Neuroscience Letters 304 (2001) 4952

www.elsevier.com/locate/neulet

Temporal facilitation of the exor reex induced by C-ber activity:

comparison between adult and aged rats
K. Kanda a,*, H. Sato b, T. Kemuriyama a, K. Iwata c
a

Department of Central Nervous System, Tokyo Metropolitan Institute of Gerontology, 352, Sakaecho, Itabashi-Ku, Tokyo
1730015, Japan
b
Department of Physiology, Tokyo Medical University, Tokyo 1608402, Japan
c
Department of Oral Physiology, School of Dentistry, University of Osaka, Osaka 5650871, Japan
Received 20 January 2001; received in revised form 13 March 2001; accepted 15 March 2001

Abstract
We investigated the wind-up phenomenon of the exor reex in adult and aged rats. The sural nerve was stimulated at
C-ber strength and reex activity was recorded from the semitendinosus muscle. The wind-up rate, the increment rate
of the C-ber response (i.e. activity from 100 to 600 ms after stimulation) by successive stimuli (ve train pulses), was
decreased exponentially with increasing stimulus intervals from 3 to 20 s. The time constant of the decay for the aged
rats was 9.2 ^ 3.2 s (mean ^ SD), which was signicantly longer than for the adult rats (6.4 ^ 2.9 s). The ndings indicate
that the effects of C-ber activation on the spinal nociceptive pathways attenuate more slowly in aged rats as compared
with adult rats. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Wind-up; Flexor reex; C-ber; Pain; Aging; Rat

It has been reported that unmyelinated bers in the

peripheral nerve show little change in number and conduction velocity with advancing age [14,18]. The density of
substance P and CGRP bers in the spinal dorsal horn
declines slightly in aged rats [1]. Psychophysical studies
in humans have revealed that pain threshold slightly
decreases or even remains unaltered in the elderly as
compared with young individuals [7]. Therefore, it appears
that central pain mechanisms are well maintained during
aging. In our recent study, however, it was demonstrated
that in aged rats, spontaneous activity and response to
noxious stimuli of dorsal horn neurons is signicantly
higher compared to adult rats [10]. It was also found that
noxious stimuli more often produced after-discharges
following the cessation of the stimulus, and its frequency
was signicantly higher in aged rats than in adult rats [10].
Wind-up is an activity dependent temporal facilitation
which is induced mainly by activation of C-bers [8].
Thus, to further clarify the difference between adult and
aged animals in the persistent effect following C-ber activation of the nociceptive pathways, we investigated the
* Corresponding author. Tel.: 181-33964-3241 ext. 3083; fax:
181-33579-4776.
E-mail address: kandak@tmig.or.jp (K. Kanda).

wind-up phenomenon of the exor reex in the present

study.
Experiments were performed using male Fischer 344/
DuCjr rats of two age groups (adult: 11.3 ^ 2.0 months
old, n 15; and aged: 29.4 ^ 1.5 months old, n 15)
raised under pathogen-free conditions and fed ad libitum.
The mean survival time of rats in the colony was about 28
months. Under halothane anesthesia (1.52% in N2O and
O2), the trachea, a common carotid artery and an external
jugular vein were cannulated. The rats were articially
ventilated. A small incision was made in the left hind leg,
and the sural nerve was dissected out from the surround
tissues and sectioned at the mid-belly level of the lateral
gastrocnemius muscle. The rats were next xed in a metal
frame using pins for the hip and a clamp for the foot, then
the halothane was reduced to 0.81.2% in oxygen. The sural
nerve was then placed on a pair of stimulating electrodes. A
pair of wire electrodes (Teon-coated S-S wires with a 100mm diameter and a bared 2-mm tip) were inserted into the
semitendinosus muscle to record exor reex activity. The
exposed portion was covered with warmed mineral oil.
The sural nerve was stimulated using ve electrical trains.
Each train consisted of ve constant current pulses (0.55.0
mA for 2 ms), with the interval between trains being 1 min.
This set of stimuli was repeated at various train intervals

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S03 04 - 394 0( 0 1) 01 76 1- X

50

K. Kanda et al. / Neuroscience Letters 304 (2001) 4952

with a 5-min pause. The train intervals and the order of

examinations were as follows: 3, 20, 5, 15, 8, 12 and 10 s.
In some experiments, the nerve was stimulated using a
random order of trains with different stimulus intervals,
where the total number of trains for each interval was
three. To evaluate the magnitude of the C-ber response,
which was the reex response induced by C-ber activity,
the electromyogram (EMG) was digitized, rectied and
integrated from 100 to 600 ms after each individual stimulation. No apparent difference in the results was found for the
two different stimulation paradigms. Thus, the data were
pooled and analyzed together. At the end of the experiment,
activation of C-bers was conrmed by recording action
potentials at the sciatic nerve.
In the present experiment, in which a wide (2 ms) duration of stimulus pulse was used, C-ber action potentials in
the sciatic nerve and late EMG activities in the hamstring
muscles were detected at the 0.2 mA stimulus intensity, and
these responses were augmented with increased stimulus
intensity up to 25 mA. Fig. 1A shows that the late component of the exor reex (i.e. C-response) progressively
increases during successive stimulation at the 3 s interval
and 0.5 mA intensity. The magnitude of the response
seemed to increase linearly. The slope of the linear regres-

sion line was designated as the wind-up rate. The wind-up

rate declined when the train interval was lengthened (Fig.
1B). The decay of the wind-up rate with increasing stimulus
intervals ts well to an exponential function (Fig. 1C). The
time constant of the wind-up decay for aged rats (9.2 ^ 3.2
s, n 15; 1.0% halothane; 0.5 mA stimulus intensity) was
signicantly longer compared to that for the adult rats
(6.4 ^ 2.9 s, n 15; 1.0% halothane; 0.5 mA stimulus
intensity) (t-test, P , 0:05) (Fig. 2).
Wind-up is an activity-dependent plasticity. Most of the
previous in vivo experiments applied 0.5 or 1.0-s interval
stimulation to the peripheral nerve in order to investigate
wind-up [8]. Price reported that the exor reex increased
progressively when C-bers were activated and when the
stimulus frequency exceeded 0.3/s [15,19]. The frequency
range which can produce wind-up seems to be relatively
narrow. In the present experiments, however, clear temporal
facilitation of exor reex activity could be observed when
C-bers were stimulated at 3 s or even longer intervals. In
aged rats, the effect of C-ber stimulation lasted for more
than 1 min (unpublished data). Woolf and King [20] have
described that in some dorsal horn neurons, depolarization
lasts for tens of seconds following C-ber stimulation. Thus,
the present ndings seem to well reect these slow excita-

Fig. 1. (A) Rectied electromyograms showing progressive increases in reex activity of semitendinosus muscle during a train stimulation with ve pulses at 3 s intervals to the sural nerve. (B) C-response increases linearly. Each value is the mean of ve responses.
Stimulus intervals were as follows: 3 s (W), 5 s (X), 8 s (B), 10 s (O), 12 s (S), 15 s (P), and 20 s (X). The slope of the linear regression line
was designated as the wind-up rate. (C) The wind-up rate seems to decline exponentially with increasing train intervals. Note that the
ordinate is log scale. The time constant for the decay of this case is 5.65 s.

K. Kanda et al. / Neuroscience Letters 304 (2001) 4952

Fig. 2. Histograms showing the distribution of the decay time

constant obtained under 1.0% halothane anesthesia and at a
stimulus strength of 0.5 mA. The time constant of the decay
for aged rats (9.2 ^ 3.2 s) was signicantly longer compared to
that of adult rats (6.4 ^ 2.9 s) (t-test, P , 0:05).

tory postsynaptic potentials (EPSPs). The extent of surgery

used for the experimental preparation might have affected
the frequency range of stimulation which produces wind-up.
We made only a small incision of the skin over the sural
nerve, and sectioned the nerve at the mid-belly level of the
lateral gastrocnemius muscle. After prolonged stimulation,
the exor reex response sometimes became smaller and the
time constant of decay tended to become shorter. The
response itself often could not be evoked, even when the
stimulus intensity was increased to very high levels, such as
510 mA and when the interval was short (i.e. 3 s). This
seems to be due to the inhibition of pathways mediating the
exor reex. This inhibition is probably mediated supraspinally through the descending system [4,5] because the
response reappeared after spinal transection (unpublished
observation). More invasive procedures, such as a laminectomy, were performed in some previous studies, which might
induce stronger inhibition. However, the reason for the
difference in ndings between the present and former
experiments in terms of the range of stimulus interval
which induced wind-up of the exor reex should be studied
further.
It has been reported that many transmitters, modulators
and receptors are involved in producing the wind-up
phenomenon [8]. Among them, there are AMPA and
NMDA receptors for glutamate, substance P, 5-HT, noradrenaline, and VIP. The bistable property of the neuronal
membrane may also play a role in producing the phenomenon [16,17,21]. It seems premature to discuss in detail the
mechanisms underlying the present nding that the time
constant of the wind-up decay with increasing stimulus

51

interval was signicantly longer in aged rats than in adult

rats. However, it might be worthwhile to discuss the possibility that a change in the descending control system takes
part in the excitability change in the spinal dorsal horn
neurons. It has been suggested that wind-up is affected by
the descending inhibitory system [4,5]. In our previous
experiments, spinal cord block resulted in a signicant
increase in responses of spinal nociceptive neurons to
noxious stimulation as well as their background activity in
adult rats. However, neuronal activity in aged rats was not
affected following spinal cord block. It has been demonstrated that there are abnormal proles and a decreased
density of 5-HT-immunoreactive (ir) and DBH-ir bers in
the dorsal horn in aged rats [10]. It has also reported that 5HT and norepinephrine content in the rat spinal cord
declines with age [9,12]. It is known that 5-HT and norepinephrine play an important role in descending inhibitory
systems. These ndings indicate that the descending inhibitory system is impaired in the aged rat. Thus, it is likely that
a decreased inhibitory action in the spinal nociceptive pathways may contribute to the hyperactivity of spinal neurons
which in turn prolongs the after-effects of stimulation.
Our previous study has shown hyperactivity of nociceptive spinal dorsal horn neurons in aged rats. The present
ndings provide further evidence for considerable change
in nociceptive pathways in the aged. It has been reported
that chronic pain is common among elderly people [2,3,6].
Animal studies have also revealed that partial peripheral
nerve injury induces more sever or prolonged hyperalgesia
in aged rats as compared to adult rats [11,13]. It has been
suggested that wind-up and central sensitization share
common mechanisms, although it has been shown that
these are separate phenomenon [8]. Thus, the alteration in
mechanisms producing wind-up with advancing age
deserves further study to seek adequate treatment and
prevention measures for chronic pain in the elderly.
This study was supported by a grant from the Ministry of
Health and Welfare (H11-Choju-006).
[1] Bergman, E., Johson, H., Zhang, X., Hokfelt, T. and Ulfhake,
B., Neuropeptides and neurotrophin receptor mRNAs in
primary sensory neurons of aged rats, J. Comp. Neurol.,
375 (1996) 303320.
[2] Brena, S. and Bonica, J.J., Nerve blocks for managing pain
in the elderly, Postgrad. Med., 47 (1970) 215220.
[3] Ferrell, B.A., Pain management in elderly people, J. Am.
Geriatr. Soc., 39 (1991) 6473.
[4] Gozariu, M., Bouhassira, D., Willer, J.C. and LeBars, D., The
inuence of temporal summation on a C-bre reex in the
rat: effects of lesions in the rostral ventromedial medulla
(RVM), Brain Res., 792 (1998) 168172.
[5] Gozariu, M., Bragard, D., Willer, J.-C. and LeBars, D.,
Temporal summation of C-ber afferent inputs: Competition between facilitatory and inhibitory effects on C-ber
reex, J. Neurophysiol., 78 (1997) 31653179.
[6] Harkins, S.W., Kwentus, J. and Price, D.D., Pain and suffering in the elderly, In J.J. Bonica (Ed.), Clinical Management
of Pain, Lea and Fabiger, New York, 1988, pp. 552559.

52

K. Kanda et al. / Neuroscience Letters 304 (2001) 4952

[7] Harkins, S.W. and Scott, R.B., Pain and presbyalgos, In J.E.
Birren (Ed.), Encyclopedia of Gerontology, Academic Press,
San Diego, 1996, pp. 247260.
[8] Herrero, J.F., Laird, J.M.A. and Lopez-Garcia, J.A., Wind-up
of spinal cord neurones and pain sensation: much ado
about something? Prog. Neurobiol., 61 (2000) 169203.
[9] Johnson, H., Ulfhake, B., Dagerlind, A., Bennett, G.W.,
Fone, K.C.F. and Hokfelt, T., The serotoninergic bulbospinal
system and brainstem-spinal cord content of serotoninTRH-, and substance P-like immunoreactivity in the aged
rat with special reference to the spinal cord motor nucleus,,
Synapse, 15 (1993) 6389.
[10] Kanda, K., Iwata, K., Tsuboi, Y., Nomoto, E. and Sumino, R.,
Aging effects on nociceptive mechanisms in the rat spinal
cord: Immunohistochemical, electrophysiological and
behavioral studies, Soc. Neurosci. Abstr., 23 (1997) 443.
[11] Kim, Y.I., Na, H.S., Yoon, Y.W., Nahm, S.H., Ko, K.H. and
Hong, S.K., Mechanical allodynia is more strongly manifested in older rats in an experimental model of peripheral
neuropathy, Neurosci. Lett., 199 (1995) 158160.
[12] Ko, M.L., King, M.A., Gordon, T.L. and Crisp, T., The effects
of aging on spinal neurochemistry in the rat, Brain Res.
Bull., 42 (1997) 9598.
[13] Novak, J.C., Lovell, J.A., Stuesse, S.L., Cruce, W.L.R.,
McBurney, D.L. and Crisp, T., Aging and neuropathic pain,
Brain Res., 833 (1999) 308310.

[14] Ochoa, J. and Mair, W.G.P., The normal sural nerve in man.
II. Changes in the axons and Schwann cells due to ageing,
Acta Neuropathol., 13 (1969) 217239.
[15] Price, D.D., Characteristics of second pain and exion
reexes indicative of prolonged central summation, Exp.
Neurol., 37 (1972) 371387.
[16] Russo, R.E. and Hounsgaard, J., Short-term plasticity in
turtle dorsal horn neurons mediated by L-type Ca2 1 channels, Neuroscience, 61 (1994) 191197.
[17] Russo, R.E. and Hounsgaard, J., Plateau-generating
neurones in the dorsal horn in an in vitro preparation of
the turtle spinal cord, J. Physiol., 493 (1996) 3954.
[18] Sato, A., Sato, Y. and Suzuki, H., Aging effects on conduction velocities of myelinated and unmyelinated bers of
peripheral nerves, Neurosci. Lett., 53 (1985) 1520.
[19] Schouenborg, J., Functional and topographical properties
of eld potentials evoked in rat dorsal horn by cutaneous Cbre stimulation, J. Physiol., 365 (1984) 169192.
[20] Woolf, C.J. and King, A., Physiology and morphology of
multireceptive neurons with C-afferent ber inputs in the
deep dorsal horn of the rat lumbar spinal cord, J. Neurophysiol., 58 (1987) 460479.
[21] Woolf, C.J. and Salter, M.W., Neuronal plasticity: increasing the gain in pain, Science, 288 (2000) 17651768.