Faber 1990
). This
decline is reversed and in fact EPSPs become larger than normal when
the axotomized nerve is treated with NT-3 (Mendell et al.
1999
; Munson et al. 1997a
). The apparent specificity of these actions (NGF: nociceptors; NT-3: spindle afferents
and motoneurons; BDNF, NT-4/5: motor axons) is in keeping with the
expression of the corresponding trk receptors on subpopulations of
sensory neurons and on motoneurons (see reviews in Phillips and
Armanini 1996
; Sendtner et al. 1996
).
The present work was undertaken to explore the role of neurotrophins in
the development of the Ia afferent/hindlimb motoneuron system and
associated afferent pathways. In a recent paper, the Ia/motoneuron
synapse was shown to undergo substantial developmental changes during
the first eight postnatal days (Seebach and Mendell 1996a). These changes consisted of increases in motoneuron size (decrease in input resistance, increase in rheobase) and increases in
synaptic strength as determined by measurement of EPSP amplitude and
susceptibility to synaptic depression during high-frequency stimulation. Also noted were changes in the polysynaptic potentials recorded from the motoneuron after electrical stimulation of the dorsal
root (DR) at different ages. BDNF, NT-3, or both may play a role in the
specification of motoneuron and afferent-motoneuron synaptic properties
during this period, as trkB receptors (activated by BDNF or NT-4/5) are
expressed by motoneurons (Mu et al. 1993
), and muscle
spindle afferents are known to depend on NT-3 for survival (Ernfors et al. 1994
; Hory-Lee et al.
1993
). The mRNA for each neurotrophin is expressed at maximal
levels in skeletal muscle at birth and then declines (Funakoshi
et al. 1995
; Griesbeck et al. 1995
).
We have examined the effect of neurotrophins on the development of this
system in two ways. First, we provided exogenous neurotrophins by
systemic administration throughout the first neonatal week to see
whether the developmental timetable of this system would be affected.
Second, we depleted the system of endogenous neurotrophins by
administering a trk immunoadhesin (Ashkenazi et al.
1993), which, depending on the particular trk immunoadhesin
that is used (trkA, trkB, or trkC), binds NGF, BDNF and NT-4 or NT-3,
respectively. These immunoadhesins have been shown in vitro
(Shelton et al. 1995
) and in vivo (Munson et al.
1997b
) to block the biological activity of the neurotrophin
corresponding to their trk receptor. The results have been reported in
abstract form (Seebach and Mendell 1996b
) and are
compared with observations of the normal maturation of these synaptic
and motoneuron properties that were published in a previous report
(Seebach and Mendell 1996a
).
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METHODS |
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Pregnant Sprague-Dawley rats were monitored at 12-h intervals to establish date and approximate time of birth.
Administration of neurotrophins
Neonatal rats received subcutaneous injections of BDNF, NT-3,
trkB immunoadhesin, trkC immunoadhesin, or control vehicle every other
day beginning on the day of birth (postnatal day 0, P0). This regime
was employed because daily injections aggravated the skin in the area
of the injection site in some animals. Initial studies were carried out
at 2 µg/g based on previous work with NGF in this laboratory
(Lewin et al. 1993; Ritter and Mendell 1992
). The results at 2 µg/g were very modest and are
not included in the results. The data reported here were largely from
animals treated with 5 µg/g (in the case of BDNF, 25% of the cells
were in animals treated with 10 µg/g with no difference in results from those treated with 5 µg/g). Control animals in the same litter were given like-volume injections of phosphate-buffered saline. Animals
receiving either the trkB or trkC immunoadhesin received 10 µg in 50 µl of phosphate-buffered saline every other day from P0 through P6
(dosage suggested by Dr. R. Lindsay, personal communication). The
saline-injected control preparations were found to have values of
motoneuron and synaptic parameters similar to those obtained previously
in untreated preparations (Seebach and Mendell 1996a
), and so these groups were amalgamated.
At P8, rats were anesthetized and killed by decapitation. The lumbar
region of the spinal cord was removed after ventral laminectomy, hemisected, and placed in a recording chamber perfused with modified Krebs solution as described previously (Seebach and Mendell
1996a; Seebach and Ziskind-Conhaim 1994
;
Ziskind-Conhaim et al. 1993
). Spinal cords were
maintained in recording solution at 35°C for
12 h.
Measuring motoneuron properties and evoked potentials
Suction electrodes were attached to the L5 dorsal
and ventral roots for stimulation. Recordings were made from
antidromically identified motoneurons with stable resting membrane
potentials more negative than 55 mV using intracellular
microelectrodes (80-160 M
) filled with 3 M potassium acetate.
Motoneuron input resistance and rheobase were measured as described
previously (Seebach and Mendell 1996a
).
Measurement of responses evoked by dorsal root stimulation
Ten responses to electrical stimulation of the dorsal root (DR),
presented at a rate of 0.1 Hz and with an intensity of twice the
threshold (2×T) for evoking the monosynaptic potential, were recorded
digitally, and then averaged on-line or off-line to estimate EPSP
amplitude (Seebach and Mendell 1996a). This stimulation
rate did not produce depression of the evoked EPSP at P1-9 (see also Seebach and Ziskind-Conhaim 1994
; Ziskind-Conhaim
1988b
, 1990
). In some cases, this stimulus intensity evoked
action potentials either monosynaptically or polysynaptically, and it
was necessary to use stimulus intensities as low as 1.5T in those
instances. In those cases the maximum amplitude of the monosynaptic and
polysynaptic EPSPs probably were underestimated. This would have had a
disproportionate effect on the findings after treatments that increased
EPSP amplitude. Any lowering of stimulus intensity that caused us to
underestimate the amplitude of the monosynaptic EPSP also would have
caused an underestimate of the magnitude of the polysynaptic EPSP. The fact that we found the largest polysynaptic EPSP components in preparations that received different treatments from those in which
monosynaptic components were largest (see RESULTS) suggests that this factor did not influence the reported results to any great
extent. Latency was measured from the onset of the stimulus artifact
(adjusted to be cathodal stimulation) to the base of the monosynaptic EPSP.
The presence of polysynaptic potentials that often began close to the
peak of the monosynaptic potential complicated the measurement of the
latter. One approach to overcome this difficulty was to inject
depolarizing current through the intracellular electrode to reverse the
depolarizing disynaptic IPSP generated in neonatal motoneurons
(Seebach and Ziskind-Conhaim 1994). The time of
occurrence for the peak monosynaptic response then could be located
during off-line analysis and used to measure the amplitude of the peak response in sweeps recorded at resting membrane potential. In addition,
in many cases the polysynaptic components of the evoked response were
abolished by introduction of a
low-Ca2+/high-Mg2+ saline in the bath, which
eliminates polysynaptic evoked potentials in this preparation
(Pinco and Lev-Tov 1993
; Seebach and Mendell 1996a
; Seebach and Ziskind-Conhaim 1994
) and
allowed the confirmation of the latency of the monosynaptic peak. This
procedure reduced the amplitude of the monosynaptic potential, but did
not affect the latency of its peak (Seebach and Mendell
1996a
).
Measurement of polysynaptic responses
In addition to the monosynaptic response, we also determined an
index of the polysynaptic response to these stimuli. This was
accomplished by measuring the magnitude of the synaptic potential at
10, 20, and 30 ms after the peak of the monosynaptic response. Most of
the depolarization induced at these long latencies was produced by
higher (electrical) threshold afferents (Fig.
1), and furthermore monosynaptic EPSPs
would be expected to decay substantially during this time (as seen at
low-intensity stimulation or in low-Ca2+ solutions
(Seebach and Mendell 1996a)). Thus although we could not
establish that all of this long latency depolarization was due to
polysynaptic components, much of it was. Any change in the magnitude of
the monosynaptic response due to a treatment would necessarily alter
the magnitude of the later components. However, a change in the
amplitude of the monosynaptic component and an opposite change in the
polysynaptic component after a particular treatment could be
interpreted as independent changes in the two components.
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Depression of response to dorsal root stimulation at high frequencies
After 0.1-Hz stimulation, the DR was shocked with a series of
bursts of 10, 33, and 100 Hz stimuli (see Seebach and Mendell 1996a for details) to determine whether manipulation of
neurotrophin levels altered the improvement in the ability of this
synapse to follow high-frequency stimulation noted during the first
postnatal week (Seebach and Mendell 1996a
). The change
in EPSP amplitude during the burst was calculated by comparing the mean
amplitude of the last two EPSPs in the burst
(EPSPn
1 and EPSPn) to
the EPSP measured during low-frequency stimulation (EPSP0.1
Hz). The equation is as follows:
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Chemicals and solutions
All solutions were pH 7.2-7.3. The dissecting solution contained (in mM) 140 NaCl, 5.0 KCl, 1.0 MgCl2, 4.0 CaCl2, 11 dextrose, and 4.3 HEPES. The recording solution contained (in mM) 116 NaCl, 5.4 KCl, 4.0 CaCl2, 11 dextrose, 2.0 MgSO4, 26 NaHCO3, and 1 NaH2PO4. The low-Ca2+/high-Mg2+ recording solution was modified to contain 0.9 mM Ca2+ and 6.0 mM Mg2+.
Preparation of neurotrophin- and trk immunoadhesin-containing salines
Stock solutions of NT-3, BDNF, trkB-IgG, and trkC-IgG were diluted in phosphate-buffered saline and frozen in 400-µl aliquots. Concentrations were adjusted so that individual injections would range from 25 to 75 µl. Neurotrophins and trk immunoadhesins were received courtesy of Regeneron Pharmaceuticals.
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RESULTS |
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Animal health and weight were monitored carefully. Some animals that received the highest doses of BDNF (10 µg/g) were severely under normal weight by P8, and these animals were excluded from physiological study. The skeletal structure of the low-weight neonates appeared to be fairly normal, but their musculature was slight and they had very little body fat. There were no significant differences in animal weight or observed deviation from normal behavior in the animals used for physiology.
EPSP amplitude
The largest EPSPs in BDNF- and trkB-IgG-treated preparations are compared with the largest EPSP in controls in Fig. 2. Similar comparisons for NT-3 and trkC-IgG are displayed in Fig. 3. After supplementary BDNF the largest EPSP was considerably smaller than in normal controls of the same age, whereas after treatment with the immunoadhesion molecule trkB-IgG, the largest EPSP was considerably larger. Opposite changes also were seen after NT-3 and trkC-IgG treatment where NT-3 increased the amplitude of the largest EPSP and trkC- IgG decreased it. Distributions of EPSP amplitude for each treatment are displayed as cumulative sum histograms in Fig. 4. Note the shift to the left of the control histogram for BDNF and trkC-IgG treatments and a shift to the right for NT-3 and trkB-IgG treatments.
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Because EPSP amplitude was measured in controls and after four
different treatments with potentially 10 paired comparisons, the
Student-Newman-Keuls (SNK) test, which adjusted for multiple comparisons, was carried out. Initially a one-way ANOVA on the square
root of amplitude, a transformation required to equalize the variance
of the amplitude distributions within treatment groups, demonstrated a
significant difference in mean amplitude across treatments
[F(4, 105) = 12.2; P = 0.0001]. The post
hoc SNK test (also on amplitude) revealed that EPSP amplitude was
depressed significantly (P < 0.05) by BDNF [2.6 ± 0.4 (SE) mV (n = 20) compared with 5.7 ± 0.7 mV (n = 25) in controls] and enhanced
(P < 0.05) by trkB-IgG [9.5 ± 1.4 mV
(n = 23)]. NT-3 led to a significant increase
(P < 0.05) in EPSP amplitude (10.6 ± 1.3 mV;
n = 23), but the decrease in amplitude after trkC-IgG
(3.8 ± 0.5 mV; n = 19) failed to reach
significance (but see DISCUSSION). Amplitude of EPSPs in
trkC-IgG-treated preparations was not significantly different from
those in BDNF-treated preparations nor was a significant difference
observed between NT-3-treated preparations and trkB-IgG-treated preparations.
EPSP latency
The changes in monosynaptic EPSP amplitude were accompanied by changes in latency (time from the stimulus artifact to the onset of the EPSP) of the dorsal root-evoked EPSPs. A one-way ANOVA indicated that the five means were not equal [F(4,105) = 4.6; P < 0.002]. The post hoc SNK test at the 0.05 level revealed a significant increase in latency of EPSPs in BDNF-treated preparations with respect to the controls (5.2 ± 0.2 ms vs. 4.5 ± 0.3). No significant difference in mean latency was observed on comparing any of the other treatments with controls.
Effects on synaptic depression
Synaptic depression was measured during stimulus trains of 10, 33, and 100 Hz, and the mean values are displayed in Table 1. The mean depression increased progressively as stimulus frequency increased. At each frequency a one-way ANOVA revealed no significant difference in modulation values across treatments [10 Hz: F(4, 68) = 0.64, P > 0.6; 33 Hz: F(4, 67) = 0.09, P > 0.9; 100 Hz: F(4, 65) = 1.87, P > 0.1].
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Effects on polysynaptic potentials
Administration of supplementary BDNF and NT-3 also affected the strength of polysynaptic potentials recorded from the motoneurons after dorsal root stimulation (Fig. 5). To analyze these differences quantitatively, we calculated the difference in amplitude between the potential measured 10 ms after the peak of the monosynaptic response (see METHODS) and the peak of the monosynaptic potential itself (at time 0 in Fig. 5). A one-way ANOVA revealed that there were significant differences in the mean value of this difference over all treatments [F(4, 103) = 10.3; P < 0.001]. The post hoc SNK test revealed a significant increase in the mean difference in amplitude between the polysynaptic and monosynaptic responses after BDNF compared with the increase in controls (P < 0.01) despite the significant decrease in the amplitude of the monosynaptic EPSP. Similarly, a significant decrease in the polysynaptic potential compared with the monosynaptic peak was observed after NT-3 despite a significant increase in the amplitude of the monosynaptic EPSP (P < 0.05). No significant differences in these measures from control values were seen after trkB-IgG or trkC-IgG treatments despite their effects on the mean amplitude of the monosynaptic EPSP (Fig. 5).
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Because the polysynaptic potentials are a composite of excitatory and
inhibitory input, the larger polysynaptic component after BDNF
treatment could arise from a strengthening of excitatory input and/or a
lessening of inhibitory input. Another possibility is a change in the
reversal potential for glycine- and/or
GABAA-receptor-mediated potentials, both of which may be
depolarizing during development (Serafini et al. 1995;
Takahashi et al. 1992
). Indeed, in four of four cases
with chronic BDNF-treated motoneurons, we could not reverse the
composite polysynaptic depolarizing potentials when the cell was
depolarized in the range from
55 to
50 mV, a level at which these
potentials are normally reversed (Pinco and Lev-Tov
1993
; Seebach and Mendell 1996a
; Seebach
and Ziskind-Conhaim 1994
).
Effects on motoneuron properties
EPSP amplitude can be affected strongly by the electrical properties of the postsynaptic cell. To determine whether the changes in EPSP amplitude were in part due to differences in motoneuron development induced by the chronic treatments with BDNF or NT-3 or with their associated trk immunoadhesin, motoneuron input resistance and rheobase were measured. The mean values are presented in Table 2. A one-way ANOVA revealed significant differences in mean rheobase across treatments [F(4, 115) = 4.74; P = 0.001]; the post hoc SNK procedure revealed significant differences from controls only after trkB-IgG (Table 2). No significant differences in input resistance across treatments were observed [F(4, 121) = 2.40; P > 0.05]. We conclude that the effect of neurotrophins on motoneuron properties is much more modest than their effect on EPSP amplitude in the developing neonatal rat.
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Correlations between amplitude of the monosynaptic EPSP and motoneuron rheobase
Motoneurons in untreated P8 neonates exhibit a positive
correlation between amplitude and rheobase (Seebach and Mendell
1996a) that is reversed from what is observed in the adult rat
(Peshori et al. 1998
) or in cats (Collins et al.
1988
). We investigated whether the treatments that increased
EPSP amplitude in these preparations preserved this relationship. To
increase power, we combined the data from trkB-IgG-and NT-3-treated
preparations and from the BDNF- and trkC-IgG-treated preparations
because their mean amplitude did not differ and plotted EPSP amplitude
versus motoneuron rheobase (Fig. 6). In
carrying out statistical analysis, we transformed amplitude into
amplitude as we had in the analysis of amplitude differences among
these preparations. Using a multiple regression analysis, we found that
the slope of the linear regression for the combined NT-3, trkB-IgG data
was 0.28 ± 0.10 (SE), and for the combined BDNF, trkC-IgG data it
was 0.02 ± 0.07. The difference between these slopes barely
lacked statistical significance (P = 0.059) although
with this value of probability, it could not be concluded that the
slopes were equal. Subsequent linear regression of EPSP amplitude on
motoneuron rheobase for the combined NT-3, trkB-IgG data gave a
correlation coefficient r = 0.40, which is highly
significant (P = 0.01), indicating preservation of the positive slope of the amplitude versus rheobase relationship after neurotrophin treatments that increased EPSP amplitude. A similar analysis for data from BDNF- and trkC-IgG- treated preparations revealed no significant relationship between EPSP amplitude and rheobase (r = 0.05; P = 0.79),
indicating that the positive slope of the amplitude versus rheobase
relationship is not preserved if EPSP amplitude is diminished.
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DISCUSSION |
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BDNF and NT-3 mRNAs are expressed in skeletal muscle at the time
of birth, but their levels fall during or soon after the first
postnatal week (Funakoshi et al. 1995; Griesbeck
et al. 1995
). Assuming that levels of NT-3 and BDNF protein
also decline during this period, the present results indicate that
maintenance of higher than normal levels by provision of supplementary
neurotrophins alters the amplitude of the EPSP measured at postnatal
day 8. Surplus NT-3 resulted in elevated monosynaptic EPSP amplitude by
day 8, whereas BDNF administered in the same dosage and over the same
period diminished the mean amplitude of the monosynaptic EPSP.
We also investigated the effects of trkB and trkC immunoadhesins, which
bind any available BDNF or NT-3 (Ashkenazi et al. 1993),
in these preparations. Recent studies indicate that they act in vivo in
the rat (Munson et al. 1997b
) The effect of the trkB
immunoadhesin on synaptic function was highly specific in the sense
that its action was opposite to that of BDNF. This suggests that BDNF
acts tonically in the postnatal rat to reduce EPSP amplitude (see
discussion of potential mechanisms in the following text). TrkC-IgG
reduced the mean amplitude of the monosynaptic EPSP (Figs. 2 and 4),
which is opposite to the action of NT-3. Although the nominal
t-test comparing these two means (in controls and in
trkC-IgG treated) was significant (P < 0.05) and the
variances of their distributions were significantly different
(F test: P < 0.03), the necessity to
correct for the multiple treatments brought the result for mean
amplitude to just below statistical significance.
The weaker effect of trkC-IgG compared with that of trkB-IgG has been
noted before in vivo. In the adult rat, the effects of trkC-IgG on
peripheral axon conduction velocity were more modest than those of
trkB-IgG and required a higher dosage (Munson et al.
1997b). Thus the nonsignificant result on EPSP amplitude with trkC-IgG could be explained by the use of too low a dose and should be
reexamined with higher concentrations.
We assume IgG is stable under these conditions because of previous
findings that IgG injected systemically at days 2-4 still can be
detected immunologically in skin and in spinal cord at day 14 (Tonra and Mendell 1997). We also assume that trk
receptors are stable because trkB-IgG elicited a significant effect.
Because the effects of these 2 trk immunoadhesins are different with
respect to synaptic transmission, they are not due to a nonspecific
effect of the IgG heavy chain, which is known to penetrate the
blood-brain barrier in the neonatal period (Tonra and Mendell
1997
).
To interpret these experiments, it is necessary to consider that they
are carried out against the backdrop of normal development. Mean
amplitude of the monosynaptic EPSP does not change significantly during
this period despite substantial growth of the target motoneurons (Seebach and Mendell 1996a). This indicates that the
synaptic current produced by the group Ia afferents is increasing
throughout this period (to generate EPSPs of the same amplitude). The
changes in motoneuron input resistance produced by the various
neurotrophin treatments were not significant. Thus they could not
account for the 86% increase in the monosynaptic EPSP amplitude after
NT-3 treatment, the 67% increase after trkB-IgG, and/or a reduction to
45% of the normal value with BDNF treatment, all of which were significant. We conclude that change in motoneuron input resistance was
not the primary cause of the observed changes in monosynaptic EPSP
amplitude. Because neither trkB-IgG nor trkC-IgG prevented the decrease
in motoneuron input resistance normally observed between postnatal days
2 (41 M
) and 8 (22 M
) (Seebach and Mendell 1996a
),
we also can conclude that at this developmental stage BDNF and NT-3 are
not crucial determinants of factors determining input resistance.
If postsynaptic changes are not sufficient to account for the changes
in EPSP amplitude, we must consider either changes in the number of
afferents or alterations in the synaptic region itself, either
presynaptic or postsynaptic. Some evidence for presynaptic changes
comes from examination of the latency of the monosynaptic EPSPs. During
normal development, EPSP latency after DR stimulation decreases from a
mean of 8.5 ms (P2) to a mean of 4.7 ms (P8) (Seebach and
Mendell 1996a) as the afferent fibers grow and become
myelinated (Friede and Samorajski 1968
). In the present
experiments, we found that BDNF treatment that reduced EPSP amplitude
increased EPSP latency substantially compared with untreated controls.
In contrast, both treatments that increased EPSP amplitude (NT-3;
trkB-IgG) reduced EPSP latency, although not significantly.
Changes in latency could include changes in afferent conduction time
and/or synaptic delay. It was difficult to distinguish between these
two sources of latency because the short conduction distance from
dorsal root to spinal cord in the neonates prevented us from measuring
axonal conduction velocity accurately. Because EPSP latency was
increased by BDNF treatment, a decrease in conduction velocity of large
muscle afferents might be anticipated in BDNF-treated preparations.
Unfortunately, no direct measurements of the effects of BDNF on
conduction velocity of sensory afferents appear to be available.
However, in the transected peripheral nerve model the other trkB
agonist, NT-4/5, does not affect conduction velocity of axotomized
sensory afferents in the rat although it does prevent the decline in
conduction velocity of transected motor axons (Munson et al.
1997b). Sequestration of trkB agonists with trkB-IgG has no
effect on sensory fiber conduction velocity (Munson et al. 1997b
). In the cat, NT-4 has little effect on the decrease in conduction velocity of axotomized afferents or axotomized motor axons
(Mendell et al. 1999
). Thus it remains to be
demonstrated whether the present findings can be explained by an effect
of BDNF on the development of sensory axon conduction velocity.
When NT-3 is provided to the axotomized MG nerve in adult cats or rats,
the anticipated decrease in axonal conduction velocity of large (group
Ia) afferents projecting to motoneurons is prevented (Mendell et
al. 1999; Munson et al. 1997b
). NT-3 also
reverses the decline in conduction velocity in diabetic rats
(Tomlinson et al. 1996
). No significant change in
latency after NT-3 treatment was observed under the conditions of these
experiments. Whether this is the result of a dosage problem or the
inability of NT-3 to affect the conduction velocity of
intact afferent fibers is not presently known.
In the adult cat (Collins et al. 1988) or rat
(Peshori et al. 1998
), there is a negative correlation
between EPSP amplitude and motoneuron rheobase, such that motoneurons
with large values of rheobase generate small EPSPs on the average,
whereas those with small values of rheobase generate large EPSPs. This
was not the case in neonatal rats where the correlation between these variables was positive (Seebach and Mendell 1996a
).
Because rheobase is increasing during development (Seebach and
Mendell 1996a
), it was suggested that during the first
postnatal week, motoneurons growing the fastest, i.e., with the largest
values of rheobase, were the ones that had the most highly developed
synapses and thus produced the largest EPSPs. Our results indicate that
this relationship is preserved if EPSP amplitude is increased but not if it decreases. These data do not presently allow an unequivocal determination of whether the neurotrophin-induced changes in amplitude are confined to motoneurons of a particular range of rheobase. We
cannot, of course, be certain that the mechanisms leading to increased
EPSP amplitude after NT-3 or trkB-IgG treatment are the same despite
the similarity in the outcome.
It is difficult to be more precise about the mechanisms by which
neurotrophins increase or decrease EPSP amplitude. Previous findings
indicate a developmental decrease in the susceptibility of these
synapses to depression during high-frequency stimulation (Seebach and Mendell 1996a). This is probably due at
least in part to the maturation of some aspect of the synapse that
gains the ability to recover more completely from the effects of a
previous stimulus despite releasing more transmitter. However,
myelination of afferent fibers taking place during this period also
could contribute by making the impulses more able to be conducted into the fine terminals during high-frequency stimulation. None of the
neurotrophin manipulations had a consistent effect on synaptic depression at any frequency of stimulation. This suggests that changes
in probability of transmitter release or branch blocking in the
terminals, both of which would be expected to change the level of
depression, are not responsible for the changes in amplitude observed
here. This leaves open the possibility that the changes in EPSP
amplitude are the result of altered numbers of transmitter release
sites or altered numbers and/or sensitivity of the postsynaptic receptors. Another possibility is a change in the number of spindle afferents, which has been shown to increase in genetically altered mice
with muscles that overexpress NT-3 from E11.5 through the first 2 wk of
post natal life (Wright et al. 1997
). The number of
spindles also is elevated in rats treated with NGF before postnatal day
4 (Miyata et al. 1986
). This would indicate a potential
role for trkA receptors in this process. Because NT-3 in high doses can
activate the trkA receptor (Barbacid 1994
), this
possibility deserves serious consideration.
The effects of chronic NT-3 administration in increasing the composite
EPSP at the Ia-motoneuron synapse is consistent with recent findings in
the cat spinal cord where application of NT-3 directly to the
axotomized medial gastrocnemius (MG) nerve was found to increase the
size of the EPSPs made by the MG afferents on intact LGS motoneurons
(Mendell et al. 1999; Munson et al. 1997a
). However, the effects of BDNF obtained here are
different from those that have been noted in other systems. In the
hippocampus (Figurov et al. 1996
; Levine et al.
1995
; Patterson et al. 1996
) or at the
neuromuscular junction (Stoop and Poo 1996
), BDNF acts acutely to increase the probability of transmitter release, whereas in
the present work with chronic application, the effect was apparently to
decrease synaptic efficacy. The present result could be an effect on
the timetable of maturation of these synapses during the neonatal
period rather than a direct synaptic effect on transmitter release.
However, preliminary data indicate that BDNF applied directly to the
spinal cord also can depress the monosynaptic EPSP acutely but without
the increased response latency (Seebach and Mendell
1996b
; unpublished data).
An important caveat in evaluating the differences between this work and
previous studies of BDNF action is our inability to confirm that the
effects observed after BDNF administration were direct, i.e., not via
some other factor induced or upregulated by BDNF. We also cannot be
certain that the effects were confined to the Ia-motoneuron synapse.
For example, it is likely that trkB receptors are expressed on the
presynaptic terminals of at least some of the large diameter afferents
(McMahon et al. 1994). If so, BDNF might depolarize
these terminals (based on the ability of BDNF to increase probability
of release at synapses in the hippocampus or the neuromuscular
junction; see preceding text). Thus we can speculate that if
periodic administration of supplementary BDNF results in tonic
activation of the trkB receptor, it would cause primary afferent
depolarization and an indirect decrease in synaptic efficacy of the
Ia/motoneuron synapse due to presynaptic inhibition. In addition,
because BDNF in larger doses diminished muscle mass (see
RESULTS), it is possible that there were subtle retrograde
trophic changes in developing the Ia/motoneuron synapse even in cases
where animal weight (and muscle mass) was normal (Mendell et al.
1994
).
BDNF treatment increased the amplitude of the polysynaptic reflex, which was strikingly different from its effect on the monosynaptic reflex. It is important to note that the polysynaptic reflex differs from the monosynaptic reflex in being produced to a large extent by smaller fibers with a higher electrical threshold than those producing the monosynaptic EPSP (Fig. 1). Thus BDNF may exert different developmental or physiological actions on small diameter afferents, many of which are likely to be cutaneous, than on large muscle afferent fibers. NT-3 also elicited different effects on monosynaptic and polysynaptic potentials, suggesting different actions on large and small afferent fibers. A possible complication in this interpretation is a change in the polysynaptic component's reversal potential, which after chronic BDNF was at a more depolarized level than in controls (see RESULTS). This would make the depolarizing potential appear larger.
Because the neurotrophins and their antagonists were applied
systemically, it is impossible to determine their site of action. The
blood brain barrier is not firmly established in these neonatal rats
(Tonra and Mendell 1997), and so neurotrophins could act directly on cells in the spinal cord. However, the receptors for these
neurotrophins exist on both sensory and motor neurons (McMahon et al. 1994
), and it is possible that BDNF and NT-3 are
internalized via reaction with their trk receptor in the periphery (or
anywhere along the axon) and carried centrally to influence the cell's metabolism and subsequent growth. The foregoing statement assumes that
the interactions are solely on motor neurons and sensory neurons.
However, we cannot disregard the possibility that the interactions take
place on other neurons and/or glia in the spinal cord.
Although the mechanisms mediating these actions remains obscure for the
present, it is clear that the net effects of BDNF and NT-3
administration are relatively straightforward. Whether these effects
are restricted to the developmental period is not known. Some recent
experiments suggest that BDNF may have different effects on motoneuron
properties in the adult rat than those noted here (Gonzalez and
Collins 1997), but the effects on synaptic potentials are not
known in the adult. From a functional perspective, these results may
have important implications for development of segmental reflexes and
may provide insights on, or even tools for, how these reflexes might be
manipulated in the adult, for example to diminish the monosynaptic
reflex during spasticity.
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ACKNOWLEDGMENTS |
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Dr. Nancy Mendell of the Department of Applied Mathematics and Statistics at SUNY-Stony Brook provided help with the statistical analysis. We thank Regeneron Pharmaceuticals, for the generous gifts of BDNF, NT-3, trkB-IgG and trkC-IgG.
This work was supported by National Institute of Neurological Disorders and Stroke Grants RO1 NS-16996 (Javits Neuroscience Award to L. M. Mendell) with additional support from RO1 NS-32264 and PO1 NS-14899.
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FOOTNOTES |
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Address reprint requests to L. M. Mendell.
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 6 July 1998; accepted in final form 22 January 1999.
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REFERENCES |
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