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INTRODUCTION |
It is well known that increased or decreased activity at CNS synapses can result in both long- and short-term changes in synaptic strength. However, our understanding of the precise mechanisms responsible for such modulation in the intact organism is incomplete (Hawkins et al. 1993
). The sodium channel blocker tetrodotoxin (TTX) provides a useful tool for in vivo study of activity-related modulation of synaptic strength because it can be used to reversibly eliminate nerve impulse traffic, without nerve damage or interruption of axonal transport (Lavoie et al. 1976
).
Previous work has shown that TTX blockade of action potential conduction in muscle nerves increases synaptic efficacy centrally at the monosynaptic connection between muscle spindle (Ia) afferents and motoneurons (Gallego et al. 1979
; Manabe et al. 1989
; Webb and Cope 1992
). The observed enlargement of Ia excitatory postsynaptic potentials (EPSPs) was not associated with detectable changes in motoneuron electrical properties and does not require conduction blockade of motoneurons generating the enlarged EPSPs. This suggests that the enhanced efficacy is not attributable to postsynaptic factors, but the precise locus of change is yet to be identified. The possible explanations for the TTX-induced increase in Ia synaptic efficacy include 1) a change in postsynaptic responsivity, 2) an increased amount of transmitter released from single synapses, 3) an increase in the number of synapses subsequent to central sprouting, and 4) changes in circuitry extrinisic to the monosynaptic pathway. In the present work we investigate the latter possibility.
We have used low-frequency depression (LFD) of the extracellularly recorded monosynaptic reflex (MSR) (Lloyd and Wilson 1957
) as an indirect measure of presynaptic inhibition. On the basis that LFD is a group I phenomenon of presynaptic origin (Lloyd and Wilson 1957
) and can be reduced by a
-aminobutyric acid-B (GABAB) antagonist (Thompson et al. 1996
), we hypothesized that, if a decrease or loss of presynaptic inhibition contributes to the previously reported increase in Ia EPSP amplitude observed after nerve conduction blockade in the rat (Manabe et al. 1989
), then this loss would be expected to cause a decrease in the extent of LFD of the MSR.
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METHODS |
Male Sprague-Dawley rats (250-350 g) (Harlan, Birmingham, AL) were assigned to control, 3-day, or 10-day treatment groups (n = 10, 7, and 4, respectively). All rats were housed in similar conditions with a 12:12-h dark:light cycle and were allowed food and water ad libitum.
Nerve conduction block
Rats assigned to TTX treatment groups underwent surgery, as previously described (Gardiner et al. 1992
), for implantation of an osmotic pump (Alza, Palo Alto, CA) attached to a silastic tubing-cuff assembly. The pump delivered (at 0.5 µl/h) to the tibial branch of the left sciatic nerve, for a period of either 3 or 10 days, a solution containing 250 µg/ml TTX (Sigma, St. Louis, MO), 200 U/ml pencillin (Apothecon, Princeton, NJ) and 200µg/ml gentamicin sulfate (Elkin-Sinn, Cherry Hill, NJ). Four rats from the control group had pumps implanted that contained the solution without TTX. Surgery was carried out under aseptic conditions after the rat had been anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) given intraperitoneally.
Successful and selective tibial nerve blockade was determined by the absence of left ankle extension and presence of toe spread (mediated by the unblocked common peroneal nerve) when the animal was lifted by the tail. In all cases blockade was evident within 12-24 h. Animals with sham cuffs (containing mixture minus the TTX) showed normal ankle extension.
Acute surgical preparation
The day of the experiment, rats were initially anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) given intraperitoneally, and then maintained by chronic infusion (0.8-1.6 ml/h) of this mixture diluted in Ringer solution/dextrose (1.5:10 ml or 9.6/1.1 mg/ml, ketamine/xylazine, respectively). Arterial blood pressure and end-tidal CO2 were monitored and maintained at
65 mmHg and
4%, respectively. Finally, body temperature was monitored and maintained at37 ± 1°C.
Surgery was performed on both hindlimbs to expose the sciatic nerve. The nerves were freed from their connective tissue and the common peroneal and sural nerve branches were cut and separated from the tibial nerves. The tibial nerves were crushed at their most distal points before data collection. Further surgery exposed the vertebrae from approximately S1 to T10 and a laminectomy was performed to expose the spinal cord from approximately L6 to T12. The dura was cut and the L3, L4, and L5 ventral roots were carefully located on both sides and then cut at their most accessible caudal point.
Data collection setup and protocol
On completion of surgery the animal was moved to a stereotaxic frame for data collection. Bipolar stimulating electrodes were placed on the right and left (proximal to cuff site) tibial nerves, and a monopolar ball electrode was placed in contact with dorsal roots to record dorsal root volleys. Finally, a bipolar electrode was placed on the L5 ventral root to record the MSR. Once all electrodes were in place, group I threshold was determined with the use of the dorsal root volley and then the stimulation intensity was set at 2-2.5 times threshold for all experiments (pulse duration 0.5 ms).
In 16 of 21 experiments bilateral data were collected (n = 6, 6, and 4 for control, 3-day, and 10-day TTX, respectively). The experimental protocol consisted of recording one trial of six responses at each test frequency interspersed by one trial of six responses at the control frequency (0.1 Hz) as follows
There was a minimum of 10 s between trials. Data were amplified (×1,000) and filtered (DC, 10 kHz) and then digitized and collected at a sampling rate of 22 kHz with the use of PC-based software (Cambridge Electronics Design, Cambridge, UK).
Data analysis
Figure 1 shows control data recorded at the control frequency of 0.1 Hz and a test frequency of 1.0 Hz. To compare the extent of depression both within (right-left comparisons) and across animals, we expressed the mean MSR amplitude at a given test frequency (0.2-20 Hz) as a percentage of the mean MSR amplitude of the control frequency series that immediately preceded it. The mean amplitude of the control frequency was calculated with the use of all six traces, whereas the mean amplitude of the test frequency used only the last four traces of the series so as not to underestimate the extent of the depression (Fig. 1).

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| FIG. 1.
Monosynaptic reflex recorded from the L5 ventral root evoked by stimulation of the tibial nerve (2 times group I threshold) at 2 different frequencies (0.1 and 1.0 Hz) in a control rat. Depression was calculated as a percentage of control amplitude by dividing the mean amplitude of the test frequency monosynaptic reflex (MSR; excluding the 1st 2 pulses) by the mean amplitude of the control frequency MSR and multiplying by 100.
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In cases in which bilateral data were collected, the extent of depression at each frequency was also expressed as a right/left ratio for comparison purposes. A value of 1 indicated that the depression observed on the right and left sides within a given animal was identical.
Three of the seven test frequencies, representing the range of frequencies tested (0.4, 1.0, and 10.0 Hz), were chosen for statistical analysis with the use of the nonparametric Kruskall-Wallis analysis of variance. A probability value of <0.05 was selected for all comparisons.
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RESULTS |
A total of 21 experiments was performed and bilateral data were collected in 16. The bilateral control data include those from four rats (2 3-day, 2 10-day) that had cuffs placed on the left nerve but were attached to pumps that did not contain TTX. No functional deficit was apparent in these animals and stimulation of the tibial nerve proximal to the cuff, before acute experiments, evoked strong muscle contraction.
Our initial analysis examined only data from animals in which bilateral measures were made. In Fig. 2, the extent of depression measured on the left side (treated) is expressed as a ratio of that measured on the right side (untreated) for each animal. Our hypothesis, that the extent of LFD (% amplitude at 0.1 Hz) would be decreased by TTX-induced inactivity, would predict a decreased right/left ratio (<1) in the experimental groups. However, no systematic decrease was evident, and there were no significant differences in right/left ratios between groups for any of the frequencies (P values
0.3).

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| FIG. 2.
Bilateral data from control, 3-day, and 10-day tetrodotoxin (TTX)-treated rats (n = 6, 6, and 4, rats, respectively). Each point represents the right/left ratio of the normalized MSR amplitude (% of amplitude recorded at 0.1 Hz) from a single animal (N. B.: some points overlap). Small arrows: indicate mean values. A ratio equal to 1 indicates that the extent of the depression on the right side was equal to that observed on the left side.
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Analysis of the bilateral data confirmed only that there was no systematic change in the relative extent (right:left) of LFD observed in the TTX-treated rats. However, it did not exclude the possibility that TTX-induced inactivity resulted in some compensatory change on the untreated side, such that the right/left ratio remained constant. To address this we examined the average extent of the depression for both treated and untreated sides in the three groups. The extent of depression observed at a given frequency was remarkably similar to that found in previous work in the rat (Kaizawa and Takahashi 1970
; Thompson et al. 1992
), with the amplitude of the MSR being reduced, on average, to ~50% at a stimulation frequency of 1 Hz and then decreasing progressively to 10-15% of control amplitude at 10 Hz (Fig. 3). The depression measured in control and TTX-treated animals, was not different across groups for either the left (treated) or right (untreated) sides or between left and right sides within groups for any frequency (P
0.2 for all comparisons). These comparisons indicate that TTX-induced nerve blockade did not alter LFD on the treated side, nor did it result in any apparent compensatory change on the untreated side.

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| FIG. 3.
Comparison of depression (mean ± SE) of MSR recorded from the left (treated) and right sides of control and experimental groups (3- and 10-day conduction blockade) at stimulation frequencies of 0.4, 1.0, and 10.0 Hz. Extent of depression was similar at all frequencies. All data are shown (control: n = 10, 6 bilateral; 3-day TTX: n = 7, 6 bilateral; 10-day TTX: n = 4, all bilateral). Control data for the left side includes 4 animals that had pumps and cuffs implanted that did not contain TTX.
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One final aspect of these data was also considered. That is, we examined the interpulse decrease in MSR amplitude to determine whether it was expressed in a similar manner in the three experimental groups. As can be seen in Fig. 4, the interpulse pattern of decrease of MSR amplitude was very similar between control and TTX-treated sides for all frequencies (and for right and left sides within animals, not shown). Statistical comparison showed that the extent of depression was not significantly different among the second to the sixth pulses (P
0.3 for all comparisons). These results demonstrate that the interpulse development of LFD was unaffected by nerve conduction blockade.

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| FIG. 4.
Average normalized amplitude of the MSR for each pulse in the train at 0.4, 1.0, and 10 Hz for the left side only of control and 3-day and 10-day TTX-treated rats. Dashed line: 50% of control amplitude. Note that in all cases the majority of the depression is evident by the 3rd pulse. Comparison of mean depression for each pulse (2nd-6th) showed no differences between groups.
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In addition, in several experiments, the effect of stimulation intensity was tested by comparing the extent of LFD when the MSR amplitude was near maximal (2-2.5 times group I threshold) and when stimulation intensity was lowered to reduce the MSR amplitude by 50%. Results agreed with previous work and confirmed that the depression is related to group I afferents (Kaizawa and Takahashi 1970
; Lloyd and Wilson 1957
; Thompson et al. 1992
), and that the extent of depression in the rat is independent of stimulation intensity (Kaizawa and Takahashi 1970
; Thompson et al. 1992
).
A final experiment was performed to determine the effect of the anesthetic on LFD. MSR data were recorded under normal anesthetized conditions and then over a period of >4 h after the animal had been decerebrated and anesthetic discontinued. The traces in Fig. 5A show data recorded before and after decerebration at the control frequency (0.1 Hz) and demonstrate that absolute amplitude was unchanged by decerebration and discontinuation of the anesthetic. The traces in Fig. 5B were recorded at 1.0 Hz and show the typical pattern of depression before and after decerebration. However, it is also evident that the frequency-related depression is moderately less following decerebration and removal of anesthetic, perhaps due to altered excitability as suggested by the appearance of polysynaptic activity (Fig. 5, A and B). Nonetheless, the extent of this effect makes it unlikely that variations in the level of anesthetic, which was infused at a constant rate throughout data collection, would have significantly affected the present findings.

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| FIG. 5.
A: MSR recorded at 0.1 Hz before, 30 min after, and 4 h after decerebration ( , - - -, and - - -, respectively). B: as in A, except stimulation was at 1 Hz. Depression was present but slightly less after decerebration and discontinuation of anesthetic.
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DISCUSSION |
The primary finding of this investigation is that completely silencing afferent input from the tibial nerve to the spinal cord, for up to 10 days, does not alter the extent of LFD of the MSR. This was shown to be true both in comparisons with untreated control animals and when comparisons were made between the treated and untreated sides of the same animal. On the basis that LFD is an expression of presynaptic inhibition of group I afferents (Curtis and Eccles 1960
; Kaizawa and Takahashi 1970
; Lloyd 1957
; Lloyd and Wilson 1957
), we also conclude that the previously reported increase in the Ia EPSP amplitude after similar periods of afferent blockade (Manabe et al. 1989
) is unlikely to be the result of the removal or diminution of presynaptic inhibition onto group I afferents.
The absence of change in LFD subsequent to TTX treatment is clear from the present results, and regardless of the specific pathways responsible for this depression, it is striking that eliminating tibial afferent activity for up to 10 days in no way altered its expression. However, the more critical conclusion for our ongoing investigations, that presynaptic inhibition is unaffected by TTX-induced inactivity, is based on the premise that LFD is an expression of presynaptic inhibition of group I afferents.
There is considerable accumulated evidence associating LFD with presynaptic inhibition. LFD of the MSR was studied by Lloyd and Wilson (1957)
in the cat. They concluded that LFD (0.05-10 Hz) resided predominantly in Ia afferents and was a presynaptic phenomenon. The latter conclusion was based on the impressive degree of concurrence between the course of LFD and that of other phenomena previously demonstrated to be of presynaptic origin. In particular, Lloyd and Wilson showed that the relationship between stimulus interval and LFD followed a course identical to that of the fourth component (D.R.IV.R) of the dorsal root potential (DRP) (Lloyd and Wilson 1957
, their Fig. 11). The DRP (Barron and Matthews 1938
) represents the prolonged depolarization at terminal arborizations of primary afferent fibers and was proposed early on to be related to inhibition in the spinal cord (Gasser and Graham 1933
). Eccles et al. (1961)
confirmed this by showing a decrease in EPSP amplitude that accompanied group I afferent depolarization (PAD) and followed closely the time course of the DRP. This early work led to later investigations showing that PAD is mediated by GABAergic axoaxonic connections between interneurons and Ia afferents and can be reduced by antagonists to both GABAA and GABAB receptor types. It is now well accepted that the effectiveness of Ia-motoneuron connections can be modulated presynaptically by last-order GABAergic interneurons (reviewed in Levy 1977
; Rudomin 1990
).
Thus the early demonstration by Lloyd and Wilson (1957)
that LFD of the MSR, measured at group I strength, parallels the time course of the DRP provides, in light of subsequent evidence, strong support for the use of LFD as an indirect measure of presynaptic inhibition. The contention that LFD is, at least in part, an expression of presynaptic inhibition gains further support from the recent demonstration in the rat that the extent of LFD can be reduced by intraspinal administration of a GABAB-specific antagonist (Thompson et al. 1996
).
Therefore we interpret the lack of a detectable change in LFD as an absence of a dramatic change in presynaptic inhibition. We recognize the possibility that additional factors such as transmitter depletion or receptor desensitization might also contribute to LFD. However, to accomodate our finding that TTX-induced blockade caused no change in LFD would require either that the portion of LFD associated with presynaptic inhibition be unchanged or that other factors that contribute to LFD be altered in an exactly equal and opposite fashion by TTX treatment. We consider the latter possibility unlikely and thus the present study gives us no encouragement to extend investigation to more direct measurements of presynaptic inhibition, such as excitability testing of primary afferents (Rudomin 1990
). Nonetheless, the present results alone do not rule out subtle changes in presynaptic inhibition following afferent inactivity, or changes that might occur with longer periods of inactivity (Thompson et al. 1992
).
The conclusion that presynaptic inhibitory input onto group I afferents is unchanged with TTX-induced disuse only provides evidence that the synaptic effectiveness of the last-order interneuron that makes the axoaxonic connection with the afferent was unaffected by the afferent blockade. The presynaptic inhibitory circuitry responsible for presynaptic inhibition involves at least two interneurons (Rudomin 1990
) that also receive numerous other inputs, both descending and segmental. Thus it is possible that a TTX-induced increase in efficacy of the group I input onto the first-order interneuron is compensated for within the circuit, and/or by other inputs, such that the output of the last-order interneuron is unaltered. An intriguing alternative explanation would be that changes in synaptic efficacy can occur selectively at different connections made by the same afferent (e.g., Ia-motoneuron vs. Ia-interneuron connection). This idea requires further investigation, but receives some support from the finding that the response to high-frequency stimulation of a single afferent onto different cells of the same type (i.e., motoneurons) can be very different (Collins et al. 1986
), suggesting that the postsynaptic cell plays a role in determining the characteristics of its afferent connections. It seems plausible, then, that a similar degree of flexibility may exist among connections onto different cell types.