Morphine-sensitive late components of the flexion reflex in the neonatal rat

0I

,Yc,u~ovc,i[,rlc,c, Lc/rcr.s. 78 ( 1987) 91 -96 Elscvier Scientific Publishcrs

Ireland

Ltd.

NSL 04664

Morphine-sensitive late components of the flexion reflex in the neonatal rat Y. Hori and S. Watanabe Lkpcrr~mcwr of Pi~y.~iolo~y,Sh~ol (Rcccived Unmyelinated

hi’), n,orti.c:

The X I S-day-old Multiunit

neural

superficial

pcroneal

the hamstring dlhchargcs

fiber; Flexor reflex: Morphine;

in response

muscles).

Attempts

together

to electrical

nerve) were recorded

increment

limited afferent opiate analgesic

stimulus

Neonatal

strengths

rat

with peripheral

stimulation

between

recruited

later components was precisely

a hmdlimh. plantar

ol

nerve or nerve innervating

afferent

of the llexion reflex

fibers. The neonatal

when compared

of the tlexion

associated

fibers in the nerve. These late Rexion reflex discharges in a naloxone-reversible

nerve (sural.

the magnitude

or unmyelinated

fiber volleys were exaggerated

of the flexion reflex discharges

morphine

nerves innervating

of a cutaneous

from a flexor nerve (deep peroneal

were made IO tind relations

due to myelinated

I I March 1987)

1987; Accepted

and the sizes of the volleys in the myelinated

rat5. Higher

observed

I6 February

rat spinal cord was isolated

discharges

ion reilcx discharges adult

o/‘Medicim~, Kyorin Unirwrsit~~, Tok.w (Jupm i

reflex discharges.

with the recruitment

were shown

flex-

with those in the The

of unmyc-

to be depressed

by the

manner.

Many studies concerning the analgesia systems have been done by using flexion reflex responses as a test index [3, 4, 13, 15, 171. The flexion reflex consists of two components, (i) a low-threshold short-latency component which contributes to rhythmic responses such as locomotion and (ii) a high-threshold late component which might account for the long-lasting nociceptive flexor withdrawal reflex [ 161. An in vitro preparation of the isolated spinal cord of neonatal rats, introduced by Otsuka and Konishi [IO], has been used extensively for both pharmacological and physiological studies of pain mechanisms [5, 141. In the present study, we used the neonatal rat spinal cord isolated together with some peripheral nerves innervating a hindlimb and recorded reflex discharges evoked in a flexor nerve by electrical stimulation of a cutaneous nerve. It will be shown that a late component of the flexion reflex which could be attributed to the activity of unmyelinated cutaneous afferent fibers can be recorded. The depressive effects of bath application of morphine on the late component of the flexion reflex will be also reported. Experiments were performed on S- 15-day-old SpragueeDawley rats. Animals were anesthetized with ether inhalation. The spinal column below mid-thoracic level and (‘r,r,c,.~f~,,lc/c,~z~,~,; Y. Hori,

Department

of Physiology,

6-70-Z. Mltaka.

Tokyo

0304-3940.X7’S

03.50 @ 1987 Elsevier Scientitic

School

of Medicine,

181. Japan.

Publishers

Ireland

Ltd.

Kyorin

University.

Shlnkawa

92

a hindlimb

were removed

spinal fluid (ACSF). vating the hindlimb the peripheral

nerves.

cord and the attached

from the rest of the body and placed in an artificial

Then the spinal cord was exposed and peripheral were dissected, and the spinal cord was isolated The spinal

cord was hemisected,

peripheral

nerves

were placed

cerebro-

nerves innertogether with

and the hemisected

spinal

in a 5 ml bath. The bath was

continuously perfused with ACSF at a rate of 2-3 ml/min. The bath temperature was always maintained at 26°C by a thermistor sensor control. The ACSF consisted of (in mM): NaCl 113, KC1 4.5, NaHC03 25, NaH2P04 I, CaC12 2, MgCll 1, glucose I I, and was bubbled with 95% 02-5s COz. Multiunit neural discharges were recorded with a suction electrode from a flexor nerve (either the deep peroneal nerve or the nerve innervating the hamstring muscles). Reflex discharges evoked in the flexor nerve by electrical stimulation of a cutaneous nerve (sural nerve, plantar nerve or superficial peroneal nerve) were fed into a signal processor (NEC-San’ei 7T07). The number of spikes in the reflex discharge was counted for the analysis. Poststimulus time histograms were also formed. The time course of the reflex was tested at intervals of 5 min. A single test was performed by 5 consecutive stimuli, each being given once every 15 s. The afferent volley was usually monitored from the dorsal rootlets using a suction electrode. Attempts were made to find out any relations between the magnitude of the reflex discharge and the size of the afferent volley. Fig. 1 shows the results of one of such experiments. This example was obtained from a ICday-old rat. The sural nerve was stimulated and recordings were made from the deep peroneal nerve in this case. The stimulus strength was progressively raised to activate smaller afferent fibers. With a stimulus strength of 2.5 V. the afferent volley was restricted to myelinated fibers, and the volley evoked long-lasting neural discharges (Fig. IA). The volley in myelinated fiber groups (A volley) was maximum at this stimulus strength and it did not increase in size even when stimulus strength was raised up to 8 V (Fig. I D). A stimulus intensity of approximately 3.0 V was threshold for the unmyelinated fibers in this preparation. Increasing the stimulus strength to 8.0 V resulted in the appearance of more discrete afferent volleys in the unmyelinated fibers (C volley, Fig. 1D). Stimuli with higher intensities beyond threshold level for the unmyelinated fibers elicited larger numbers of spikes in the reflex discharge that were dispersed over a longer duration of the reflex activity (Fig. I B-D). The results of the series of observations partly illustrated in Fig. IA-D are summarized in Fig. lE, F. Rather abrupt recruitment of the A volley and gradual augmentation of the C volley are shown by the continuous and dotted lines respectively (Fig. 1E). The size of the resultant reflex activity is shown in Fig. 1F. It should be noticed here that, although a reflex discharge could be evoked by A volleys, it did increase further in parallel with the gradual recruitment of the C volley. An isolated mammalian spinal cord preparation in vitro exhibits an evoked dorsal root reflex which might travel antidromically along the peripheral nerves [l, 121. In order to confirm that the reflex discharge recorded in a flexor nerve reflects discharges of flexor motoneurons, the effects of cutting the ventral roots on the recorded reflex discharge were studied in some experiments. After cutting the ventral roots of all seg-

93

10

A

’

reflex

L

afferent

discharges

_--

i!,iilw, L+

B:

ALL i

1

I---

I

01

t

0

Fig.

volley

12

3

I A D: representaive

tion of a cutaneous reflex discharges

4

recordings

5

evoked in the deep peroneal

the stimulating

and recording

8

7

of the reflex discharges

nerve. The poststimulus

The trace on the right side shows afferent between

6

v

stimula-

on the left side in each set of records

nerve in response

volleys recorded

10

evoked in a flexor nerve by electrical

time histogram

electrodes

9

to electrical

from the dorsal

being about

stimulation rootlet,

the conduction

28 mm. The stimulus

shows

of the sural nerve. intensity

distance was 2.5 V

(A). 4.0 V (B). 6.0 V (C) and 8.0 V (D). The duration of the stimulus pulse was 0.5 ms in all cases. An arrow below each histogram indicates the time of stimulus pulse. The sizes of A and C volleys (E), and the number stimulus

of the spikes of the reflex discharge

which

and the end of the flexion reflex (F) were plotted

occurred against

during

the period

the stimulus

strength.

between

50 ms after

D

E

control (with

10

depressive

effects of morphine

B, 10 min after perfusion

solution. containing

D -F: antagonism naloxone;

i

on the late component

with solution

F. 30 min after returning

containing

of the effects of morphine

E, IO min after perfusion

sural nerve was stimulated

to normal

solution.

rIXO”ery

neloxone)

:

Fig. 2. A-C: sponse;

F

morphine (with

nsloxone)

morphine;

by naloxone.

with solution The recordings

and the reflex discharges

of the flexion reflex. A, control C, 30 min after returning D, 5 mm after perfusion

containing were obtained

were recorded

both naloxone

re-

to normal

with solution and morphine;

from an 1 l-day-old

from the nerve innervating

rat. The the ham-

string muscles.

ments in a preparation, the reflex discharge which could be attributed to the C volley was observed to disappear in all instances studied. The effects of the opiate analgesic, morphine in this study, upon the flexion reflex were investigated in 6 preparations. The late component of the flexion reflex was observed to be depressed by morphine in all cases. Fig. 2A-C shows an example of the depressive effects of morphine. Ten minutes perfusion of the preparation with morphine at a concentration of 1.3 x 10e5 M decreased not only the duration of the flexion reflex but also the number of flexion reflex spikes to approximately 50% of the control value (Fig. 2B). These depressive effects of morphine were abolished within 30 min after removal of morphine (Fig. 2C). Accurate counting of the number of spikes of the short-latency component of the reflex was difficult because of a relatively large stimulus artifact. it seemed, however, that the short-latency component of the flexion reflex discharge due to A volley, was relatively unaffected by morphine. In order to know whether the depression of the late component of the flexion reflex was opiate receptor specific, the effects of the opiate antagonist naloxone were studied. The preparation was further perfused with ACSF containing naloxone (1 .Ox low6 M) for 5 min (Fig. 2D). Then it was perfused with ACSF containing both naloxone (1 .Ox 10m6 M) and morphine (1.3 x 10m5M) for 10 min (Fig. 2E). The depression of a late component of the flexion reflex produced by morphine was observed to be reduced by naloxone. In all 15 cases in which the effects of finely graded stimulus strengths were exam-

95

ined in detail as illustrated evoke neural

discharges

fairly long compared became shorter

in Fig. 1, it was observed

that the A volley was able to

in a flexor nerve. The duration with that in the adult

as older animals

of these reflex discharges

rat. The duration

was

of the reflex activity

were used.

The tendency was also noticed in the present study that recording of the reflex discharge attributable to the C volley was rather difficult in the first week of life. This tendency is quite compatible with a previous report on the neonatal rat in vivo that application of a chemical irritant to the skin started to evoke hamstring EMG responses only after 10-l 1 days from birth [7]. The mean conduction velocities of the volleys in myelinated and unmyelinated fibers recorded in the present experiments were about 8 and 0.3 m/s, respectively. Since the temperature of the preparation was maintained at 26°C in the present study, these fiber groups could conduct more rapidly at normal body temperature. Applying the temperature coefficient (Qto) reported for mammalian nerve fibers [l I], the conduction velocities of the fiber groups are consistent with those values previously reported for neonatal rats [6]. Myelin formation in peripheral nerve fibers is still incomplete in 8-l 5-day-old neonatal rats, upon which the present experiments were performed. From anatomical and neurophysiological observations [6, 81, we are inclined to think that the unmyelinated afferent fibers which were associated with a late component of the flexion reflex discharge in the neonatal rat represent unmyelinated afferent fibers in the adult rat rather than fibers still to become myelinated. A late component of the flexion reflex evoked by the C volley has been reported previously in in vivo experiments on the adult animal [3, 4, 9, 15, 171. The depressive effects of morphine on the late component of the flexion reflex has been also reported in the in vivo experiments [2, 41. The morphine-susceptible late component of the flexion reflex in the present isoloated in vitro preparation of the neonatal rat may be consistent with those previous reports. Finally, although functional connections of primary afferent fibers in the newborn spinal cord are still incomplete and peripheral nerve fibers are not fully developed, such preparations of the neonatal rat spinal cord, isolated together with peripheral nerves, could be employed for physiological and pharmacological studies of the analgesia systems of the spinal cord. We are grateful to Professor W.D. Willis for his invaluable advice and encouragement. We are also grateful to Professor S. Homma for his continuous encouragement and to Dr. J.M. Chung for his critical reading of the manuscript.

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