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Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons
Pain, 44 (1991) 187-193 c’ 1991 Elsevier Science Publishers ADONIS 0304395991000768
187 B.V. 0304-3959/91/$03.50
PAIN 01722
Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons Janice L.K. Hylden, Richard L. Nahin, Richard .J. Traub * and Ronald Dubner Neurobiology
and Anesthesiology
Branch, National Institute
(Received
ofDental Research,
National lnstituies
16 May 1990, revision received 23 July 1990, accepted
of Health, Bethesda, MD 20892 (U.S.A.)
3 August
1990)
Spinal cord application of the K-opioid receptor agonists dyno~hin (50 nmoi) or (lS,2S)U-50,488H Summa~ (0.19-l-9 pmol) produced changes in the excitability of some superficial dorsal horn nociceptive neurons. One-third of the cells exhibited expansion of their receptive fields as defined using mechanical stimuli following a spinal K agonist (dynorphin or U-50,488H); receptive field expansions were of the same order as those observed immediately after a conditioning electrical stimulus applied to a peripheral nerve. In addition, spinal U-50,488H produced changes in mechanical and thermal thresholds of the majority of superficial dorsal horn neurons. These changes were dose-dependent. Facilitation of responses occurred at lower doses and inhibition occurred primarily at higher doses, but these effects were not reversed by subsequent administration of naloxone. The data are consistent with the hypothesis that one action of increases in spinal dyno~hin levels due to peripheral inflammation, tissue injury or nerve damage, is to contribute to enhanced neuronal excitability in superficial dorsal horn neurons. Key words: Spinal
cord; Nociception;
Dynorphin;
U-50,488H;
Introduction
Lamina
I; (Rat)
Many superficial spinal dorsai horn neurons respond to noxious or near-noxious stimulation of their relatively small and well-defined peripheral receptive fields and have been regarded as having an important role in the transmission of pain- and temperature-related information. The response properties of these cells, although characte~stically stable under controlled conditions, are not fixed, but can change under the influence of various peripheral stimuli [4,13,17,20]. We have recently reported on inflammation-induced changes in receptive fields of lamina I projection neurons and have concluded that dorsal horn mechanisms contribute to the plasticity observed in this population of cells [13]. The observed changes in receptive field and excitability of
superficial dorsal horn neurons were closely correlated with the development of behavioral hyperalgesia in this model of inflammation. We also know that there are alterations in the regulation of dorsal horn neuropeptide levels during inflammation. Most notably, there is a large increase in both dynorphin peptide and preprodynorphin mRNA in the lumbar dorsal horn ipsilateral to unilateral hind paw inflammation in the rat [15]. Therefore, we were interested in examining the possible effects of spinally administered dynorphin, or a nonpeptidergic K-opioid agonist (U-50,488H), on the receptive field properties of superficial dorsal horn neurons. A knowledge of the effects of K-opioid agonists on nociceptive dorsal horn neurons allows for a better understanding of the role of spinal dynorphin with respect to in~ammation-induced hyperalgesia.
..-
Methuds
* Current address: Department of Pharmacology, cine, University of Iowa, Iowa City, IA, U.S.A.
College
of Medi-
Correspondence to: Dr. Janice L.K. Hylden, NAB, NIDR, NIH, Building 30, Room B-20, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A.
Rats were initially anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A tracheal cannula was inserted and the femoral vein was cannulated for subsequent intravenous injection of a-chloralose (40-60
mg/kg) and gallamine triethiodide (as needed). Body temperature and expired pC0, were monitored and maintained at normal levels. The lumbar enlargement of the spinal cord was exposed by laminectomy of lumbar vertebrae 1 and 2 and the vertebral column secured by clamps. A small agar reservoir was formed around the exposed spinal cord. The dura was removed and the reservoir was filled with an artificial CSF solution (approximately 0.1 ml). The sciatic nerve was exposed in the upper thigh and suspended on a pair of silver hook electrodes. Extracellular recordings of superficial dorsal horn neurons were made with glass micropipettes (2 M NaCl, 5-10 MSZ). Electrical stimulation of the sciatic nerve (1 msec pulses at 4 mA. 0.5 Hz) was used as a search stimulus. Cells were characterized with respect to their responses to tactile and thermal stimulation of their receptive fields. Receptive fields were carefully mapped using blunt probes and small serrated forceps and drawn to scale on a standard diagram. Mechanical thresholds were determined using calibrated von Frey filaments. Thermal thresholds were determined by positioning a contact thermode at the center of the receptive field and applying a slowly increasing thermal stimulus (l3”C/sec) from a baseline of 35’C to a maximum of 50 or 52°C. Thermal inter-stimulus intervals were more than 5 min for any given receptive field. Data were stored on tape for later analysis and computer-assisted generation of histograms. Stimulation and recording techniques and procedures used for characterization of cells have been reported previously in greater detail [13]. After each superficial dorsal horn cell was identified and control responses were recorded, the CSF solution covering the spinal cord was carefully removed with absorbant cotton gauze and replaced with 50-100 ~1 of drug solution. Responses to cutaneous stimuli were recorded at various times beginning 5 min after drug administration. The test drugs were dissolved in artificial CSF solution.
antidromically activated lamina 1 projection neurons previously recorded in chloralose-anesthetized ratx [I 31. Therefore. we can assume that these cells were located in lamina I or II. All cells were activated by electrical stimulati~~n of the sciatic nerve at latencies consistent with myelinated (50%). uiln~yeiinated (X?6) or myelinated and unmyelinated (42%) afferent drive. Calculated conduction velocities of myelinated afferents were consistent with AS input. Receptive fields were located on the hindpaw - examples are illustrated in Fig. 1. We recorded from 15 superficial dorsal horn cells before and after administration of dynorphin to the spinal cord (50 ~1 of a 10-j M solution. total dose = 50 nmol, of either dynorphin A( l--8) or dynorphin A( 1 17)). In 5 of these 15 cells there was an expansion of the receptive field (as defined using mechanical stimuli) onto an adjacent toe and/or pad at 5 min after dynorphin treatment (Fig. 1A and Table I); the other 10
6.
Ubo,488H
c.
ELECTRICAL
Rest&s Data obtained from 43 superficial dorsaI horn cells were included in this study. All cells responded to high threshold mechanical stimuli (noxious or near-noxious pressure or pinch) and were classified as nociceptivespecific (n = 40) or wide-dynamic-range (n = 3). Mechanical thresholds were 36-290 mN (mean, 150 mN) for the nociceptive-specific cells and 4-15 mN for the wide-dyna~c-range cells. The majority of cells also responded to noxious thermal stimulation (n = 33; mean threshold, 47“ C; range 41-50° C). These cells had response properties and recording depths ( < 500 pm and dorsal to the A/3 fiber electrically evoked field potential observed in lamina III) similar to a population of
Fig. 1. Receptive fields of superficial dorsal horn cells that exhibited expansion after spinal dynorphin (A), spinal (lS,2S)U-50,488H (B) or electrical stimulation of the sciatic nerve (C). Shaded areas represent receptive fields prior to manipulation. Filled areas indicate the maximum extent of the expanded portion of the receptive field.
189
TABLE
I
(A) EXPANSION OF RECEPTIVE FIELDS IN RESPONSE SPINAL K AGONIST OR PERIPHE~L STIMULATION
.~ Dynorphin (IS,ZS)U-50,488H Electrical stimulation
Expanded a receptive field
W increase in receptive field size, mean (range)
s/15 8/23 12/23
52 (26-100) 31 (7- 58) 77 (2-160)
(B) EFFECT OF SPINAL (lS,2S)U-50,488H SPONSIVENESS (n = 23 cells) Facilitation
Receptive field size Mechanical Thermal
8 8 4
b
Inhibition
1 4 7
b
ON
CELL
Facilitation/ i~bition b
No change
0 0 5
14 11 5c
TO
RE-
b
’ Number of celis responding over total cells tested. h Number of cells responding out of 23 cells tested. ’ Two additional cells had no initial response to heat.
cells showed no changes in receptive field size. Receptive field changes were followed as long as 15-50 mm. There were no apparent differences in the effects of dynorphin A(l-8) and dynorphin A(l-17) with respect to receptive field expansion. No changes in mechanical thresholds were noted in superficial dorsal horn cells following spinal administration of dynorphin. Three cells were checked for thermal responsiveness after dynorphin. One of these cells demonstrated an increase in thermal threshold (from 41 to 46”C, at 10 min) that began to recover by 30 mm after dynorphin administration; this cell did not have an expanded receptive field. A separate group of 23 cells was studied before and after ad~nistration of the active isomer of the K agonist U-50,488H ((lS,2S)U-50,488H tartrate [S] was a generous gift from Dr. B. DeCosta). Nine of these 23 cells exhibited a change in receptive field size at 5-10 min after spinal administration of U-50,488H (100 ~1 of a 1.9 or 19 mM solution, total dose = 0.19 or 1.9 pmol): 8 cells had an expanded receptive field (Fig. 1B and Table I) and 1 cell showed a diminished receptive field size. Changes in receptive field size remained as long as the pool of U-50,488H was maintained (up to 40 min). Cells that were studied after application of U-50, 488H were likely to exhibit changes in responsiveness to mechanical stimuli, often in conjunction with receptive field expansion. Eight cells (5 of which had expanded receptive fields) exhibited a decreased mechanical threshold on the order of 150-200 mN and 4 cells had increased mechanical thresholds (> 50 mN) after U-50, 488H. Thus, the majority of cells that demonstrated enhanced mechanical sensitivity also had expanded receptive fields; expansion of receptive field was never
accompanied by a decreased responsiveness to mechanical stimuli. Twenty-one of the 23 cells studied before and after U-50,488H had an initial response to heat (thresholds ranged from 42 to 5O’C). Ten to 15 min after application of U-50,488H, 9 cells demonstrated a facilitation of the heat-evoked response as-evidenced by a decrease in thermal threshold and/or an increase in the number of spikes elicited by a given thermal stimulus. Facilitation of thermal responsiveness was often accompanied by facilitation of mechanical responsiveness (5 of 9 cells; an example is shown in Fig. 2), but never by inhibition of the response to mechanical stimuli. The neuron in Fig. 2A-D demonstrated an enhanced response to application of a large arterial clamp to the receptive field. This stimulus was considered to be near-noxious by the investigators. This cell also had an enhanced response to noxious heat (Fig. 2E-H). Five of the cells demonstrating decreased thermal thresholds at an early time point or following lower doses of U-50,488H demonstrated an inhibition of the thermal response at a later time point (20 min post-U-50,488H) or to a higher dose of U-50,488H (Fig. 3). Seven other cells also exhibited inhibition of the thermal response (15-20 min post-U50,488H, n = 5 at 19 FM and 2 at 1.9 PM). We were unable to reverse either the inhibitory (n = 3) or facilitatory (n = 2) effects of U-50,488H by administration of naloxone (1 or 10 mg/kg, i.v.; Fig. 2). Recovery of thermal and/or mechanical responses was only observed after removal of U-50,488H from the pool and washing the spinal cord with artificial CSF. Recovery was not observed until 45-90 min after washing (n = 3, Fig. 3). Electrical stimulation of peripheral nerves is known to produce changes in the receptive fields of superficial dorsal horn cells 141. We examined the receptive fields of 23 cells before and after electrical stimulation of the sciatic nerve in order to compare drug-induced receptive field changes to stimulation-induced receptive field changes in our preparation. Twelve of the 23 superficial dorsal horn cells given a standardized barrage of electrical pulses (0.5 msec, 5 mA at 1 Hz for 20 set) demonstrated expanded receptive fields immediately after stimulation and lasting less than 10 mm (2.5 + 1.4 min, n = 12). The fields expanded onto adjacent toes or pads, as was also observed after drug treatment (Fig. 1). The majority of cells (> 75%) showing expanded receptive fields after electrical stimulation or drug treatment increased the area of their receptive field by 25-100%. The mean percent increase in receptive field (Table I), due to electrical stimulation of the sciatic nerve (77%), was slightly higher than that observed after dynorphin (52%) or U-50,488H (31%). The degree of expansion due to electrical stimulation or drug was somewhat less than that observed by Cook et al. [4] after stimulation of a muscle nerve and less than we
IYll
A. Control response to mechanical stimuli. 60
! PO 3 E 20 cn
F. 13 min after U50,4f8 (787 nmol). 6o
B.
5s@c
11 min affer U50,468 (187 nmol). 60
25
60~ C.
22 min after ~50,466.
10.
L 8.
D.
1
26 min after U50,468. /7
s
Nabxone (t mg&g, i.v.) at 30 min after U5UA86.
01
I
1 H. Naioxone (I rn~g)
25 a25 min after ~50,466. r55
60 45
Fig 2. Mechanical and thermal facilitation. Standard mechanical stimuli (A-D) consisted of applying a large arterial clamp (L) to the receptive field for 6 set followed by a similar application of a small arterial clamp (S). Some responses to probing with von Frey filaments (85 and 120 mN. arrows below abscissa) are also included on records A-D. Histograms E-H represent responses to a thermal stimulus. The thermal trace is indicated in each panel and consisted of a controlled ramp of 3”C/sec delivered by a contact thermode from a baseline of 35 o C to a maximum of 52°C (2 set plateau) with a rapid return to baseline. The ordinate on the right indicates the temperature at the thermodefskin interface). Thermal thresholds (temperature at which neuronal activity exceeded background) are indicated with arrows and temperature labels. Both the mechanical (response to L in B and C) and thermal (F and G) responses of this nociceptive-specific cell were facilitated at 11-26 min after spinal application of 187 nmol U-50.488H. This facilitation did not appear to be reversed by naloxone (D and H).
191
have seen during hind paw inflammation
[13]. We have never seen receptive field changes of this magnitude over a 5-60 min period while recording from lamina I cells under control conditions in rat or cat [12,13].
Discussion The observed changes in receptive fields to mechanistimuli and in mechanical and thermal thresholds after spinal U-50,488H or dynorphin indicated that there were 2 general responses on the part of superficial dorsal horn cells to K agonists: (1) a net increase in responsiveness to mechanical and/or thermal stimuli, decreased threshold(s) that were often accompanied by an expansion in receptive field; (2) a net decrease in responsiveness, increase in thermal threshold that was accompanied by an increased mechanical threshold in some cells. Dual effects of K agonists on dorsal horn neurons have also been reported by Knox and Dickenson 1161; they observed that spinally administered dynorphin and U-50,488H produced facilitation or inhibition of C-fiber
cal
8. I 5 min after UF~”
(1~ rmot).
55 I-
60 45 40 35 20 25 0 55
35
25
D. 15 mtn after ~50,433
(1 .g
r
rmot).
-45
40-
-.-
20-
01
55
I
I
35
25
60qE. 60 mln aflerwash. 4o-
20-,
0
I
Fig. 3. U-50,488H-induced facilitation/inhibition. The dose-dependent nature of facilitation and inhibition due to W50,488H are illustrated in the responses of this nokeptive-specific neuron. The response to a SOT heat pulse was enhanced by a low dose (B, 187 nmol) and inhibited by higher doses (C and D, 560 nmol and 1.9 pmol) of U-50,488H. The thermal response returned after washing the exposed spinal cord with artificial CSF (E).
evoked responses in cells that responded to both innocuous and noxious stimulation. Inhibition of C-fiber evoked activity is the apparent mechanism by which spinally administered is p- and b-opioid receptor agonists [6] produce an inhibition of dorsal horn neurons
and may similarly be responsible for the decreased response to noxious stimuli observed in some cells in the present study after a K-opioid agonist. This antinociceptive action of opioids has been associated with a reduction in receptive field size of dorsal horn spinothalamic cells [lo]. Conversely, facilitation of C-fiber evoked responses would be expected to produce a net increase in responsiveness to noxious stimuli [16] and may be associated with the decreased mechanical and thermal thresholds and expanded receptive fields observed in some superficial dorsal horn cells after spinal administration of K-opioid agonists. Caudle and Isaac
[2] have also observed a short-term facilitatory effect of dynorphin on C-fiber evoked reflex activity. This facilitation was followed by a profound in~bition. Their data suggest that the facilitation of spinal reflexes involves excitation of N-methyl-D-aspartate (NMDA) receptors. Facilitation of nociceptive input may be responsible for the antianalgesic effect of spinal dynorphin observed by Fujimoto and Rady [9]. It is possible that the two types of responses to K agonists represent activation of two functionally different populations of cells in the superficial dorsal horn. Alternatively, the two responses may reflect interaction of the agonists at more than one receptor type. In this study, we were unable to reverse the effects of U-50,488H by a moderate or high dose of naloxone. This may indicate that the observed effects of U-
lY2
50,488~I were not mediated by classical opioid receptors or that the rather large doses of U-50,488H in a concentrated location could not be sufficiently displaced by post-administration of the antagonist. Using similar routes of administration, naloxone did not consistently reverse the inhibitory effects of morphine 1141. In addition, Knox and Dickenson also found that the effects of intrathecal IC agonists were not antagonized by intrathecal naloxone [16]. These authors suggested that excitation and inhibition of dorsal horn neurons by U-50,488H and dyno~hin are mediated by K-receptor activation, which has a low affinity for naloxone, or are partly non-opioid in nature. Caudle and Isaac [2] have suggested that dynorphin-induced potentiation and subsequent loss of C-fiber reflexes involves an excitotoxic event, likely mediated at NMDA sites. A similar mechanism may account for the excitability changes observed in the present study. Application of K agonists to the spinal cord may initiate the release of neurotransmitters that mediate the observed facilitation/inhibition of dorsal horn neurons, perhaps by an excitotoxic event. Short-term administration of opioid antagonists after initiation of this event would have little or no effect. The neurotoxic effect of dynorphin, as indicated by hind limb flaccidity, is not consistently prevented or reversed by naloxone [11,19,21,22]. The selective Kopioid agonist U-50,488H, even when administered at a high dose, does not mimic the paralytic actions of dynorphin [19,22]. Therefore, it has been suggested that this particular pathophysiologic consequence of intrathecal dynorphin administration is not mediated by rc-opioid receptors and in fact may involve alterations in blood flow resulting from non-opioid or combined opioid and non-opioid actions of dynorphin 1181. The effects of dynorphin and U-50.488H on individual dorsal horn neurons, as observed in the present study and observed by Knox and Dickenson 1161, were similar and may not be directly related to the ultimate expression of hind limb paralysis by only one of these agents. A conditioning volley from C fiber afferents can have profound effects on receptive field size [4], as can peripheral tissue injury due to punctate burns to the skin [20] or inflammation [13]. We have now shown that K-opioid receptor agonists also effect the receptive fields of superficial dorsal horn neurons. Since there is a large increase in dorsal horn levels of dynorphin in animals with peripheral inflammation [15] or following partial peripheral nerve damage [I], peripheral nerve transection [3] or spinal cord injury [8], we postulate that dynorphin levels are related to enhanced excitability and development of expanded receptive fields following various types of injurious stimulation. Increased nociceptive activity in the periphery may lead to excessive depolarization via excitation at NMDA receptor sites. Dynorphin may enhance this excitability and, in conjunction with other neuropeptides, promote excitotoxic-
ity. This hypothesized chain of events would have irnportant consequences with respect to central mechanisms of inflammation or injury-induced hyperalgesia [7]. Unchecked excitotoxicity in the spinal cord likely effects small inhibitory neurons to a greater extent than larger cells. A net loss of small inhibitory interneurons would produce a shift in the excitability of remaining neurons. Such an excitability shift would contribute to the observed phenomena of receptive field expansion and behavioral hyperalgesia.
References 1
2
3
4
5
6
13
14
Bennett, G.J.. Kajander, K.C., Sahara,
Y., Iadarola, M.J. and Sugimoto, T., Nemochemical and anatomical changes in the dorsal horn of rats with an experimental painful peripheral ncuropathy. In: F. Cervero, G.J. Bennett and P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 463-471. Caudle, R.M. and Isaac, L., Influence of dynorphin(l-13) on spinal reflexes in the rat, J. Phannacol. Exp. Ther., 246 (1988) 5088513. Cho. H.J. and Basbaum, A.I., increased staining of immunoreactive dynorphin cefl bodies in the deafferented spinal cord of the rat, Neurosci. Lett., 84 (1988) 125-130. Cook, A.J., Woolf, C.J., Wall. P.D. and McMahon, S.B.. Dynamic receptive field plasticity in rat spinal cord dorsal horn following C-primary afferent input, Nature, 325 (1987) 151-153. DeCosta, B., George, C., Rothman, R.B., Jacobson, A.E. and Rice, K.C., Synthesis and absolute configuration of optically pure enantiomers of a k-opioid receptor selective agonist, Fed. Eur. Biothem. Sot. Lett., 223 (1987) 335-339. Dickenson, A.H., Sullivan, A.F., Knox, R., Zajac, J.M. and Roques, B.P., Opioid receptor subtypes m the rat spinal cord: electrophysiological studies with p- and I-opioid receptor agonists in the control of nociception, Brain Res. 413 (1987) 36-44. Dubner, R., Neuronal plasticity and pain foflowing peripheral inflammation or nerve injury. In: M. Bond (Ed.), Proc. VIth World Congress on Pain. Elsevier, Amsterdam, in press. Faden, A.I., Mohneaux, C.J.. Rosenberger, J.G., Jacobs, T.P. and Cox, B.M., Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury, Ann. Neural., 17 (1985) 386-390. Fujimoto, J.M. and Rady, J.J., Intracerebroventricular physostigmine-induced analgesia: enhanced by naloxone, B-funaltrexamine and nor-binaltorphimine and antagonism by dynorphin A(l-17), J. Pharmacol. Fxp. Ther., 251 (1989) 1045-1052. Hayes, R.L.. Price, D.D., Ruda, M. and Dubner, R., Suppression of nociceptive responses in the primate by e&trial stimulation of the brain or morphine administration: behavioral and electrophysioiogical comparisons, Brain Res.. 167 (1979) 417-421. Herman, B.H. and Goldstein, A., Antin~iception and paralysis induced by intrathecal dynorphin A. J. Pharmacol. Exp. Ther., 232 (1985) 27-32. Hylden, J.L.K., Hayashi, H., Dubner, R. and Bennett, G.J., Physiology and morphology of the lamina I spinomesencephahc projection, J. Comp. Neural., 247 (1986) 505-515. Hylden, J.L.K., Nabin, R.L., Traub, R.J. and Dubner, R., Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms, Pain, 37 (1989) 229-243. Hylden, J.L.K. and Wilcox. G.L., Antinociceptive effect of morphine on rat spinothalamic tract and other dorsal horn neurons, Neuroscience, 19 (1986) 393-401.
193 15 Iadarola, M.J., Brady, L.S., Draisci, G. and Dubner, R., Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding, Pain, 35 (1988) 313-326. 16 Knox, R.J. and Dickenson, AH., Effects of selective and nonselective s-opioid receptor agonists on cutaneous C-fibre-evoked responses of rat dorsal horn neurones, Brain Res., 415 (1987) 21-29. 17 Laird, J. and Cervero, F., A comparison of the receptive field plasticity of nocireceptive and multireceptive dorsal horn neurones following noxious mechanical stimulation. In: F. Cervero, G.J. Bennett and P.M. Headley @is.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 473-476. 18 Long, J.B., Kinney, R.C., Malcolm, D.S., Graeber, G.M. and Holaday, J.W., Intrathecal dynorpbin Ai_is and dynorphin A,_,, reduce rat spinal cord blood flow by non-opioid mechanisms, Brain Res., 436 (1987) 374-379.
19 Long, J.B., Petras, J.M., Mobley, W.C. and Holaday, J.W., Neurological dysfunction after intrathecal injection of dynorphin A(l-13) in the rat. II. Nonopioid mechanisms mediate loss of motor, sensory and autonomic function, J. Pharmacol. Exp. Ther., 246 (1988) 1167-1174. 20 McMahon, S.B. and Wall, P.D., Receptive fields of rat lamina I projection cells move to incorporate a nearby region of injury, Pain, 19 (1984) 235-247. 21 Spamipinato, S. and Candeletti, S., Characterization of dynorphin A-induced antinociception at spinal levels, Eur. J. Pharmacol., 110 (1985) 21-30. 22 Stevens, C.W. and Yaksh, T.L., Dynorphin A and related peptides administered intrathecally in the rat: a search for putative kappa opiate receptor activity, J. Pharmacol. Exp. Ther., 238 (1986) 833-838.
187 B.V. 0304-3959/91/$03.50
PAIN 01722
Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons Janice L.K. Hylden, Richard L. Nahin, Richard .J. Traub * and Ronald Dubner Neurobiology
and Anesthesiology
Branch, National Institute
(Received
ofDental Research,
National lnstituies
16 May 1990, revision received 23 July 1990, accepted
of Health, Bethesda, MD 20892 (U.S.A.)
3 August
1990)
Spinal cord application of the K-opioid receptor agonists dyno~hin (50 nmoi) or (lS,2S)U-50,488H Summa~ (0.19-l-9 pmol) produced changes in the excitability of some superficial dorsal horn nociceptive neurons. One-third of the cells exhibited expansion of their receptive fields as defined using mechanical stimuli following a spinal K agonist (dynorphin or U-50,488H); receptive field expansions were of the same order as those observed immediately after a conditioning electrical stimulus applied to a peripheral nerve. In addition, spinal U-50,488H produced changes in mechanical and thermal thresholds of the majority of superficial dorsal horn neurons. These changes were dose-dependent. Facilitation of responses occurred at lower doses and inhibition occurred primarily at higher doses, but these effects were not reversed by subsequent administration of naloxone. The data are consistent with the hypothesis that one action of increases in spinal dyno~hin levels due to peripheral inflammation, tissue injury or nerve damage, is to contribute to enhanced neuronal excitability in superficial dorsal horn neurons. Key words: Spinal
cord; Nociception;
Dynorphin;
U-50,488H;
Introduction
Lamina
I; (Rat)
Many superficial spinal dorsai horn neurons respond to noxious or near-noxious stimulation of their relatively small and well-defined peripheral receptive fields and have been regarded as having an important role in the transmission of pain- and temperature-related information. The response properties of these cells, although characte~stically stable under controlled conditions, are not fixed, but can change under the influence of various peripheral stimuli [4,13,17,20]. We have recently reported on inflammation-induced changes in receptive fields of lamina I projection neurons and have concluded that dorsal horn mechanisms contribute to the plasticity observed in this population of cells [13]. The observed changes in receptive field and excitability of
superficial dorsal horn neurons were closely correlated with the development of behavioral hyperalgesia in this model of inflammation. We also know that there are alterations in the regulation of dorsal horn neuropeptide levels during inflammation. Most notably, there is a large increase in both dynorphin peptide and preprodynorphin mRNA in the lumbar dorsal horn ipsilateral to unilateral hind paw inflammation in the rat [15]. Therefore, we were interested in examining the possible effects of spinally administered dynorphin, or a nonpeptidergic K-opioid agonist (U-50,488H), on the receptive field properties of superficial dorsal horn neurons. A knowledge of the effects of K-opioid agonists on nociceptive dorsal horn neurons allows for a better understanding of the role of spinal dynorphin with respect to in~ammation-induced hyperalgesia.
..-
Methuds
* Current address: Department of Pharmacology, cine, University of Iowa, Iowa City, IA, U.S.A.
College
of Medi-
Correspondence to: Dr. Janice L.K. Hylden, NAB, NIDR, NIH, Building 30, Room B-20, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A.
Rats were initially anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A tracheal cannula was inserted and the femoral vein was cannulated for subsequent intravenous injection of a-chloralose (40-60
mg/kg) and gallamine triethiodide (as needed). Body temperature and expired pC0, were monitored and maintained at normal levels. The lumbar enlargement of the spinal cord was exposed by laminectomy of lumbar vertebrae 1 and 2 and the vertebral column secured by clamps. A small agar reservoir was formed around the exposed spinal cord. The dura was removed and the reservoir was filled with an artificial CSF solution (approximately 0.1 ml). The sciatic nerve was exposed in the upper thigh and suspended on a pair of silver hook electrodes. Extracellular recordings of superficial dorsal horn neurons were made with glass micropipettes (2 M NaCl, 5-10 MSZ). Electrical stimulation of the sciatic nerve (1 msec pulses at 4 mA. 0.5 Hz) was used as a search stimulus. Cells were characterized with respect to their responses to tactile and thermal stimulation of their receptive fields. Receptive fields were carefully mapped using blunt probes and small serrated forceps and drawn to scale on a standard diagram. Mechanical thresholds were determined using calibrated von Frey filaments. Thermal thresholds were determined by positioning a contact thermode at the center of the receptive field and applying a slowly increasing thermal stimulus (l3”C/sec) from a baseline of 35’C to a maximum of 50 or 52°C. Thermal inter-stimulus intervals were more than 5 min for any given receptive field. Data were stored on tape for later analysis and computer-assisted generation of histograms. Stimulation and recording techniques and procedures used for characterization of cells have been reported previously in greater detail [13]. After each superficial dorsal horn cell was identified and control responses were recorded, the CSF solution covering the spinal cord was carefully removed with absorbant cotton gauze and replaced with 50-100 ~1 of drug solution. Responses to cutaneous stimuli were recorded at various times beginning 5 min after drug administration. The test drugs were dissolved in artificial CSF solution.
antidromically activated lamina 1 projection neurons previously recorded in chloralose-anesthetized ratx [I 31. Therefore. we can assume that these cells were located in lamina I or II. All cells were activated by electrical stimulati~~n of the sciatic nerve at latencies consistent with myelinated (50%). uiln~yeiinated (X?6) or myelinated and unmyelinated (42%) afferent drive. Calculated conduction velocities of myelinated afferents were consistent with AS input. Receptive fields were located on the hindpaw - examples are illustrated in Fig. 1. We recorded from 15 superficial dorsal horn cells before and after administration of dynorphin to the spinal cord (50 ~1 of a 10-j M solution. total dose = 50 nmol, of either dynorphin A( l--8) or dynorphin A( 1 17)). In 5 of these 15 cells there was an expansion of the receptive field (as defined using mechanical stimuli) onto an adjacent toe and/or pad at 5 min after dynorphin treatment (Fig. 1A and Table I); the other 10
6.
Ubo,488H
c.
ELECTRICAL
Rest&s Data obtained from 43 superficial dorsaI horn cells were included in this study. All cells responded to high threshold mechanical stimuli (noxious or near-noxious pressure or pinch) and were classified as nociceptivespecific (n = 40) or wide-dynamic-range (n = 3). Mechanical thresholds were 36-290 mN (mean, 150 mN) for the nociceptive-specific cells and 4-15 mN for the wide-dyna~c-range cells. The majority of cells also responded to noxious thermal stimulation (n = 33; mean threshold, 47“ C; range 41-50° C). These cells had response properties and recording depths ( < 500 pm and dorsal to the A/3 fiber electrically evoked field potential observed in lamina III) similar to a population of
Fig. 1. Receptive fields of superficial dorsal horn cells that exhibited expansion after spinal dynorphin (A), spinal (lS,2S)U-50,488H (B) or electrical stimulation of the sciatic nerve (C). Shaded areas represent receptive fields prior to manipulation. Filled areas indicate the maximum extent of the expanded portion of the receptive field.
189
TABLE
I
(A) EXPANSION OF RECEPTIVE FIELDS IN RESPONSE SPINAL K AGONIST OR PERIPHE~L STIMULATION
.~ Dynorphin (IS,ZS)U-50,488H Electrical stimulation
Expanded a receptive field
W increase in receptive field size, mean (range)
s/15 8/23 12/23
52 (26-100) 31 (7- 58) 77 (2-160)
(B) EFFECT OF SPINAL (lS,2S)U-50,488H SPONSIVENESS (n = 23 cells) Facilitation
Receptive field size Mechanical Thermal
8 8 4
b
Inhibition
1 4 7
b
ON
CELL
Facilitation/ i~bition b
No change
0 0 5
14 11 5c
TO
RE-
b
’ Number of celis responding over total cells tested. h Number of cells responding out of 23 cells tested. ’ Two additional cells had no initial response to heat.
cells showed no changes in receptive field size. Receptive field changes were followed as long as 15-50 mm. There were no apparent differences in the effects of dynorphin A(l-8) and dynorphin A(l-17) with respect to receptive field expansion. No changes in mechanical thresholds were noted in superficial dorsal horn cells following spinal administration of dynorphin. Three cells were checked for thermal responsiveness after dynorphin. One of these cells demonstrated an increase in thermal threshold (from 41 to 46”C, at 10 min) that began to recover by 30 mm after dynorphin administration; this cell did not have an expanded receptive field. A separate group of 23 cells was studied before and after ad~nistration of the active isomer of the K agonist U-50,488H ((lS,2S)U-50,488H tartrate [S] was a generous gift from Dr. B. DeCosta). Nine of these 23 cells exhibited a change in receptive field size at 5-10 min after spinal administration of U-50,488H (100 ~1 of a 1.9 or 19 mM solution, total dose = 0.19 or 1.9 pmol): 8 cells had an expanded receptive field (Fig. 1B and Table I) and 1 cell showed a diminished receptive field size. Changes in receptive field size remained as long as the pool of U-50,488H was maintained (up to 40 min). Cells that were studied after application of U-50, 488H were likely to exhibit changes in responsiveness to mechanical stimuli, often in conjunction with receptive field expansion. Eight cells (5 of which had expanded receptive fields) exhibited a decreased mechanical threshold on the order of 150-200 mN and 4 cells had increased mechanical thresholds (> 50 mN) after U-50, 488H. Thus, the majority of cells that demonstrated enhanced mechanical sensitivity also had expanded receptive fields; expansion of receptive field was never
accompanied by a decreased responsiveness to mechanical stimuli. Twenty-one of the 23 cells studied before and after U-50,488H had an initial response to heat (thresholds ranged from 42 to 5O’C). Ten to 15 min after application of U-50,488H, 9 cells demonstrated a facilitation of the heat-evoked response as-evidenced by a decrease in thermal threshold and/or an increase in the number of spikes elicited by a given thermal stimulus. Facilitation of thermal responsiveness was often accompanied by facilitation of mechanical responsiveness (5 of 9 cells; an example is shown in Fig. 2), but never by inhibition of the response to mechanical stimuli. The neuron in Fig. 2A-D demonstrated an enhanced response to application of a large arterial clamp to the receptive field. This stimulus was considered to be near-noxious by the investigators. This cell also had an enhanced response to noxious heat (Fig. 2E-H). Five of the cells demonstrating decreased thermal thresholds at an early time point or following lower doses of U-50,488H demonstrated an inhibition of the thermal response at a later time point (20 min post-U-50,488H) or to a higher dose of U-50,488H (Fig. 3). Seven other cells also exhibited inhibition of the thermal response (15-20 min post-U50,488H, n = 5 at 19 FM and 2 at 1.9 PM). We were unable to reverse either the inhibitory (n = 3) or facilitatory (n = 2) effects of U-50,488H by administration of naloxone (1 or 10 mg/kg, i.v.; Fig. 2). Recovery of thermal and/or mechanical responses was only observed after removal of U-50,488H from the pool and washing the spinal cord with artificial CSF. Recovery was not observed until 45-90 min after washing (n = 3, Fig. 3). Electrical stimulation of peripheral nerves is known to produce changes in the receptive fields of superficial dorsal horn cells 141. We examined the receptive fields of 23 cells before and after electrical stimulation of the sciatic nerve in order to compare drug-induced receptive field changes to stimulation-induced receptive field changes in our preparation. Twelve of the 23 superficial dorsal horn cells given a standardized barrage of electrical pulses (0.5 msec, 5 mA at 1 Hz for 20 set) demonstrated expanded receptive fields immediately after stimulation and lasting less than 10 mm (2.5 + 1.4 min, n = 12). The fields expanded onto adjacent toes or pads, as was also observed after drug treatment (Fig. 1). The majority of cells (> 75%) showing expanded receptive fields after electrical stimulation or drug treatment increased the area of their receptive field by 25-100%. The mean percent increase in receptive field (Table I), due to electrical stimulation of the sciatic nerve (77%), was slightly higher than that observed after dynorphin (52%) or U-50,488H (31%). The degree of expansion due to electrical stimulation or drug was somewhat less than that observed by Cook et al. [4] after stimulation of a muscle nerve and less than we
IYll
A. Control response to mechanical stimuli. 60
! PO 3 E 20 cn
F. 13 min after U50,4f8 (787 nmol). 6o
B.
5s@c
11 min affer U50,468 (187 nmol). 60
25
60~ C.
22 min after ~50,466.
10.
L 8.
D.
1
26 min after U50,468. /7
s
Nabxone (t mg&g, i.v.) at 30 min after U5UA86.
01
I
1 H. Naioxone (I rn~g)
25 a25 min after ~50,466. r55
60 45
Fig 2. Mechanical and thermal facilitation. Standard mechanical stimuli (A-D) consisted of applying a large arterial clamp (L) to the receptive field for 6 set followed by a similar application of a small arterial clamp (S). Some responses to probing with von Frey filaments (85 and 120 mN. arrows below abscissa) are also included on records A-D. Histograms E-H represent responses to a thermal stimulus. The thermal trace is indicated in each panel and consisted of a controlled ramp of 3”C/sec delivered by a contact thermode from a baseline of 35 o C to a maximum of 52°C (2 set plateau) with a rapid return to baseline. The ordinate on the right indicates the temperature at the thermodefskin interface). Thermal thresholds (temperature at which neuronal activity exceeded background) are indicated with arrows and temperature labels. Both the mechanical (response to L in B and C) and thermal (F and G) responses of this nociceptive-specific cell were facilitated at 11-26 min after spinal application of 187 nmol U-50.488H. This facilitation did not appear to be reversed by naloxone (D and H).
191
have seen during hind paw inflammation
[13]. We have never seen receptive field changes of this magnitude over a 5-60 min period while recording from lamina I cells under control conditions in rat or cat [12,13].
Discussion The observed changes in receptive fields to mechanistimuli and in mechanical and thermal thresholds after spinal U-50,488H or dynorphin indicated that there were 2 general responses on the part of superficial dorsal horn cells to K agonists: (1) a net increase in responsiveness to mechanical and/or thermal stimuli, decreased threshold(s) that were often accompanied by an expansion in receptive field; (2) a net decrease in responsiveness, increase in thermal threshold that was accompanied by an increased mechanical threshold in some cells. Dual effects of K agonists on dorsal horn neurons have also been reported by Knox and Dickenson 1161; they observed that spinally administered dynorphin and U-50,488H produced facilitation or inhibition of C-fiber
cal
8. I 5 min after UF~”
(1~ rmot).
55 I-
60 45 40 35 20 25 0 55
35
25
D. 15 mtn after ~50,433
(1 .g
r
rmot).
-45
40-
-.-
20-
01
55
I
I
35
25
60qE. 60 mln aflerwash. 4o-
20-,
0
I
Fig. 3. U-50,488H-induced facilitation/inhibition. The dose-dependent nature of facilitation and inhibition due to W50,488H are illustrated in the responses of this nokeptive-specific neuron. The response to a SOT heat pulse was enhanced by a low dose (B, 187 nmol) and inhibited by higher doses (C and D, 560 nmol and 1.9 pmol) of U-50,488H. The thermal response returned after washing the exposed spinal cord with artificial CSF (E).
evoked responses in cells that responded to both innocuous and noxious stimulation. Inhibition of C-fiber evoked activity is the apparent mechanism by which spinally administered is p- and b-opioid receptor agonists [6] produce an inhibition of dorsal horn neurons
and may similarly be responsible for the decreased response to noxious stimuli observed in some cells in the present study after a K-opioid agonist. This antinociceptive action of opioids has been associated with a reduction in receptive field size of dorsal horn spinothalamic cells [lo]. Conversely, facilitation of C-fiber evoked responses would be expected to produce a net increase in responsiveness to noxious stimuli [16] and may be associated with the decreased mechanical and thermal thresholds and expanded receptive fields observed in some superficial dorsal horn cells after spinal administration of K-opioid agonists. Caudle and Isaac
[2] have also observed a short-term facilitatory effect of dynorphin on C-fiber evoked reflex activity. This facilitation was followed by a profound in~bition. Their data suggest that the facilitation of spinal reflexes involves excitation of N-methyl-D-aspartate (NMDA) receptors. Facilitation of nociceptive input may be responsible for the antianalgesic effect of spinal dynorphin observed by Fujimoto and Rady [9]. It is possible that the two types of responses to K agonists represent activation of two functionally different populations of cells in the superficial dorsal horn. Alternatively, the two responses may reflect interaction of the agonists at more than one receptor type. In this study, we were unable to reverse the effects of U-50,488H by a moderate or high dose of naloxone. This may indicate that the observed effects of U-
lY2
50,488~I were not mediated by classical opioid receptors or that the rather large doses of U-50,488H in a concentrated location could not be sufficiently displaced by post-administration of the antagonist. Using similar routes of administration, naloxone did not consistently reverse the inhibitory effects of morphine 1141. In addition, Knox and Dickenson also found that the effects of intrathecal IC agonists were not antagonized by intrathecal naloxone [16]. These authors suggested that excitation and inhibition of dorsal horn neurons by U-50,488H and dyno~hin are mediated by K-receptor activation, which has a low affinity for naloxone, or are partly non-opioid in nature. Caudle and Isaac [2] have suggested that dynorphin-induced potentiation and subsequent loss of C-fiber reflexes involves an excitotoxic event, likely mediated at NMDA sites. A similar mechanism may account for the excitability changes observed in the present study. Application of K agonists to the spinal cord may initiate the release of neurotransmitters that mediate the observed facilitation/inhibition of dorsal horn neurons, perhaps by an excitotoxic event. Short-term administration of opioid antagonists after initiation of this event would have little or no effect. The neurotoxic effect of dynorphin, as indicated by hind limb flaccidity, is not consistently prevented or reversed by naloxone [11,19,21,22]. The selective Kopioid agonist U-50,488H, even when administered at a high dose, does not mimic the paralytic actions of dynorphin [19,22]. Therefore, it has been suggested that this particular pathophysiologic consequence of intrathecal dynorphin administration is not mediated by rc-opioid receptors and in fact may involve alterations in blood flow resulting from non-opioid or combined opioid and non-opioid actions of dynorphin 1181. The effects of dynorphin and U-50.488H on individual dorsal horn neurons, as observed in the present study and observed by Knox and Dickenson 1161, were similar and may not be directly related to the ultimate expression of hind limb paralysis by only one of these agents. A conditioning volley from C fiber afferents can have profound effects on receptive field size [4], as can peripheral tissue injury due to punctate burns to the skin [20] or inflammation [13]. We have now shown that K-opioid receptor agonists also effect the receptive fields of superficial dorsal horn neurons. Since there is a large increase in dorsal horn levels of dynorphin in animals with peripheral inflammation [15] or following partial peripheral nerve damage [I], peripheral nerve transection [3] or spinal cord injury [8], we postulate that dynorphin levels are related to enhanced excitability and development of expanded receptive fields following various types of injurious stimulation. Increased nociceptive activity in the periphery may lead to excessive depolarization via excitation at NMDA receptor sites. Dynorphin may enhance this excitability and, in conjunction with other neuropeptides, promote excitotoxic-
ity. This hypothesized chain of events would have irnportant consequences with respect to central mechanisms of inflammation or injury-induced hyperalgesia [7]. Unchecked excitotoxicity in the spinal cord likely effects small inhibitory neurons to a greater extent than larger cells. A net loss of small inhibitory interneurons would produce a shift in the excitability of remaining neurons. Such an excitability shift would contribute to the observed phenomena of receptive field expansion and behavioral hyperalgesia.
References 1
2
3
4
5
6
13
14
Bennett, G.J.. Kajander, K.C., Sahara,
Y., Iadarola, M.J. and Sugimoto, T., Nemochemical and anatomical changes in the dorsal horn of rats with an experimental painful peripheral ncuropathy. In: F. Cervero, G.J. Bennett and P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 463-471. Caudle, R.M. and Isaac, L., Influence of dynorphin(l-13) on spinal reflexes in the rat, J. Phannacol. Exp. Ther., 246 (1988) 5088513. Cho. H.J. and Basbaum, A.I., increased staining of immunoreactive dynorphin cefl bodies in the deafferented spinal cord of the rat, Neurosci. Lett., 84 (1988) 125-130. Cook, A.J., Woolf, C.J., Wall. P.D. and McMahon, S.B.. Dynamic receptive field plasticity in rat spinal cord dorsal horn following C-primary afferent input, Nature, 325 (1987) 151-153. DeCosta, B., George, C., Rothman, R.B., Jacobson, A.E. and Rice, K.C., Synthesis and absolute configuration of optically pure enantiomers of a k-opioid receptor selective agonist, Fed. Eur. Biothem. Sot. Lett., 223 (1987) 335-339. Dickenson, A.H., Sullivan, A.F., Knox, R., Zajac, J.M. and Roques, B.P., Opioid receptor subtypes m the rat spinal cord: electrophysiological studies with p- and I-opioid receptor agonists in the control of nociception, Brain Res. 413 (1987) 36-44. Dubner, R., Neuronal plasticity and pain foflowing peripheral inflammation or nerve injury. In: M. Bond (Ed.), Proc. VIth World Congress on Pain. Elsevier, Amsterdam, in press. Faden, A.I., Mohneaux, C.J.. Rosenberger, J.G., Jacobs, T.P. and Cox, B.M., Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury, Ann. Neural., 17 (1985) 386-390. Fujimoto, J.M. and Rady, J.J., Intracerebroventricular physostigmine-induced analgesia: enhanced by naloxone, B-funaltrexamine and nor-binaltorphimine and antagonism by dynorphin A(l-17), J. Pharmacol. Fxp. Ther., 251 (1989) 1045-1052. Hayes, R.L.. Price, D.D., Ruda, M. and Dubner, R., Suppression of nociceptive responses in the primate by e&trial stimulation of the brain or morphine administration: behavioral and electrophysioiogical comparisons, Brain Res.. 167 (1979) 417-421. Herman, B.H. and Goldstein, A., Antin~iception and paralysis induced by intrathecal dynorphin A. J. Pharmacol. Exp. Ther., 232 (1985) 27-32. Hylden, J.L.K., Hayashi, H., Dubner, R. and Bennett, G.J., Physiology and morphology of the lamina I spinomesencephahc projection, J. Comp. Neural., 247 (1986) 505-515. Hylden, J.L.K., Nabin, R.L., Traub, R.J. and Dubner, R., Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms, Pain, 37 (1989) 229-243. Hylden, J.L.K. and Wilcox. G.L., Antinociceptive effect of morphine on rat spinothalamic tract and other dorsal horn neurons, Neuroscience, 19 (1986) 393-401.
193 15 Iadarola, M.J., Brady, L.S., Draisci, G. and Dubner, R., Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding, Pain, 35 (1988) 313-326. 16 Knox, R.J. and Dickenson, AH., Effects of selective and nonselective s-opioid receptor agonists on cutaneous C-fibre-evoked responses of rat dorsal horn neurones, Brain Res., 415 (1987) 21-29. 17 Laird, J. and Cervero, F., A comparison of the receptive field plasticity of nocireceptive and multireceptive dorsal horn neurones following noxious mechanical stimulation. In: F. Cervero, G.J. Bennett and P.M. Headley @is.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 473-476. 18 Long, J.B., Kinney, R.C., Malcolm, D.S., Graeber, G.M. and Holaday, J.W., Intrathecal dynorpbin Ai_is and dynorphin A,_,, reduce rat spinal cord blood flow by non-opioid mechanisms, Brain Res., 436 (1987) 374-379.
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