Neuroscience Vol. 43, No. 2/3, pp. 593-600, 1991

0306-4522/91 $3.00+ 0.00 Perpmon Prim pie © 1991 IBRO

Printed in Great Britain

INTRASPINAL RELEASE OF SUBSTANCE P A N D CALCITONIN GENE-RELATED PEPTIDE D U R I N G OPIATE DEPENDENCE A N D WITHDRAWAL C. R. MORTON,* W. D. HlYr~N~ and I. A. I-IE~RY Division of Neuruscience, The John Curtin School of Medical Retearch, The Australian National University, Canberra, A.C.T. 2601, Australia Almtraet---The antibody microprobe technique was used to study the release ofimmunoreacfive substance P and immtmorenctive calcitonin gen~related peptide within the lower lumbar spinal cord of anaetthetized spinaliz~ cats pretreated twice daffy for 3.5 days with increa~ng doses of morphine hydrochloride (2-20 mg/k8, i.p.). Both peptides were released in the region of the substantia gelatinoss during noxious cutaneous thermal stimulation or hiSh-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreacUve substance P or immtlnorea~ve cak'itonin 8erie-related peptide in the superficial gray matter dorul to these neurons. In addition, the rekat¢ of both peptid~ was not si~miflcantlydifferent to that detected under similar experimental conditions in opioid-nalve cats. The results m q l ~ t that alterations m nonropeptid¢ rekmse from the central terminah of nociccptive primary afferent neurons do not occur during ~at~ of opiate depen~nce and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.

It is well documented that chronic administration of an opiate such as morphine induces a state of dependent, whereupon discontinuation of the opiate, or administration of an opioid antagonist such as naloxone, evokes a characteristic w i t h d r a w a l o r abstinence syndrome (see Ref. 27 for a review). In the CNS of animals chronically treated with opiates, the precipitation of withdrawal has been associated with neuronal excitahifity exceeding that observed in the prewithdrawal state, l'z2-~41a phenomenon which could be mediated by increased release of excitatory substances, including transmitters, from neurons. Relevant in this context are reports that the levels of several neuropeptides in various CNS regions are altered during states of dependence and withdrawal. Thus, in rats,

There are difficulties with the interpretation of such observations in that local tissue levels of peptides are determined by several variables including synthesis, release and degradation, variations in any of which could influence the content in a particular CNS location. Moreover, measurements on microdissected brain regions are of fimited usefulness in determining the precise anatomical site(s) of altered peptide levels. It is clearly important to study neuropeptide release directly during dependence and withdrawal. Such experiments have been performed with #1 vitro preparations of rodent spinal cord. Naloxone increased the potassium- or capsaicin-cvoked release of IR-SP from spinal cord slice preparations from morphinedependent (but not morphine-naive) mice47and rats. Is To date, however, there have been no reports of localized central neuropeptide release under /n vivo conditions of opiate dependence and withdrawal. The present experiments, therefore, sought to examine the intraspinal release of IR-SP and I R - C G R P in morphine-dependent cats prior to, and subsequent to, precipitation of withdrawal by administration of naloxone. The method employed was the antibody microprobe technique, the use of which has demonstrated the localized release of IR-SP Is~°~l and IRC G R P 3s in the superficial dorsal horn, in the region of the substantia gelatinosa, evoked by nociceptive afferent input to the lumbar cord in this species.

the induction of opiate dependence by chronic administration of morphine has been associated with elevated concentrations of immunoreactive (IR)-substanee P (SP), 4"~'~°'~ IR-cholecystokinin, IR-somatostatin, IR-neurotensin, ;4 and IR-calcitonin gene-related peptide (CGRP) 4s in a variety of microdissected CNS regions. In tlmse studies, opiate withdrawal has usually reversed the effect of morphine on central peptide levels. One possible interpretation of these findings is that the release of neuropeptides is inhibited in the presence of the opiate but increased during withdrawal, with the enhanced release contributing to the manifestation of the abstinence syndrome. *To whom c o m = t ~ should be addressed. tPretent addrem: Physiologitches Institut der Universitit, D-g700 Wfirzburg, F.R.G. AbbrevlaNons: CGRP, calcitonin gene-related peptide; IR, immunoreactive; SP, substance P.

EXPtilMih~AL PI{g-'IDUilS Animal preparation

Morphine hydrochloride was administered to 15 cats (2.73.8 kg) twice daily with doses increasing in the sequence 2,

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5, 5, 10, 10, 20, 20mg/kg, i.p. (calculated as free morphine base). 26 The seventh injection (20mg/kg), given on the fourth day, was followed by induction of anaesthesia (pentobarbitone sodium 20-35 mg/kg, i.p., initially, supplemented during surgery with 2-3 mg/kg, i.v., as assessed by frequent evaluation of the level of anaesthesia). A cephalic vein, a carotid artery and the trachea were cannulated, and body temperature, blood-pressure and end-tidal CO: were continuously monitored. Following a lumbar laminectomy and spinal transection at the thoracolumbar junction, the exposed cord was covered with a thin layer of Ringer-agar. In eight experiments the left and right tibial nerves were exposed and mounted on bipolar platinum electrodes under warm mineral oil. Cats were artificially ventilated with air following neuromuscular paralysis with gallamine triethiodide (4 mg/kg, i.v.), then constantly infused with a pentobarbitone-gaIlamiue mixture (3 and 4 mg/kg per h, i.v., respectively), with end-tidal CO: levels maintained at 4%. Adequacy of anaosthesia was regularly oonlirmed by the absence of a hypertensive response to noxious cutaneous stimulation cephalic to the first lumbar segment. Microprobe preparation Antibody microprobes were prepared to detect either IR-SP or IR-CGRP as previously desea'ibed. 19~°.~ Glass micropipettes were evenly coated with a siloxane polymer by several incubations in a 10% solution of 3-aminopropyltriethoxysilane (Aldrich) in toluene. The microp/pettes were immersed in 2.5% glutaraldehyde (Merck) and the tips then incubated (4°C, 24-48 h) in 2 mgtml Protein A (Sigma). Antibodies to SP or to CGRP were subsequently bound to the Protein A by incubation (4°C, 24-48 h) of the tips in dilutions of rabbit antiserum (SP, 1:400, a gift from Prof. R. Helme, Monash University, Melbourne, or CGRP, 1:1500, Milab, Maim6, Sweden). Data from the supplier indicated no known cross-reactivity of the CGRP antiserum with structurally related peptides. The SP antiserum did not cross-react with neurokinin A at 10 -s M. Methods The Ringer-agar overlying the lower lumbar spinal cord was removed and the dorsal cord surface thus exposed was covered with sterile artificial cerebrospinal fluid at 37°C, pH 7.4. A platinum ball electrode was used to record cord dorsum potentials in response to innocuous mechanical stimulation of the ipsilateral hind paw or to low intensity electrical stimulation of the ipsilateral tibial nerve, thereby determining the appropriate somatotopic regions for microprobe insertion and the electrical thresholds of the nerves. To permit microprobe entry, small perforations were made in the dura and pin mater about midway between the median and lateral dorsal sulci. Prior to microprobe insertion, the suitability of each site was confirmed by recording, with a 4 M NaCl-filled microelectrode, excitatory responses of neurons in laminae IV or V to light mechanical stimulation of the cutaneous receptive field or to low intemity nerve stimulation. Microprobes were inserted in pairs to a depth of 3.0 mm from the dorsal surface. To evoke neuropeptide release, nociceptive afferent stimulation was applied ipfilaterally, either by hind paw immersion in water at 50 or 52°C or by electrical stimulation of the tibial nerve (trains of three pulses at 333 Hz, 0.5 ms duration, repeated at 10 Hz) at intensities sutf~ent to excite myelinated (A) and unmyelinated (C) fibres (200-300 × threshold for the lowest threshold fibres). Microprobes were inserted, and stimuli applied, for selected periods within the range 10-20 rain, times over which consistent release of IRSP hs~° and IR-CGRP 3s has been detected in the substantia gelatinosa region of the feline lumbar cord with this technique. Several microprobes were used to measure release during a particular nociceptive stimulation procedure applied for a particular time, both prior to and subsequent to naloxone administration. Thus, only one type of stimulation

(thermal or electrical) and one period of stimulation (min) were used throughout any one experiment. Previous studies with antibody microprobes in this experimental preparation have detected, in the substantia gelatinosa region, a basal presence of IR-SP and IR-CGRP which is unaffected by non-noziceptive alTerent stimulation but increased by nociceptive stimulation, indicating release of these peptides by impulses in nociceptive afferent fibres?°-" In the present experiments, some microprobes were also inserted in the absence of peripheral stimulation or during non-nociceptive afferent stimulation [water at 35°C or lowintensity (1,5 × threshold) tibial nerve stimulation], to confirm that the nociceptive stimuli used did evoke release of IR-SP and IR-CGRP. To ensure continued high concentrations 0f morphine in the spinal cord during release measurements, m3an additional dose of morphine hydtochloride ( > 10 mg/kg, i.v., calculated as free base) was administered just prior to the first microprobe insertions in each experiment, approximately 5 6 h after the final i.p. administration of morphine. Naloxone hydrochiofide was administered by slow i.v. injection in divided doses within the range 0.1-3.8mg/kg (calculated as free naloxone base). When tibia] nerve stimulation was employed, excitatory responses of single laminae IV/V neurons to electrical excitation (0.5-ms pulses, 0.2--0.35 HZ) of unmyelinatod tibial afferent fibres were recorded extracellularly with a 4 M NaCl-filled microelectrode during the period of administration of morphine (four experiments) or naloxone (all eight experiments). A counter was gated to count action potentials with lateacies and stimulus thresholds indicating excitation by impulses in C fibres, with an analogue of each count plotted on a pen-recorder. Naloxone was administered several times over the course of each experiment, and microprobes were inserted 0-56 rain following the most recent naloxone injection. In the majority of experiments, a pair of microprobes was in place in the cord prior to the first naloxone injection, to detect any transient enhanced release of IR-SP or IR-CGRP following morphine withdrawal. After removal from the cord, the microprobe tips (15 ram) were incubated for 36 h in a solution of the appropriate radiolabelled peptide, either [I:~I]SP (2000mCi/#mol, Amersham) or [l~ I]CGRP (rat) (1700 mCi/#mol, Peninsula, or 2000 mCi/#mol, Auspep), diluted to 2 #Ci/ml, and subsequently mounted on paper next to single emulsion X-ray film (Kodak NMC) for an appropriate time to obtain optimal silver grain density. In each experiment, parallel/n vitro assays of sensitivity were performed by incubating microprobe tips at 37°C in known concentrations of SP (Sigma) or CGRP (Peninsula) for 10-20 rain, prior to incubation in appropriate radiolabeUed peptide for 36 h. The radioactivity bound to the tips was then measured in a gamma counter. Data analysis, Computerized microdensitometric analysis of each autoradiographic image produced an image density scan for each microprobe, consisting of digitized optical density values plotted with respect to distance from the tipY Regions of in vivo peptide release along the length of each microprobe were identifiable as relative deficits of tracer binding, producing localized zones of reduced optical density (peaks) on the sloping scan line. Mean image density scans were calculated for selected groups of microprobes subjected to the same experimental procedure, and differences in release between such groups were assessed by computerized analyses of the differences between their mean scans. 2°~' For defined peaks on individual scans, peptide release was also quantified by measurement of the area delineated by the peak and the baseline taper of the microprobe on the scan. Such areas are a measure of the total amount of peptide released at a particular site, and mean areas for selected groups of microprobes were statistically compared by the Student's t-test for unpaired data. 3s'3.'39 The units of area were grey scale values x mm.

Release of neuropeptides in dependence and withdrawal BgSULTS

Effects of morphine and naloxone on neuronal responses The morphine pretreatment schedule employed in these experiments has previously been shown to induce tolerance and dependence in feline lumbar dorsal horn neurons. 26Thus, in opiate-naive cats, the excitation of these spinal neurons by impulses in unmyelinated primary afferents has been shown to be reduced by the intravenous administration of morphine) 6"3m~zbut seldom affected by similar administration of naloxone. 1~'32In contrast, in morphine-pretreated cats, these excitatory neuronal responses have not been reduced by even large doses of morphine (10 mg/kg, i.v.), but have been markedly increased by naloxone administered microelectrophoretically into the substantia gelatinosa region. 2~ These observations, which have been interpreted as indicative of opiate tolerance and dependence, 26 were confirmed in those of the present experiments where tibial nerve stimulation was used (see Fig. 1). The gated C fibre responses of laminae IV/V neurons in the morphine-pretreated cats were not influenced by the administration of morphine in doses of 1.0-10.0 mg/kg, i.v. (four/four experiments), but were increased by naioxone administration (0.11.0 mg/kg, i.v.) to a mean of 199% of pre-naloxone values (range 138--277%) (eight/eight experiments). Effect of naloxone on release of immunoreactivesubstance P and immunoreactive-calcitonm gene-related peptide Of the 496 antibody microprobes used in this study, 316 were used to detect peptide release/n vivo (186 for IR-SP, 130 for IR-CGRP), and 180 for parallel/n vitro assays. For both SP and CGRP, an/n vitro concentration of unlabelled peptide of 10-TM or greater

595

consistently suppressed the subsequent binding of tracer. The nociceptive stimulation paradigms employed produced release of both IR-SP and IR-CGRP in the substantia gelatinosa region of the superficial dorsal horn, as previously described. Is~o'3s The localization and extent of this release along the length of microprobes inserted 3 mm into the spinal cord are exemplified in Fig. 2. The zones of severely reduced tracer binding on each microprobe indicate pronounced release of IR-SP in response to noxious cutaneous heating (Fig. 2, left) and of IR-CGRP in response to high-intensity electrical stimulation of the tibial nerve (Fig. 2, right), before and after administration of naloxone. In the quantitative data analysis for each peptide, mean image density scans were calculated for prenaloxone microprobes and compared with mean scans of microprobes inserted following naloxone administration. No differences in the evoked release of either peptide were observed between the two types of nociceptive stimulation, so scans of microprobes detecting release during thermal and electrical stimulation procedures were pooled. The mean scans of microprobes detecting IR-SP release and IR-CGRP release prior to naloxone administration are illustrated in Fig. 3A and B, respectively. Both mean scans feature a peak centred 1.1 mm ventral to the dorsal cord surface, representing release of these peptides in the substantia gelatinosa region in response to the nociceptive stimulation. The mean scans of microprobes detecting IR-SP release and IR-CGRP release following administration of naloxone (0.1-3.8 mg/kg, i.v.) are illustrated in Fig. 3C and D, respectively. For both IR-SP and IR-CGRP, there were no significant differences between the mean scan of

40-

J lIME ( m )

Fig. 1. The effects of intravenous morphine and naloxone on the responses of a lamina V dorsal horn neuron in the lower lumbar cord of a cat pretreated with morphine (see Expering.ntal Procedures). The pen-recordings are the numbers of action potentinh evoked by impulses in nnmyclinAtedprimary afferent fibres following elemical stimulation of the ip,ilateral tibial necve (0.5 ms pulses, 0.35 I ~ 300 × thmdmid). The timea of administration of morphine (1.0 mg/kg) and naloxone (0.5 mg/k$), indicated by arrows, were approximately 10 h after the last morphine pretreatment injection (20 mg/kg i.p.) and 4 h after a further dose of morphine (10 mg/k8, i.v.). The tip of the recordin8 electrode was located 0.5 mm ventral to the centre of the zone of IR-SP release detected in this cat (see Fig. 2).

596

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CGRP

NALOXONE POST-

NALOXONE PRE-

POb~-

Fig. 2. Localization and release of IR-SP and 1R-CGRP in the superficial lumbar dorsal horn in cats pretreated with morphine. Radiolabell©d peptide hound to the microprobes shows as white on the photographic enlargements of the autoradiographic imams of microprobes. Release of IR-SP was detected during il~ilateral noxious cutaneous thermal stimulation (50°C, 15rain); release of IR-CORP during high-intensity electrical stimulation (see Experimental Procedures) of the ipralateral tibial nerve (10 min). With both peptides, reduced tracer binding (representing endogenous peptide release) was observed in the substantia gelatinosa region on microprobes inserted before and after naloxone administration (I .5-1.8 mg/kg, i.v., in divided doses). microprobes inserted after naloxone and that of the corresponding pre-naloxone microprobes, indicating a lack of effect of the opioid antagonist on the release of the two neuropeptides in the substantia gelatinosa region. The effect of naloxone on the noxiously evoked release of IR-SP and I R - C G R P was also assessed by calculation of the area of the peak of release in the substantia gelatinosa on individual scans. The mean areas (and S.E.M.) corresponding to each group of microprobes in Fig. 3 A - D are shown in Fig. 3. For each peptide, the similar areas measured pre- and post-naloxone confirm comparable release prior to. and during, naloxone-precipitated opiate withdrawal. Microprobes inserted in the spinal cord in the absence of peripheral stimulation or during nonnociceptive afferent (thermal or electrical) stimulation detected a basal presence of IR-SP and of I R - C G R P in the substantia gelatinosa region, as found in previous work. 2°'35 The extent of this basal presence of the two peptides was unchanged by naloxone administration (not illustrated). The release of both neuropeptides in this spinal region was significantly greater following nociceptive stimulation, confirming the efficacy of the stimulation procedures in evoking release.~s'2°'3~

less than that from cord slices from opiate-naive rats. '5 It was therefore conceivable that release in the lower lumbar cord of dependent cats might differ from that occurring under the same conditions in cats not exposed to opiates. For each peptide, this question was addressed by matching, in terms of type of nociceptive stimulation and duration of insertion, the microprobes used to detect release in dependent cats with an equal number of microprobes used to detect release in cats which had not been pretreated with morphine. These mean scans of microprobes which detected IR-SP release and IR-CGRP release in the lumbar cord of opiate-naive cats during nociceptive stimulation are illustrated in Fig. 3E and F, respectively. For both peptides, the peak of release centred On the region of the substantia gelatinosa was not significantly different to that detected in the morphine-dependent cats, either prior to or subsequent to administration of naloxone (Fig. 3A-D). This was also confirmed by comparisons of the mean areas of the peaks of release in the substantia gelatinosa on microprobes used in naive and dependent animals (Fig. 3). Thus, the induction of morphine dependence by the present experimental protocol was not associated with alterations in the noxiously evoked release of IR-SP and IR-CGRP in the dorsal horn.

Comparison of release in dependent and naive cats

In recent experiments measuring capaaicin-evoked release of IR-SP from rat dorsal cord slices, release during continued exposure to morphine prior to naloxone administration was found to be significantly

DISCUSSION

These experiments have shown that in morphinedependent cats, opiate withdrawal precipitated by

Release of neuropeptides in dependence and withdrawal

DEPENDENTCATS PltE-N&(EIIE

597

NAIVE CATS I~ST-NALOX(IE

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Fig. 3. lntraspinal release of IR-SP and IR-CGRP in morphine-dependent cats before and after administration of naloxone, and in opiate-naive cats. The solid fines are mean image demity scans of autoradiographs of antibody microprobes; the broken lines are the S.E.M. for each mean scan. Ordinates, digitized optical density values expressed as an arbitrary grey scale (see Experimental Procedures). The limits defining the area of optical density values to be integrated transversely across autorsdiographs were narrower for CGRP than for SP microprobes, resulting in mailer grey scale values for CGRP scans. Abscissae, depth of insertion into lower lumbar spinal cord (nun), with the cord surface at 0. The plotted mean scan line, slope from low optical density values (in the tip region) to high optical demity values due to the geometric taper of the mia'oprobes, with zones of release depicted as upward deflections(peaks) occurring on the sloping mean scan line. Microprobes were inserted to a depth of 3.0 mm for 10 min (CGRP microprobes) or 10-20min (SP microprobes) during activation of nociceptive primary afferent fibres. (A, B) The mean scans (and S.E.M.) of microprobes detecting release of IR-SP (A, n = 44) and release of IR-CGRP 0l, n = 33) during nociceptive afferent (noxious cutancom thermal or high-intensity fibial nerve) stimulation in morphine-pretreated cats prior to naloxone administration. (C, D) The mean scans (and S.E.M.) of microprobes detecting release of IR-SP (C, n = 44) and release of IR-CGRP (D, n = 33) during nociceptive afferent stimulation in morphine-pretnmted cats subsequent to naloxone administration (0.1-3.8 mg/kg, i.v.). (E, F) The mean scans (and S.E.M.) of microprobes detecting releaJe of IR-SP (E, n -44) and release of IR-CGRP (F, n ffi 33) during nociceptive afferent stimulation in opiate-naive cats. The mean values (and S.E.M.) for the area under the peak of neuropeptide r e l ~ in the subttantia ge]atinosa on single image density scans of microprobes are shown for each group of microprobes in A-F. The units of area are grey scale valuta × ram. S t a ~ comparisons by the Student's t-test for unpaired data showed no sianificant differences in mean areas A, C and E, nor in mean areas B, D and F.

administration of naloxone does not alter the nociceptively evoked release of IR-SP and of IR-CGRP in the substantia gelatmosa of the lumbar spinal cord, despite concomitant large increases in the nociccptive excitation of dorsal horn neurons located ventral to the region of release. Moreover, the release of these neuropeptides in the dependent cats was similar to that detected in opiate-naive cats. The IR-SP in the superficial dorsal horn is derived largely from smalldiameter primary afferent neurons, although there is some contribution from IR-SP-containing intrinsic spinal neurons.~ j ~ Although the release observed could conceivably originate from either source, there is substantial other evidence that IR-SP release in the

superficial dorsal horn during acute nociception is derived from the central terminals of primary afterents (see Ref. 20). The origin of the released IRCGRP is less equivocal since this peptide is contained within a much larger proportion of dorsal root ganglion neurons, preferentially within the smalldiameter cells, but appears not to be present in dorsal horn cell bodies. 7~3~129Therefore, the present results suggest that the changes in CNS function which characterize states of opiate toicmlz~ dependence and withdrawal are not prodL_u~_ by alterations in the release of either IR-SP or IR-CGRP from the intraspinal terminals of -nmyelinated primary afferent fibres.

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The morphine pretreatment experimental protocol employed in these experiments to induce dependence was chosen in the light of previous work which highlighted potential problems in the interpretation of neuropharmacological studies of opiate tolerance and dependence. 26 In the morphine-pretreated cats of the present experiments, the excitation of lumbar dorsal horn neurons by impulses in unmyelinated primary afferents was unaffected by further administration of morphine but increased by naloxone administration. However, in animals or tissues chronically exposed to opiates, the absence of a depressant effect of an opiate on neuronal firing does not necessarily represent tolerance since it could equally result from a lack of unoccupied receptors due to the presence of continued high concentrations of the opiate. Moreover, an excitant effect of naloxone on neuronal firing in such preparations may not indicate withdrawal hyperexcitability but merely a return to the "'normal" discharge rate, which may be impossible to recognize due to large variations among the individual units in the neuronal pool. In the present barbiturateanaesthetized, spinalized cat preparation, however, it has been established that the mean temperature of a noxious cutaneous heat stimulus producing 50% of the maximal increase in firing (T~0) is a relatively constant feature of lumbar dorsal horn neurons. 26 In cats pretreated with morphine as in the present experiments, rs0 values do not differ significantly from those in naive cats but are reduced following naloxone administration, indicating the presence of tolerance and dependence in this experimental model. 26 Thus, it is highly probable that microprobes were detecting release of neuropeptides in spinal cords which had undergone the adaptive changes responsible for opiate tolerance and dependence. Hypotheses which have implicated altered intraspinal neuropeptide release in the phenomena of opiate dependence and withdrawal are based largely on indirect evidence. Thus, the 12-25% increase in IR-SP levels found in the spinal cord of dependent animals has been ascribed to chronic inhibition of release, which is alleviated during withdrawal. ° ° ' o Peptide content, however, is also influenced by factors other than release, such as synthesis and degradation. Even when spinal release per se has been measured, there have been important differences in results. In the experiments of Ueda e t al., 47 the potassium-evoked release of IR-SP from minced spinal cord slices taken from morphine-dependent mice was enhanced by approximately 3 2 0 by incubation with naloxone. However, both basal and evoked release from dependent cords were much greater than from naive control cords, a finding which seems incongruous with chronic inhibition of release in the dependent state. In contrast, Donnerer t5 noted that capsaicin-evoked IR-SP release from dorsal spinal cord slices of morphine-dependent rats was reduced compared with opiate-naive controls. Withdrawal produced by superfusion with naloxone did increase this release, but only to levels normally

seen in naive rats. ~ There are obvious difficulties in reconciling these and the present observations made with different species under such differing experimental conditions. With release evoked from m t , itro spinal cord preparations by such severe stimulation procedures as high potassium or capsaicin, however, it is difficult, firstly, to assess the extent to which such release mimics that produced by impulses invading the central terminals of primary afferents in vivo, and. secondly, to predict which neuronal types in the spinal cord might be affected by such stimuli and release neuropepddes. The present experiments detected no differences in the intraspinal release of IR-SP and IR-CGRP under in vivo conditions of opioid naivety, dependence and withdrawal. There is increasing evidence that the adaptive changes underlying dependence are invoked rapidly, initiated even by brief exposure to an opioid. Studies of the depressant effect of morphine on the noeiceptive excitation of feline dorsal horn neurons have shown that naloxone, administered only a few minutes after the opiate, restored and then increased the nociceptive responses to levels substantially greater than those observed prior to morphine administration. -~5'''~0 Rapid development of dependence following a very brief exposure to opioids has also been documented in studies of withdrawal responses in the guinea-pig ileum s and in whole animal behaviour. ~° In this context, it is noteworthy that the noxiously evoked release of IR-SP and of IR-CGRP in the substantia gelatinosa of the feline lumbar cord following acute administration of morphine alone or of morphine followed by naloxone is similar to that detected prior to morphine administration. 36'37This provides further evidence that although dependence may develop rapidly after exposure to opioids, the states of dependence and withdrawal are present without detectable changes in neuropeptide release from primary afferent fibres. Although opioid dependence and withdrawal may not affect neuropeptide release from sensory neurons, this may not necessarily be the case for neurons within the CNS. In fact, recent work has shown that brief exposure to morphine actually stimulated IR-SP release from guinea-pig brain slices (with a further increase following exposure to naloxone), ~° a finding which could explain the reduction in IR-SP levels in various CNS regions of this species 2 h following a single systemic administration of morphine." This process, if maintained during chronic morphine administration, might stimulate synthesis of SP by central neurons, resulting in the observed increases in IR-SP content in the CNS of dependent animals. 4"~'~°'a Interestingly, morphine has previously been found to increase the synthesis of other substances (eatecholamines, serotonin) in the brain, r4"43'~As some of the behavioural and autonomic signs of opioid withdrawal are mimicked by the intracerebroventricular administration of SP, ~'6'~2 this hypothesis requires that the effect of ongoing morphine-induced SP release in

Release of neuropeptides in ~ c e dependent animals be masked by other concomitant

depressant actions of the opiate until revealed by withdrawal, a proposal which warrants further investigation. Insofar as central neurons are concerned, however, the po~hility remains that opioid dependence and withdrawal may be associated with changes

in both synthesis and release of neuropeptides. The possible participation of primary afferent neurons in opioid dependence and withdrawal has alSO been examined in animals with a capsaicininduced deficit in small-diameter senSOry neurons, but the results have been somewhat equivocal. In such animals, SOme signs of opioid withdrawal (salivation, lacrimation, rhinorrhea) were less pronounced, while others (wet-dog shak~ 0 were increased.42 In the isolated, ~ spinal cord of neonatal rats, the capsaidn-induced depolarization of ventral roots was augmented during morphine withdrawal.~ Tsou et al. ~ reported that capsaicin attenuated the withdrawal re~q~ome of guinea-pig ileum, but this was not confirmed in a subsequent study.9 The present m/croprob¢

and withdrawal

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results do not definitively exclude the participation of other primary afferent substances, peptides or amino acids, in dependence sad withdrawal. However, in view of the widespread distribution of IR-CGRP in small sensory neurons and the lack of detectable changes in release of this peptide and also IR-SP during dependence and withdrawal, the results of the present experiments suggest that altered reimse from the ~lltral terminals of unmyelirmted primgry aff~'ent fibres does not contribute to the ~nnnif,~_J_tion of the opiate withdrawal syndrome. It is likely, therefore, that the adaptive changes underlyin8 the phenomena of opiate dependence and withdrawal occur at a point beyond the primary affercat neuron.

Acknowledsonents--Thiswork was supported by grants from the National Health and Medical Research Council of Australia and the Wenkart Foundation. The authors wish to thank A. Turudic and S. Ford for expert Mmtsace with various stages of this work and R. M. Crane for word processing.

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