Vol. 4. pp. 1485 lo 1491 PergamonPressttd 1979.Printedin Great Britain
Neuroscience
NALOXONE EXCITES NOCICEPTIVE UNITS IN THE LUMBAR DORSAL HORN OF THE SPINAL CAT J. L. HENRY Department of Research in Anaesthesia, McGill University, 3655 Drummond Street, Montreal, Quebec, H3G 1Y6, Canada Abstract-Intravenous doses of 0.05-0.15 mgikg of naloxone caused an increase in the spontaneous discharge rate of single dorsal horn units in lumbar spinal segments 5-7 in the spinal cat. This effect was seen with all of 21 units responding to noxious cutaneous stimulation. In contrast, the 7 units classified as non-nociceptive cutaneous were all unaffected by naloxone, even at doses of 0.4mgjkg. Of two proprioceptive units, one was excited by naloxone, Naloxone also increased the response of nociceptive units to noxious radiant heat applied to the skin. Section of the ipsilateral dorsal roots of segments L4-S, failed to abolish the excitatory effect of naloxone on the spontaneous activity of nociceptive units. Responses to naloxone were observed in laminae I, IV, V and VI. These results suggest that the effects of naloxone are not general and that nociceptive units in particular are excited. They also demonstrate that this effect occurs within the central nervous system and that its action is at the spinal level. It is suggested that the algesic effects of naloxone reported by others may be due, at least in part, to this action of naloxone in the spinal cord. While the results of this study support an hypothesis that an endogenous opioid agonist is being released continuously, and it is the prevention of the action of this endogenous substance on ‘opiate receptors’ at the spinal level which is observed as an excitation, it is also possible that naloxone may have an action at the spinal level independently of the actions of another agent, and that perhaps this action is on non-opiate receptors. A third possibility, raised from other recent work, is that the excitatory effects of naloxone may have been due to an antagonism of the effects of y-aminobutyrate in the spinal cord.
NAIOXONE has generally been considered to be a specific antagonist of morphine and related opiates (WOODY, 1956; MARTIN, 1967; LEWIS, BENTLEY& COWAN, 1971; MAUGH, 1972; BRAUDE, HARRIS,MAY, SMil-H 8~ VILLARREAL, 1974). However, recent evidence has been accumulating which demonstrates that naloxone may have actions independent of the antagonism to exogenously administered opiates. Some of this evidence will be described briefly in the
Discussion. In another series of experiments on substance P, which is reported elsewhere (HENRY, 1978), it was observed that the intravenous administration of naloxone caused an increase in the spontaneous rate of firing of some spinal units. This observation prompted the present study, which is a more thorough examination of the effects of the intravenous administration of naloxone on the discharge rate of single units in the dorsal horn of the acutely spinalized cat. A preliminary report of these results appears elsewhere (HENRY, 1977). EXPERIMENTAL PROCEDURES Cats were anaesthetized with alpha chloralose (60 mgjkg) intravenously after induction with halothane/ oxygen. In one case, an unanaesthetized cat was used; it Ahbreuiutions: GABA, y-aminobutyrate
was decerebrated under halothane anaesthesia, and after surgery, anaesthesia was discontinued. Spinal segments Ls-L, were exposed for recording and covered with warm mineral oil to prevent drying. To eliminate the influence of supraspinal structures on the excitability of the units studied and to ensure that changes in the discharge rate of these units upon the administration of naloxone were not due to actions on supraspinal structures, the cords were transected at the level of the first lumbar vertebra; before cutting, the site was cooled with ethyl chloride to minimize spinal shock. The core temperature was maintained at 38°C. Carotid arterial pressure was monitored on a Grass polygraph and mean pressure was always above IOOmmHg. Spinal circulation was checked periodically throughout the experiment using a Carl Zeiss zoom dissecting stereomicroscope. Extracellular unit spikes were recorded with multibarrelled mi~opi~ttes. The central recording barrel was filled with 2.7 M NaCI. One peripheral barrel was filled with Na t-glutamate (1 M, pH 7.4, Sigma), another contained naloxone HCI (25 mt.r, pH 6.2, Endo) and another was filled with Pontamine Sky Blue (2% in 0.5 M sodium acetate, Gurr) for histological identification of sites of recording (for details of method see HENRY,1976). Units were classified according to their responses to natural stimulation; the means of classification is described elsewhere (HENRY, 1976). In brief, stimuli used were blowing with an air stream, brushing with a camel hair brush, gentle to heavy pressure with a blunt glass probe, pinching with forceps or clamps, burning with an infrared bulb and passive movement of the limb.
1485
1486
J. L.
Naloxone was administered via a catheter in the Jugular vein. In most cases. a dose of 0.1 mg;kg was given. but with units which were unaffected b! this dose. 0.3-0.4 mg,‘kg were given.
RESULTS Results were obtained from 30 cats. As naloxone was given by the intravenous route, only one unit was studied in each experiment to avoid the possibility of persisting effects of earlier doses which might modify the response. Carotid arterial pressure was unaffected by the intravenous administration of naloxone. Character
of response to intravenous
naloxone
The intravenous administration of naloxone caused excitation of 22 out of the 30 single spinal units. Typically, this excitation began within 1 min of its administration and reached its maximum after 2-5 min. The discharge rate remained elevated usually for more than 1 h, and occasionally failed to return to its original level even after 2 h. A typical response to naloxone is shown in Fig. 1. While a dose of 0.1 mg/kg was given in most cases, excitation was elicited with as little as 0.05 mg/kg. In two early experiments 0.15 mg/kg were given, one of these being with the decerebrated cat. In cases where excitation failed to occur, additional doses up to 0.364mg/kg were given. yet excitation still failed to occur even with these higher doses. Correlation
of
naloxone
excitation
with
adequate
stimulus
A correlation of the effects of naloxone with the classification of the 30 units on the basis of their responses to natural stimulation is presented in Table 1. All 21 nociceptive units were excited by naloxone; this includes 17 ‘wide dynamic range’ units (PRICE & DUBNER, 1977) and four ‘nociceptive specific’ units. Two proprioceptive units were studied; naloxone excited one and failed to affect the other. The five hair-sensitive units and two touch units were unaffected by naloxone. To determine whether the excitabilities of the nociceptive versus the nonnociceptive units by naloxone are significantly different the x2 test for unequal expectations was calculated according to the method outlined by DAVIES (1949). x2 was 6.79. and thus the differences between the re-
HI~KI
sponses of the two classifications cantly different at the I”,, level.
i5flkct.sof n&\-one
of units are slgmti-
on the response
to no.\iiou., heut
In 15 cases the response to noxious radiant heat was compared before and after the intravenous administration of naloxone. In 13 cases. naloxone enhanced the response to this noxious stimulus. A typical response is illustrated in Fig. 2. In one additional case. a unit which failed to respond to noxious heat before the administration of naloxone was excited by the noxious stimulus after its administration. The time course of these effects on the heat response was similar to the above-described effect on the spontaneous discharge rate. EfJ;cts @‘dorsal
rhizotomy
on response to intrurenous
naloxone
As an attempt to determine whether the responses to naloxone were due to actions on afferent neurones peripherally or to actions in the spinal cord, the effects on the excitatory response to naloxone of cutting the ipsilateral dorsal roots were studied in five cats. Four of these cats were prepared as before. but additionally paralysed with pancuronium bromide i.v. and artificially ventilated. Paralysis was necessary because dorsal rhizotomy induced massive reflex muscular contractions which. in one discarded experiment, dislodged the electrode from the vicinity of the unit under study. During the surgical preparation of each cat the dorsal roots of segments L4-Sz on the side of the recording were looped with fine surgical thread. Later, while recording, when a unit was found which responded to noxious cutaneous stimulation, an initial dose of 0.1 mg’kg of naloxone was given intravenously; in each case, as expected from the results of previous experiments, this initial dose of naloxone had an excitatory effect. The dorsal roots on the side of recording were then cut one segment at a time. This normally induced changes in the discharge rate of the unit being studied, usually consisting of transient excitation upon section of the roots of the same and of adjacent segments and transient depression upon section of the roots of more remote segments. As expected, noxious stimulation of the skin failed to alter the discharge rate. When the discharge rate of the unit returned to a constant level a second dose of 0.1 mg!kg of naloxone was given intravenously.
FIG. 1. Pen recorder tracing of ratemeter output (cumulative counts per unit time) of a single unit in the dorsal horn showing an increase in the spontaneous discharge rate induced by the intravenous administration of naloxone. Period of drug administration indicated by horizontal bar. The record on the right was taken 1 h later. The ordinate shows the rate of discharge in impulses;s.
1487
Naloxone excites nociceptive units in lumbar dorsal horn TABLE 1. EFFECT OF THE INTRAVENOUS ADMINISTRATIONOF NAWXONE ON NUMBERS OF SlNGLE SPINAL UNITS CLASSIFIED ACCORDING TO THEIR RESPONSES TO NATURAL STIMULATION
Excited/Tested Nociceptive wide dynamic range nociceptive specific Non-nociceptive cutaneous proprioceptive
Range of increases* (%)
17117 414
X-1450 150-463
O/7 l/2
26
*Calculated as the change in spontaneous discharge rate expressed as a per cent of the rate prior to naloxone administration; units without spontaneous activity prior to naloxone administration are excluded from this calculation.
In all five cases, this administration of naloxone following section of the dorsal roots caused a prolonged increase in the discharge rate, with a similar time course to that observed before section. In four cases, a third dose of 0.1 mg/kg (the second sub-
sequent to root section) produced yet another increase in discharge rate. Figure 3 shows the excitatory responses of one of the four units to the administra-
tion of two doses of naloxone, each 0.1 mg/kg, subsequent to root section.
D 250
FIG. 2. Ratemeter record of a single unit in the drosal horn showing naloxone-induced increase in discharge rate and in the magnitude of the response to automatically controlled regular applications of noxious heat (indicated by short horizontal bars). B, C and D: 15, 30 and amin, respectively, after A. In B, C and D some responses to heat reached peak discharge rates beyond the maximal excursion of the recorder pen. The thick horizontal bar represents the period of administration of 0.1 mg/kg of naloxone HCl.
the spontaneous
FIG. .3. Ratemeter record of a single unit in the dorsal horn of segment L, after section of ipsilateral dorsal roots of segments L&S,. Upper record, 10 min after rhizotomy; lower, 20 min after rhizotomy. Two doses of naloxone, 0.1 mg/kg each, indicated by the horizontal brackets, each induced an increase in the discharge rate of this unit. In parts of the lower record the peak discharge rate passes the maximal excursion of the recorder pen.
15flkT.s of iorrtophorrtic
applicatim
oj’ naloxone
In four cases naloxone was applied by iontophoresis with positive currents of 100 nA. No reproducible effect of this application was obscrvcd with either nociceptive or non-nociceptive units. These observations are consistent with earlier unpublished data from previous studies. It is this failure to obtain consistent effects with the iontophoretic application of naloxone which led to the decision to use intravenous administration in this study. In addition, in view of the fact that with iontophoresis the tissue concentrations of substances are unknown. it is difficult to relate the effects of the iontophoretic application of naloxone to the opiate antagonizing effects of naloxone that is administered intravenously at normal pharmacological doses.
Most units studied were located in regions of the grey matter corresponding to Rexed‘s laminae IV and V. All those excited by naloxone were in Laminae I (II = 2), IV (n = 8). V (n = 1I) and VI (n = I). The unaffected proprioceptive unit was in lamina VI.
When this study was nearly complete a paper appeared which was based on the same experimental protocol (DuG(;AK. HALI.. HI-AIILEY & GRI~:RSMIT~~, 1977). These authors reported that in one decerebrated and twelve chloralose-anaesthetired cats the intravenous administration of naloxone. in doses of 0. I?- 3.3 mgjkg. elicited excitation of only two units out of 13. Each of these units was excited by the application of a noxious radiant heat stimulus to the skin; naloxone increased both the nociceptive and the non-nociceptive responses of one unit, and increased only the nociceptive response of the other unit. The failure of these authors to see excitation in more cases is difficult to explain, especially in view of the extremely high doses of naloxone they used, and their results are inconsistent with earlier reports that the systemic administration of naloxone increases spinal reflexes (GLDFARB & Hu. 1976; JACOB, TREMBLAY& COLOMB~:L.1974) and reverses the analgesia produced by acupuncture (MAYER, PRICE & RAPII, 1977: POMERANZ.CHANG & LAW, 1977) and by stimulation at supra-spinal sites (AKIL, MAYER & LIEBESKIND, 1976; OLIVERAS, Hosot~~c~rr. R~DJEMI, GUILHAIJD & BESSON, 1977), reports suggesting a possible role for endorphins in the spinal cord.
DISCUSSION It has been found that the intravenous administration of naloxone causes an increase in the discharge rate of all spinal nociceptive units tested while leaving unaffected all cutaneous non-nociceptive units tested. The two proprioceptive units, on the other hand, showed different effects with naloxone: one was excited, the other was unaffected. In addition, naloxone facilitated the excitatory response of nociceptive units to noxious cutaneous heat. The observation that these excitatory effects of naloxone could be elicited following dorsal rhizotomy of segments L,-S2 suggests that the effects of naloxone were due to an action within the central nervous system. Furthermore. the effects of naloxone must have been due to an action at the spinal level because the cords were transected just rostra1 to the level of recording. thus eliminating any influence of supraspinal structures on the neurones studied. A similar claim of an ‘opiate-unrelated’ action of naloxone at the spinal level has been reported by G~LDFARR & Hu (1976) who found that the intravenous administration of 0. I -2.0 mg/kg of naloxone increased the amplitude of both monosynaptic and polysynaptic reflexes in the spinal cat. In addition, B~:LL & MARTIN (1976) found that 0.05 mg/kg of naloxone given intravenously increased the ventral root reflex evoked by radiant heat in the decerebrated, low spinal cat. Finally, GRAIN, PETERSON, GRAIN & SIMON (1977) have recently shown that in dorsal horn regions of spinal cord tissue cultures naloxone increased the amplitude and duration’ of sensory-evoked negative slow-wave potentials even when introduced without previous exposure to opiates.
Comparison
with iontophoretic
application
of naloxonr
The excitatory effects of naloxone in the present study were not mimicked by its iontophoretic application to the same neurones, although this latter method of application has been shown to reverse depression of single units by iontophoretically or intravenously administered opiates (CALVILLO, HENRY & NEUMAN. 1974; ZIEGLG.KNSBER(;ER& BAYERL. 1976). Whether or not related to the actions of naloxone in the present study, its iontophoretic application has been reported to have variable effects on neural activity in supraspinal structures (BIOULAC. LUND & PUIL, 1975; BRADLEY,BRIGC;S.GAYTON 8~ LAMBERT, 1976; NICOLI., SKGINS. LING, BLOOM 8~ GUILLEMIN. 1977). The only observed action of the iontophoretic application of naloxone alone on spinal units has been a depression; while CALVILLO et ai. (1974) reported that one of 11 units studied was depressed, this observation has not been substantiated by further experiments (unpublished observations), and ZIE~.\io/. ~h~~~~~,~trc. 53.
BRADLEY P. B.. BRIGGS 1.. GAYTON R. J. & LAMBERT L. A. (1976) Effects of microiontophoretically enkephalin on single neurones in rat brainstem. Riaturr. Lond. 261. 425~ 426.
applied
methionine-
BRAUDE M. C., HARRIS L. S., MAY E. L.. SMITH J. P. & VILLARR~AL J. E. (1974) ,YUX,XI~ Alrtagoni,~r,s;Adt,a,lce~ in Biochemical Psychopharmacology. Raven Press, New York. CALVILLO 0.. HENRY J. L. & NEUMAN R. S. (1974) Effects of morphine and naloxone on dorsal horn neurones in the cat. Gun. J. Physiol. Phurmac. 52, 1207-121 I. CRAIN S. M., PETERSON E. M.. GRAIN B. & SIMON E. J. (1977) Selective opiate depression of sensory-evoked synaptic networks in dorsal horn regions of spinal cord cultures. Bruin Res. 133, 162 166. DAVIES 0. L. (1949) S!afisticul Methods in Reseurch and Production. Oliver & Boyd. London. DINGLEDINE R., I~ERSENL. L. & BREUKER E. (1978) Naloxone as GABA antagonist: evidence from iontophoretic, receptor binding and convulsant studies. Eur. J. Pharmacol. 47, 19-27. DUGGAN A. W., HALL J. G., HEADLEY P. M. & GRIERSMI~H B. T. (1977) The effect of naloxone on the excitation of dorsal horn neurones of the cat by noxious and non-noxious cutaneous stimuli. Bruin Rrs. 138, 185-189. ELDE R., H~~KFELTT., JOHANSSON0. & TERENIUS L. (1976) Immunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat. Neuroscience 1, 349-351. EL-SOBKY A., DOS~ROVSKY J. 0. & WALI. P. D. (1976) Lack of effect of naloxone on pain perception in humans, Nature.
Lond.
263, 783-784.
FREIIERICKSON R. C. A.. BLJRGIS V. & EDWARDS J. D. (1977) Diurnal rhythm in endorphin activity in mouse brain-effects of naloxone. pilocarpine and baclofen. Fedn Proc. Fedn Am. Sots esp. Biol. 36, 965. GOLDFARB J. & Hu J. W. (1976) Enhancement of reflexes bq naloxone in spinal cats. ,Yf,uropharmccco/ogq 15, 785-792, GREVERT P. & GOLDSTEIN A. (1977) Effects of naloxone on experimentally induced ischemic pain and on mood in human subjects. Proc. narn. Acud. Sci. U.S.A. 74, 1291-1294. HENRY J. L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Brcrrn Rex 114, 439451. HENRV J. L. (1977) Naloxone excitation of dorsal horn units in the spinal cat. ,Yeurosciencr Ahs 3, 293. HENRY J. L. (1978) Effects of morphine and naloxone on substance P-induced excitation in cat spinal cord. Proc. Can. Fed. Biol. Sot. 21, 69. HOSOBUCHI Y., ADAMS J. E. & LINCHITZ R. (1977) Pain relief by electrical stimulation of the central grey matter in humans and its reversal by naloxone. Science, N.E 197, 183-186. HUGHES J., SMITH T. B., MORGAN B. & FO~HERGILL L. (1975) Purification and properties of enkephalinthe possible endogenous ligand for the morphine receptor. Lij? Sci. 16, 1753 1758. JACOB J. J.. TREMRLAY E. C. & COLOMBEL M. (1974) Facilitation de reactions nociceptives par la naloxone cher la souris et chez le rat. Psychopharmucologia 37. 217.~223. KING C. D.. HUGHEY L. J.. MASSERAND J. M., CODD E. E. & B~RN~ W. L. (1977) Endorphin and naloxone; insomnia and seizures in cats and hyperalgesia in rats. Fedn Proc. Frdn Am. Sots ezp. Biol. 36, 965. KRNJEV~ K. (1974) Chemical nature of synaptic transmission in vertebrates. Physiol. Rec. 54, 41X--540. L,A.MOTT~C.. PERT C. B. & SNYDER S. H. (1976) Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section. Bruin Rrs. 112, 407-412. LEWIS J. W., BENTLEYK. W. & COWAN A. (1971) Narcotic analgesics and antagonists. .4. Rev Phurmuc. 11, 741-270. MARTIN W. R. (1967) Opioid antagonists. Pharmac. Rev. 19, 463-521. MA[ C;H T. H. (1972) Narcotic antagonists: the search accelerates. Scicww. h’. Y. 177. 249 250. MAYER D. J.. PRICI. D. D. & RAFII A. (1977) Antagonism of acupuncture analgesia in man by the narcotic antagonist naloxone.
Bruin
Res. 121, 368-372.
G. R., LING. N.. BLOOM F. E. & GIXLEMIN R. (1977) Neuronal actions of endorphins and cnkephaiins among brain regions: a comparative microiontophoretic study. Proc. nufn. .kud. Sri. U.S.A. 74, 2584 -2588. OLJLERASJ. L.. HOSOBUCXI Y.. REIJJEMI F., GUILBAUD G. & BE:SSONJ. M. (1977) Opiate antagonist naloxone strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior). Brain Rrs. 120, 221~ 229. PASE:RNAK G. W., G~DMAN R. & SNYDER S. H. (1975) An endogenous morphine-like factor in mammalian brain.
NICOLI.
R.
A..
SIGGINS
Ltfe Sci. 16, 1765.. 1769. P~M~RANZ B., CHANG R. & LAW P. (1977) Acupuncture reduces electrophysiological noxious stimuli: pituitary is implicated. Evpl Nrurol. 54, I77- 178. PRI(.I., D. D. & D~J~~NERR. (1977) Neurons that subserve the sensory-discriminative TE&C.IS A. K. (1974) Trrm,snritrers Bristol. Tt:~~:~llis L. & WAHLsTRiiM A. 94. 74 Xl.
and Idtwtified
Ncwrons
in the ~ammulicm
(1975)Search for an endogenous
ligand
and aspects
Cenrrul
for the opiate
behavioural of pain.
R’errow
rcccptor.
responses
to
f’ui!I 3. 307 338.
S~,.sron. Scien technica.
Acttr /J/f \‘\IO/. \c’tIilt/.
Naloxone excites nociceptive units in lumbar dorsal horn
1491
TERENIUSL. 8~ WAHLSTR~~M A. (1976) A method for site selectivity analysis applied to opiate receptors. Eur. J. Pharmacol. 40, 241-248. TE~CHEMACHER H., OPHEIMK. E., Cox B. M. & GOLDSTEINA. (1975) peptide-like substance from pituitary that acts like morphine-I. Isolation. Life Sci. 16, 1771-1776. Wools L. A. (1956) The pharmacology of nalorphine (n-allynormorphine). Pharmac. Rev. 8, 175-198. YAKSH T. L., HUANG S. P., RUDY T. A. & FREDERICK~ON R. C. A. (1977) The direct and specific opiate-like effect of Met’-enkephalin and analogues on the spinal cord. Neuroscience 2, 593-596. ZIEGLG~NSBERGER W. & BAYERLH. (1976) The mechanism of inhibition of neuronal activity by opiates in the spinal cord of cat. Brain Res. 115. 111-128. (Accepted 26 March 1979)
Neuroscience
NALOXONE EXCITES NOCICEPTIVE UNITS IN THE LUMBAR DORSAL HORN OF THE SPINAL CAT J. L. HENRY Department of Research in Anaesthesia, McGill University, 3655 Drummond Street, Montreal, Quebec, H3G 1Y6, Canada Abstract-Intravenous doses of 0.05-0.15 mgikg of naloxone caused an increase in the spontaneous discharge rate of single dorsal horn units in lumbar spinal segments 5-7 in the spinal cat. This effect was seen with all of 21 units responding to noxious cutaneous stimulation. In contrast, the 7 units classified as non-nociceptive cutaneous were all unaffected by naloxone, even at doses of 0.4mgjkg. Of two proprioceptive units, one was excited by naloxone, Naloxone also increased the response of nociceptive units to noxious radiant heat applied to the skin. Section of the ipsilateral dorsal roots of segments L4-S, failed to abolish the excitatory effect of naloxone on the spontaneous activity of nociceptive units. Responses to naloxone were observed in laminae I, IV, V and VI. These results suggest that the effects of naloxone are not general and that nociceptive units in particular are excited. They also demonstrate that this effect occurs within the central nervous system and that its action is at the spinal level. It is suggested that the algesic effects of naloxone reported by others may be due, at least in part, to this action of naloxone in the spinal cord. While the results of this study support an hypothesis that an endogenous opioid agonist is being released continuously, and it is the prevention of the action of this endogenous substance on ‘opiate receptors’ at the spinal level which is observed as an excitation, it is also possible that naloxone may have an action at the spinal level independently of the actions of another agent, and that perhaps this action is on non-opiate receptors. A third possibility, raised from other recent work, is that the excitatory effects of naloxone may have been due to an antagonism of the effects of y-aminobutyrate in the spinal cord.
NAIOXONE has generally been considered to be a specific antagonist of morphine and related opiates (WOODY, 1956; MARTIN, 1967; LEWIS, BENTLEY& COWAN, 1971; MAUGH, 1972; BRAUDE, HARRIS,MAY, SMil-H 8~ VILLARREAL, 1974). However, recent evidence has been accumulating which demonstrates that naloxone may have actions independent of the antagonism to exogenously administered opiates. Some of this evidence will be described briefly in the
Discussion. In another series of experiments on substance P, which is reported elsewhere (HENRY, 1978), it was observed that the intravenous administration of naloxone caused an increase in the spontaneous rate of firing of some spinal units. This observation prompted the present study, which is a more thorough examination of the effects of the intravenous administration of naloxone on the discharge rate of single units in the dorsal horn of the acutely spinalized cat. A preliminary report of these results appears elsewhere (HENRY, 1977). EXPERIMENTAL PROCEDURES Cats were anaesthetized with alpha chloralose (60 mgjkg) intravenously after induction with halothane/ oxygen. In one case, an unanaesthetized cat was used; it Ahbreuiutions: GABA, y-aminobutyrate
was decerebrated under halothane anaesthesia, and after surgery, anaesthesia was discontinued. Spinal segments Ls-L, were exposed for recording and covered with warm mineral oil to prevent drying. To eliminate the influence of supraspinal structures on the excitability of the units studied and to ensure that changes in the discharge rate of these units upon the administration of naloxone were not due to actions on supraspinal structures, the cords were transected at the level of the first lumbar vertebra; before cutting, the site was cooled with ethyl chloride to minimize spinal shock. The core temperature was maintained at 38°C. Carotid arterial pressure was monitored on a Grass polygraph and mean pressure was always above IOOmmHg. Spinal circulation was checked periodically throughout the experiment using a Carl Zeiss zoom dissecting stereomicroscope. Extracellular unit spikes were recorded with multibarrelled mi~opi~ttes. The central recording barrel was filled with 2.7 M NaCI. One peripheral barrel was filled with Na t-glutamate (1 M, pH 7.4, Sigma), another contained naloxone HCI (25 mt.r, pH 6.2, Endo) and another was filled with Pontamine Sky Blue (2% in 0.5 M sodium acetate, Gurr) for histological identification of sites of recording (for details of method see HENRY,1976). Units were classified according to their responses to natural stimulation; the means of classification is described elsewhere (HENRY, 1976). In brief, stimuli used were blowing with an air stream, brushing with a camel hair brush, gentle to heavy pressure with a blunt glass probe, pinching with forceps or clamps, burning with an infrared bulb and passive movement of the limb.
1485
1486
J. L.
Naloxone was administered via a catheter in the Jugular vein. In most cases. a dose of 0.1 mg;kg was given. but with units which were unaffected b! this dose. 0.3-0.4 mg,‘kg were given.
RESULTS Results were obtained from 30 cats. As naloxone was given by the intravenous route, only one unit was studied in each experiment to avoid the possibility of persisting effects of earlier doses which might modify the response. Carotid arterial pressure was unaffected by the intravenous administration of naloxone. Character
of response to intravenous
naloxone
The intravenous administration of naloxone caused excitation of 22 out of the 30 single spinal units. Typically, this excitation began within 1 min of its administration and reached its maximum after 2-5 min. The discharge rate remained elevated usually for more than 1 h, and occasionally failed to return to its original level even after 2 h. A typical response to naloxone is shown in Fig. 1. While a dose of 0.1 mg/kg was given in most cases, excitation was elicited with as little as 0.05 mg/kg. In two early experiments 0.15 mg/kg were given, one of these being with the decerebrated cat. In cases where excitation failed to occur, additional doses up to 0.364mg/kg were given. yet excitation still failed to occur even with these higher doses. Correlation
of
naloxone
excitation
with
adequate
stimulus
A correlation of the effects of naloxone with the classification of the 30 units on the basis of their responses to natural stimulation is presented in Table 1. All 21 nociceptive units were excited by naloxone; this includes 17 ‘wide dynamic range’ units (PRICE & DUBNER, 1977) and four ‘nociceptive specific’ units. Two proprioceptive units were studied; naloxone excited one and failed to affect the other. The five hair-sensitive units and two touch units were unaffected by naloxone. To determine whether the excitabilities of the nociceptive versus the nonnociceptive units by naloxone are significantly different the x2 test for unequal expectations was calculated according to the method outlined by DAVIES (1949). x2 was 6.79. and thus the differences between the re-
HI~KI
sponses of the two classifications cantly different at the I”,, level.
i5flkct.sof n&\-one
of units are slgmti-
on the response
to no.\iiou., heut
In 15 cases the response to noxious radiant heat was compared before and after the intravenous administration of naloxone. In 13 cases. naloxone enhanced the response to this noxious stimulus. A typical response is illustrated in Fig. 2. In one additional case. a unit which failed to respond to noxious heat before the administration of naloxone was excited by the noxious stimulus after its administration. The time course of these effects on the heat response was similar to the above-described effect on the spontaneous discharge rate. EfJ;cts @‘dorsal
rhizotomy
on response to intrurenous
naloxone
As an attempt to determine whether the responses to naloxone were due to actions on afferent neurones peripherally or to actions in the spinal cord, the effects on the excitatory response to naloxone of cutting the ipsilateral dorsal roots were studied in five cats. Four of these cats were prepared as before. but additionally paralysed with pancuronium bromide i.v. and artificially ventilated. Paralysis was necessary because dorsal rhizotomy induced massive reflex muscular contractions which. in one discarded experiment, dislodged the electrode from the vicinity of the unit under study. During the surgical preparation of each cat the dorsal roots of segments L4-Sz on the side of the recording were looped with fine surgical thread. Later, while recording, when a unit was found which responded to noxious cutaneous stimulation, an initial dose of 0.1 mg’kg of naloxone was given intravenously; in each case, as expected from the results of previous experiments, this initial dose of naloxone had an excitatory effect. The dorsal roots on the side of recording were then cut one segment at a time. This normally induced changes in the discharge rate of the unit being studied, usually consisting of transient excitation upon section of the roots of the same and of adjacent segments and transient depression upon section of the roots of more remote segments. As expected, noxious stimulation of the skin failed to alter the discharge rate. When the discharge rate of the unit returned to a constant level a second dose of 0.1 mg!kg of naloxone was given intravenously.
FIG. 1. Pen recorder tracing of ratemeter output (cumulative counts per unit time) of a single unit in the dorsal horn showing an increase in the spontaneous discharge rate induced by the intravenous administration of naloxone. Period of drug administration indicated by horizontal bar. The record on the right was taken 1 h later. The ordinate shows the rate of discharge in impulses;s.
1487
Naloxone excites nociceptive units in lumbar dorsal horn TABLE 1. EFFECT OF THE INTRAVENOUS ADMINISTRATIONOF NAWXONE ON NUMBERS OF SlNGLE SPINAL UNITS CLASSIFIED ACCORDING TO THEIR RESPONSES TO NATURAL STIMULATION
Excited/Tested Nociceptive wide dynamic range nociceptive specific Non-nociceptive cutaneous proprioceptive
Range of increases* (%)
17117 414
X-1450 150-463
O/7 l/2
26
*Calculated as the change in spontaneous discharge rate expressed as a per cent of the rate prior to naloxone administration; units without spontaneous activity prior to naloxone administration are excluded from this calculation.
In all five cases, this administration of naloxone following section of the dorsal roots caused a prolonged increase in the discharge rate, with a similar time course to that observed before section. In four cases, a third dose of 0.1 mg/kg (the second sub-
sequent to root section) produced yet another increase in discharge rate. Figure 3 shows the excitatory responses of one of the four units to the administra-
tion of two doses of naloxone, each 0.1 mg/kg, subsequent to root section.
D 250
FIG. 2. Ratemeter record of a single unit in the drosal horn showing naloxone-induced increase in discharge rate and in the magnitude of the response to automatically controlled regular applications of noxious heat (indicated by short horizontal bars). B, C and D: 15, 30 and amin, respectively, after A. In B, C and D some responses to heat reached peak discharge rates beyond the maximal excursion of the recorder pen. The thick horizontal bar represents the period of administration of 0.1 mg/kg of naloxone HCl.
the spontaneous
FIG. .3. Ratemeter record of a single unit in the dorsal horn of segment L, after section of ipsilateral dorsal roots of segments L&S,. Upper record, 10 min after rhizotomy; lower, 20 min after rhizotomy. Two doses of naloxone, 0.1 mg/kg each, indicated by the horizontal brackets, each induced an increase in the discharge rate of this unit. In parts of the lower record the peak discharge rate passes the maximal excursion of the recorder pen.
15flkT.s of iorrtophorrtic
applicatim
oj’ naloxone
In four cases naloxone was applied by iontophoresis with positive currents of 100 nA. No reproducible effect of this application was obscrvcd with either nociceptive or non-nociceptive units. These observations are consistent with earlier unpublished data from previous studies. It is this failure to obtain consistent effects with the iontophoretic application of naloxone which led to the decision to use intravenous administration in this study. In addition, in view of the fact that with iontophoresis the tissue concentrations of substances are unknown. it is difficult to relate the effects of the iontophoretic application of naloxone to the opiate antagonizing effects of naloxone that is administered intravenously at normal pharmacological doses.
Most units studied were located in regions of the grey matter corresponding to Rexed‘s laminae IV and V. All those excited by naloxone were in Laminae I (II = 2), IV (n = 8). V (n = 1I) and VI (n = I). The unaffected proprioceptive unit was in lamina VI.
When this study was nearly complete a paper appeared which was based on the same experimental protocol (DuG(;AK. HALI.. HI-AIILEY & GRI~:RSMIT~~, 1977). These authors reported that in one decerebrated and twelve chloralose-anaesthetired cats the intravenous administration of naloxone. in doses of 0. I?- 3.3 mgjkg. elicited excitation of only two units out of 13. Each of these units was excited by the application of a noxious radiant heat stimulus to the skin; naloxone increased both the nociceptive and the non-nociceptive responses of one unit, and increased only the nociceptive response of the other unit. The failure of these authors to see excitation in more cases is difficult to explain, especially in view of the extremely high doses of naloxone they used, and their results are inconsistent with earlier reports that the systemic administration of naloxone increases spinal reflexes (GLDFARB & Hu. 1976; JACOB, TREMBLAY& COLOMB~:L.1974) and reverses the analgesia produced by acupuncture (MAYER, PRICE & RAPII, 1977: POMERANZ.CHANG & LAW, 1977) and by stimulation at supra-spinal sites (AKIL, MAYER & LIEBESKIND, 1976; OLIVERAS, Hosot~~c~rr. R~DJEMI, GUILHAIJD & BESSON, 1977), reports suggesting a possible role for endorphins in the spinal cord.
DISCUSSION It has been found that the intravenous administration of naloxone causes an increase in the discharge rate of all spinal nociceptive units tested while leaving unaffected all cutaneous non-nociceptive units tested. The two proprioceptive units, on the other hand, showed different effects with naloxone: one was excited, the other was unaffected. In addition, naloxone facilitated the excitatory response of nociceptive units to noxious cutaneous heat. The observation that these excitatory effects of naloxone could be elicited following dorsal rhizotomy of segments L,-S2 suggests that the effects of naloxone were due to an action within the central nervous system. Furthermore. the effects of naloxone must have been due to an action at the spinal level because the cords were transected just rostra1 to the level of recording. thus eliminating any influence of supraspinal structures on the neurones studied. A similar claim of an ‘opiate-unrelated’ action of naloxone at the spinal level has been reported by G~LDFARR & Hu (1976) who found that the intravenous administration of 0. I -2.0 mg/kg of naloxone increased the amplitude of both monosynaptic and polysynaptic reflexes in the spinal cat. In addition, B~:LL & MARTIN (1976) found that 0.05 mg/kg of naloxone given intravenously increased the ventral root reflex evoked by radiant heat in the decerebrated, low spinal cat. Finally, GRAIN, PETERSON, GRAIN & SIMON (1977) have recently shown that in dorsal horn regions of spinal cord tissue cultures naloxone increased the amplitude and duration’ of sensory-evoked negative slow-wave potentials even when introduced without previous exposure to opiates.
Comparison
with iontophoretic
application
of naloxonr
The excitatory effects of naloxone in the present study were not mimicked by its iontophoretic application to the same neurones, although this latter method of application has been shown to reverse depression of single units by iontophoretically or intravenously administered opiates (CALVILLO, HENRY & NEUMAN. 1974; ZIEGLG.KNSBER(;ER& BAYERL. 1976). Whether or not related to the actions of naloxone in the present study, its iontophoretic application has been reported to have variable effects on neural activity in supraspinal structures (BIOULAC. LUND & PUIL, 1975; BRADLEY,BRIGC;S.GAYTON 8~ LAMBERT, 1976; NICOLI., SKGINS. LING, BLOOM 8~ GUILLEMIN. 1977). The only observed action of the iontophoretic application of naloxone alone on spinal units has been a depression; while CALVILLO et ai. (1974) reported that one of 11 units studied was depressed, this observation has not been substantiated by further experiments (unpublished observations), and ZIE~.\io/. ~h~~~~~,~trc. 53.
BRADLEY P. B.. BRIGGS 1.. GAYTON R. J. & LAMBERT L. A. (1976) Effects of microiontophoretically enkephalin on single neurones in rat brainstem. Riaturr. Lond. 261. 425~ 426.
applied
methionine-
BRAUDE M. C., HARRIS L. S., MAY E. L.. SMITH J. P. & VILLARR~AL J. E. (1974) ,YUX,XI~ Alrtagoni,~r,s;Adt,a,lce~ in Biochemical Psychopharmacology. Raven Press, New York. CALVILLO 0.. HENRY J. L. & NEUMAN R. S. (1974) Effects of morphine and naloxone on dorsal horn neurones in the cat. Gun. J. Physiol. Phurmac. 52, 1207-121 I. CRAIN S. M., PETERSON E. M.. GRAIN B. & SIMON E. J. (1977) Selective opiate depression of sensory-evoked synaptic networks in dorsal horn regions of spinal cord cultures. Bruin Res. 133, 162 166. DAVIES 0. L. (1949) S!afisticul Methods in Reseurch and Production. Oliver & Boyd. London. DINGLEDINE R., I~ERSENL. L. & BREUKER E. (1978) Naloxone as GABA antagonist: evidence from iontophoretic, receptor binding and convulsant studies. Eur. J. Pharmacol. 47, 19-27. DUGGAN A. W., HALL J. G., HEADLEY P. M. & GRIERSMI~H B. T. (1977) The effect of naloxone on the excitation of dorsal horn neurones of the cat by noxious and non-noxious cutaneous stimuli. Bruin Rrs. 138, 185-189. ELDE R., H~~KFELTT., JOHANSSON0. & TERENIUS L. (1976) Immunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat. Neuroscience 1, 349-351. EL-SOBKY A., DOS~ROVSKY J. 0. & WALI. P. D. (1976) Lack of effect of naloxone on pain perception in humans, Nature.
Lond.
263, 783-784.
FREIIERICKSON R. C. A.. BLJRGIS V. & EDWARDS J. D. (1977) Diurnal rhythm in endorphin activity in mouse brain-effects of naloxone. pilocarpine and baclofen. Fedn Proc. Fedn Am. Sots esp. Biol. 36, 965. GOLDFARB J. & Hu J. W. (1976) Enhancement of reflexes bq naloxone in spinal cats. ,Yf,uropharmccco/ogq 15, 785-792, GREVERT P. & GOLDSTEIN A. (1977) Effects of naloxone on experimentally induced ischemic pain and on mood in human subjects. Proc. narn. Acud. Sci. U.S.A. 74, 1291-1294. HENRY J. L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Brcrrn Rex 114, 439451. HENRV J. L. (1977) Naloxone excitation of dorsal horn units in the spinal cat. ,Yeurosciencr Ahs 3, 293. HENRY J. L. (1978) Effects of morphine and naloxone on substance P-induced excitation in cat spinal cord. Proc. Can. Fed. Biol. Sot. 21, 69. HOSOBUCHI Y., ADAMS J. E. & LINCHITZ R. (1977) Pain relief by electrical stimulation of the central grey matter in humans and its reversal by naloxone. Science, N.E 197, 183-186. HUGHES J., SMITH T. B., MORGAN B. & FO~HERGILL L. (1975) Purification and properties of enkephalinthe possible endogenous ligand for the morphine receptor. Lij? Sci. 16, 1753 1758. JACOB J. J.. TREMRLAY E. C. & COLOMBEL M. (1974) Facilitation de reactions nociceptives par la naloxone cher la souris et chez le rat. Psychopharmucologia 37. 217.~223. KING C. D.. HUGHEY L. J.. MASSERAND J. M., CODD E. E. & B~RN~ W. L. (1977) Endorphin and naloxone; insomnia and seizures in cats and hyperalgesia in rats. Fedn Proc. Frdn Am. Sots ezp. Biol. 36, 965. KRNJEV~ K. (1974) Chemical nature of synaptic transmission in vertebrates. Physiol. Rec. 54, 41X--540. L,A.MOTT~C.. PERT C. B. & SNYDER S. H. (1976) Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section. Bruin Rrs. 112, 407-412. LEWIS J. W., BENTLEYK. W. & COWAN A. (1971) Narcotic analgesics and antagonists. .4. Rev Phurmuc. 11, 741-270. MARTIN W. R. (1967) Opioid antagonists. Pharmac. Rev. 19, 463-521. MA[ C;H T. H. (1972) Narcotic antagonists: the search accelerates. Scicww. h’. Y. 177. 249 250. MAYER D. J.. PRICI. D. D. & RAFII A. (1977) Antagonism of acupuncture analgesia in man by the narcotic antagonist naloxone.
Bruin
Res. 121, 368-372.
G. R., LING. N.. BLOOM F. E. & GIXLEMIN R. (1977) Neuronal actions of endorphins and cnkephaiins among brain regions: a comparative microiontophoretic study. Proc. nufn. .kud. Sri. U.S.A. 74, 2584 -2588. OLJLERASJ. L.. HOSOBUCXI Y.. REIJJEMI F., GUILBAUD G. & BE:SSONJ. M. (1977) Opiate antagonist naloxone strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior). Brain Rrs. 120, 221~ 229. PASE:RNAK G. W., G~DMAN R. & SNYDER S. H. (1975) An endogenous morphine-like factor in mammalian brain.
NICOLI.
R.
A..
SIGGINS
Ltfe Sci. 16, 1765.. 1769. P~M~RANZ B., CHANG R. & LAW P. (1977) Acupuncture reduces electrophysiological noxious stimuli: pituitary is implicated. Evpl Nrurol. 54, I77- 178. PRI(.I., D. D. & D~J~~NERR. (1977) Neurons that subserve the sensory-discriminative TE&C.IS A. K. (1974) Trrm,snritrers Bristol. Tt:~~:~llis L. & WAHLsTRiiM A. 94. 74 Xl.
and Idtwtified
Ncwrons
in the ~ammulicm
(1975)Search for an endogenous
ligand
and aspects
Cenrrul
for the opiate
behavioural of pain.
R’errow
rcccptor.
responses
to
f’ui!I 3. 307 338.
S~,.sron. Scien technica.
Acttr /J/f \‘\IO/. \c’tIilt/.
Naloxone excites nociceptive units in lumbar dorsal horn
1491
TERENIUSL. 8~ WAHLSTR~~M A. (1976) A method for site selectivity analysis applied to opiate receptors. Eur. J. Pharmacol. 40, 241-248. TE~CHEMACHER H., OPHEIMK. E., Cox B. M. & GOLDSTEINA. (1975) peptide-like substance from pituitary that acts like morphine-I. Isolation. Life Sci. 16, 1771-1776. Wools L. A. (1956) The pharmacology of nalorphine (n-allynormorphine). Pharmac. Rev. 8, 175-198. YAKSH T. L., HUANG S. P., RUDY T. A. & FREDERICK~ON R. C. A. (1977) The direct and specific opiate-like effect of Met’-enkephalin and analogues on the spinal cord. Neuroscience 2, 593-596. ZIEGLG~NSBERGER W. & BAYERLH. (1976) The mechanism of inhibition of neuronal activity by opiates in the spinal cord of cat. Brain Res. 115. 111-128. (Accepted 26 March 1979)
