Application of morphine prior to noxious stimulation differentially modulates expression of Fos, Jun and Krox-24 proteins in rat spinal cord neurons

Ne~~usc~enceVol. 58, No. 2, pp. 305-32 I. 1994 Printed in Great Britain

0304-45~2/94 $6.00 - 0.00 Pergamon Press Ltd G 1993 IBRO

APPLICATION OF MORPHINE PRIOR TO NOXIOUS STIMULATION DIFFERENTIALLY MODULATES EXPRESSION OF Fos, Jun AND G-ox-24 PROTEINS IN RAT SPINAL CORD NEURONS T. R.

TULLE,*? T. HERDEGEN,~J. SCHADRACK,’ R. BRAVO& Ivf. ZIM~E~A~~~

and W. Z~GLG~~SBERGER* *Clinical Neuropharmacology,

Max-Planck-Institute of Psychiatry-Clinical Institute, 80804 Miinchen, F.R.G. $Institute of Physiology, University of Heidelberg, 6900 Heidelberg, F.R.G. $Bristol-Myers Squibb Pharmaceutical Research Institute, Molecular Biology Department, Princeton, New Jersey, U.S.A. Abstract-The expression of Fos, Jun and Krox-24 proteins was investigated in spinaf cord neurons of the rat 2, 4 and 8 h following noxious thermal stimulation of one hind-paw and pre-treatment with morphine. The number of neurons expressing c-Fos, c-Jun, Jun B and Krox-24 were maximal after 2 h and thereafter declined. The number of Fos B and Jun D immunoreactive neurons increased constantly for up to 8 h with Jun D showing expression above baseline only after 4 h following stimulation. Intravenous application of morphine (5 and 10 m&kg) 20 min before noxious heat stimulation decreased the expression of all six proteins at any time-point with a predilective effect on neurons of deeper laminae of the dorsal horn. The suppressive effects of morphine were more pronoun~d with the higher dose of morphine and completely reversed by intravenous naloxone (I and 10 mgfkg}. The temporospatial patterns of expression following morphine were similar to those seen without morphine, but in a much smaller number of neurons and with a shorter time-course. However, despite the high dose of morphine and continuous halothane anaesthesia during the whole experimental procedures, a considerable number of neurons expressing the various genes remained in all laminae of the spinal cord. At 2 h following noxious heat stimulation morphine had decreased the number of labellecl neurons for c-Fos, Fos B, Krox-24, cJun and Jun B to 3&60% of control levels in laminae I-II and to IO-30% in laminae III-VII,X of the spinal cord. At 4 h the level of reduction had further increased while Jun D was only moderately reduced to 75% in all laminae of the spinal cord. Eight hours following noxious heat &us morphine application we did not detect noxious evoked immunore&tivity for c-Fos, I&ox-24, c-Jun and Jun B,&while there was residual labelling for Fos B in the superficial dorsal horn and for Jun D in laminae I--VII and X of the spinal cord. The different temporospatial pattern of immediate early gene expression in neurons of the spinal cord dorsal horn following noxious stimulation suggest that variable transcription complexes may interact with DNA regulatory sequences and could thus activate alternative secondarv response genes, even under protection of a high dosage of morphine applied before noxious stimula~on.

In the spinal cord of the rat a number of immediateearly genes (IEGs) are rapidly and transiently induced in subsets of neurons by physiological and pathophysiological stimuli from skin and viscera.‘~9~‘0~‘7~J0~~4~‘7~‘1~76 Depending on the individual IEG investigated and the peripheral stimulus applied, the pattern of expression shows considerable differences in terms of the number of neurons, the intraspinal anatomjcal dist~bution, the peak time of expression and the steady-state persistence of the IEG signa1.‘~27~29~‘79~81~*4 IEGs may be viewed as “third messen-

gers” in a stimulus transcription cascade which transfer extracellular information via second messengers, such as calcium, to long-term changes in gene transcription by forming dimeric transcription regulatory DNA binding complexes. While Jun proteins can form both homo- and heterodimers, members of the Fos family require dimerization with Jun proteins for function 6’,68.88 (for review see Refs 8, 54). The various Jun-Fos complexes possess different binding affinities for DNA consensus sequences and variable transcriptional activities.6~‘5~40*48@*7’~‘8 Current research suggests that the expression and complex interaction of several IEG products together with concomittant target gene inductions or repressions may be related to the neuron’s ability to convert short-term synaptic stimulation into long-lasting responses and thus contribute to the adaptive alterations involved in neuronal plasticity (for review see Ref. 54). Pharmacological experiments unravel the

~To whom correspondence should be addressed at: Max-Planck-Institute of Psychiatry, Clinical Institute, Clin. Neuropha~acology, Kraepelinstr. 2, D-80804 Miinehen, F.R.G. Abbreaiutions: dDH, deep dorsal horn; IEG, immediateearly gene; n/s, neurons per section; sDH, superficial dorsal horn. 305

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particular importance of the early phase of central hyperexcitatio~ for initializing the stimulus-transcription cascade. In the spinal cord, morphine suppressed c-Fos induction only when injected prior to acute noxious stimulation but was ineffective even when

without prior noxious stimulation. The spinal cord lumbar enlargements were postfixed for 12 h in the same fixative and then washed for 48 h in phosphate buffer with 30% sucrose before being sectioned.

applied immediately after noxious stimuIation,i8 Morphine was also not able to suppress the steady-state expression of c_Fos in “non-acutely stimulated” polyarthritic rats.’ During the development of arthritis, non-steroidal antiphlogistic drugs did not significantly reduce c-Fos expression when administered two to three weeks after inoculation of the disease, but possessed a substantial suppressive effect if treatment was started early after the time of the induction2 Up to now, pharmacological modulation of IEG induction in the spinal cord has been described only for c-FOS. Although c-Fos expression induced by acute inllammation and noxious thermal or mechanical stimulation was equally decreased by pretreatthe N-methyl-D-aspartate ment with morphine, 3~M*7b antagonist dizocilpine molecule (MK-801) reduces c-Fos positive neurons only following acute inflammation.3g.78In order to examine the effect of a single administration of an analgesic drug on the putative composition of transcriptionally active complexes in spinal cord neurons following noxious stimulation, we compare here the differential pha~acologi~al modulation of a set of six IEG encoded proteins that belong to IEG subfamilies with a leucine zipper domain (c-Fos, Fos B, c-Jun, Jun B, Jun D) or a zinc-finger domain (Krox-24, also termed NGFl-A, Egr-1, Zif/268 (for review see Refs 8, 31, 54). Data have been partly described in a preliminary report.”

All antibodies were generated in rabbits immunized with bact~~ally expressed fusion proteins. The following sequence of each protein was used for fusion: Krox-24, amino acids 15-322: c-Fos. amino acids 7-380: Fos B. amino acids 4-338; c-J&, amino acids 80-334; Jun B, ‘amino acids 46344; Jun D. amino acids l-102. Krox-24, c-Fos, Fos B and Jun D were expressed and fused to MS2 polymerase. c-Jun was expressed as fusion protein with /3galactosidase and with MS2 polymerase. Jun B was expressed in bacteria fused to /?-galactosidase.

Animal experimenrs The experiments were performed with male Sprague Dawley rats (230_28Og), provided by a local animal supplier, anaesthetized by spontaneously breathing a mixture of 1.5 ~01% halothane and 70% ambient airj30% oxygen. For noxious heat stimulation the left hind foot was immersed in 52°C hot water (10 times for 20 s, with a 90-s interval). After the stimulation was terminated the anaesthesia was continued with 0.7 ~01% of halothane until the animals were intracardially perfused 2, 4 or 8 h later with saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Twenty minutes before noxious stimulation, animals serving as controls for morphine-treatment received 0.3 ml saline intravenously (iv.). Morphine-treated animals received 10 mgjkg morphine hydr~hloride i.v. 20 min before stimulation. The dose-related suppressive effects of morphine (5 and lOmg/kg iv.) and its naloxone-reversibility were investigated at the time-points of maximal expression of the various IEGs, i.e. animals were perfused 2 h following noxious stimulation for c-FOS, KroxI24, c-Jun and Jun B and at 8 h for Fos B and Jun D. Naloxone (1 ma/kg and lOmg/kg) were injected iv. 10 mm prior to morphine administration and repeated each hour until perfusion. All substances were dissolved in saline and intravenously injected in a volume of 0.3 ml. To assess the level of basal expression of the various IEGs and to estimate effects of handling the animals and anaesthesia per se, two other groups of halothane-anaesthetized animals received either saline or morphine lOmg/kg 2 or 8 h before perfusion

Generallon of anlisera

Cryostat coronal serial sections (25 pm) were obtained from the entire spinal cord from lumbar segments L, to L,, and every consecutive sixth section was incubated for 36 h with one of the six different polyclonal antibodies. Immunocytochemistry was performed on free-floating sections. Sections were incubated in normal goat serum, 2% in phosphate-buffered saline and 0.2% Triton X-100, for 1 h followed by the primary antisera for 36 h at 4°C. Immunoreactivity was visualized by an avidin-biotin complex method (Vectastain, Vector Laboratories, U.S.A.). Sections were developed in 0.02% diaminobenzidine with 0.02% hydrogen peroxide. The specificity of the polyclonal antibodies has been documented by preabsorption experiments and by immunopr~ipitation,4~ Thus the possibility of cross-reactivity between related antigens is minimal and the immunoreactivities described can be considered as specific, as the antibodies demonstrate a highly selective staining of related proteins. For preabsorption. 1 and 1OnM fusion protein (in 12.5 mM Tris base, 12.5 mM glycine, 0.01% sodium dodecylsulfate, and 30 PM phenylmethyIsulfonylfluoride) were incubated for 24 h with antisera diluted as for immunocytochemistry: anti-Krox-24 and anti-c-Fos 1:4000, antiFos B I : 1000, anti-c-Jun and antiJun B I :3000 and anti-Jun D 1: 7500. Thereafter, the complex of antiserum and fusion protein was processed for immun~yt~hemistry. In all cases, immunoreactivity was abolished by 1 nM of the respective antigene. Incubation with 10 nM of other related fusion proteins did not affect immunoreactivity.

The whole experimental design included 78 animals. Small numerals in italics in Table I indicate the number of animals with particular treatment for the various IEGs (minimal n = 3, maximal n = 11). For some experimental groups a single animal was sim~~neously processed for more than one IEG. From approximately 30 sections for each animal and IEG from the L, region, the five most stained sections were quantified for the number of nuclei in the superficial dorsal horn (sDH; laminae I-II) and deep laminae of the dorsal horn (Iaminae III-VI. including neurons of laminae VII and X; termed in the following as deep dorsal horn or dDH). Counterstaining with Cresyl Violet was used to assign the immunostained neurons to Rexed’s taminae. Control animals were always processed simultaneously with drug-treated animals to ensure the quality of the immunostaining technique. For statistical analysis the average number of IEG-positive neurons over the Eve most stained sections, de&red as average number of IEG-positive neurons per section (njs), was taken into consideration. Group expressions in the figures and table represent the mean of numbers of IEGpositive n/s i S.E.M. or the mean of per cent changes of total number of neurons per section relative to the averaged number of neurons of control animals processed in the same

Application of morphine prior to noxious stimulation series + S.E.M. For testing the effects of drug-treatment (0, 5 and 10mg/kg morphine iv.; morphine 10 mg/kg i.v. and prior application of naloxone I or 10 mg/kg i.v.) on the overall number of IEG-positive neurons in sDH and dDH a one-way analysis of variance (ANOVA) was performed for each IEG investigated. The differences between the various drug-treated groups and between various drugtreated groups and the control group were tested about significance with the Duncan posr hoc test. As a level of significance a = 0.01 or a = 0.05 was accepted. The timecourse of IEG expression following noxious stimulation (basal expression = 0, 2, 4, 8 h) without morphine or after 10 mg/kg iv. morphine application was tested about significance for both anatomical regions (sDN and dDH) and for each IEG using two-way ANOVA with drug-treatment and time as the two factors. Again Duncan-test were performed for multipleposr hoc comparisons in control animals following noxious stimulation (2 vs 0 h, 4 vs 2 h and 8 vs 4 h) and control vs morphine animals (0, 2,4 and 8 h). During the experimentai procedures and counting-phase, the investigators were blind to the treatment of the animals. RESULTS For the different IEGs investigated, (i) the absolute number of neurons, (ii) the pattern of laminar distri-

bution, (iii) the time-course of expression, and (iv) the modification by morphine, showed considerable differences following noxious stimulation. In the spinal cord of unstimulat~ rats only a few neurons with immunoreactivity for c-Fos, Fos B and Jun B could be detected, mostly in superficial laminae I-II of the dorsal horn (Figs la, 2a; Table 1). c-Jun showed staining in some nuclei of motoneuro~s (not shown). Krox-24 and Jun D showed basal expression in 10-20 neurons in superficial and deep laminae of the spinal cord dorsal horn (Figs 3a, 4a, Table 1). No significant differences in basal expression were observed between animals anaesthetized for 2 or 8 h with halothane (not shown). Temporospatial pattern of immediate early gene expression

Noxious heat stimulation evoked an ipsilateral expression of all IEG encoded proteins tested. Immunoreactive neurons were found to the greatest extent in the medial half of the sDH and were less frequently seen in the dDH. The maximal number of immunoreactive neurons was found at lumbar

segments L,-L, and extended rostrocaudally in agreement with the known central projection of primary endings from the hind paw.” The two-way analysis of variance showed a significant main effect time for neurons located in sDH and dDH for all IEGs investigated (F-values and corresponding significances in Figs 5 and 6). c-Fos, Krox-24, cJun and Jun B expression in the sDH characteristically peaked after 2 h and steadily declined for the following 6 h (Figs 5, 6; P-values of Duncan-tests in Table 1). Compared to the expression after 2 h, the amount of neurons after 8 h had significantly declined to relative values ranging between 10 and 50%. All IEGs tested showed a more pronounced expression in the sDI-I (Figs 2, 3;

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Fig. 1. Photomicrographs illustrating the distribution of c-Fos immunostamed neurons in lumbar spinal cord sections of 25 pm. The samples depicted were taken at various time points (basal expression = 0. 2, 4, 8 h) following noxious thermal stimulation of one hind foot (a,c,e,g) and prior intravenous application of morphine (IO mg/kg iv.; b.d,f,h). Scale bar = lOO/lm. 308

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Fig. 2. Photomicrographs illustrating the distribution of Fos B immunostained neurons in lumbar spinal cord sections of 25 pm. The samples depicted were taken at various time points (basal expression = 0, 2, 4, 8 h) following noxious thermal stimulation of one. hind foot (a,c,e,g) and prior intravenous application of morphine (10 mg/kg i.v.; b,d,f,h). Scale bar = 100 pm.

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Rg. 3. Photomicrographs illustrating the distribution of Krox-24 immunostained neurons in lumbar spinal cord sections of 25 km. The samples depicted were taken at various time points (basal expression = 0, 2, 4, 8 h) following noxious thermal stimulation of one hind foot (a,c,e,g) and prior intravenous application of morphine (10 mg/kg i.v.; b,d,f,h). Scale bar = 100 pm. 310

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Fig. 4. Photomicrographs illustrating the distruption of Jun D immunostained neurons in lumbar spinal cord sections of 25 pm. The samples depicted were taken at various time points (basal expression = 0, 2, 4, 8 h) following noxious thermal stimulation of one hind foot (a,c,e,g) and prior intravenous application of morphine (10 mg/kg i.v.; b,d,f,h). Scale bar = 100 pm. 311

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Fig. 5. Time-course of expression of c-Fos, Fos B and Krox-24 proteins in laminae I-II and laminae III-VII and X neurons in the ipsilateral rat spinal cord following noxious thermal stimulation of one hind foot (basal expression = 0, 2, 4, 8 h; solid line) and modulation by prior application of intravenous morphine (10 mg/kg i.v.; dashed line). Values given represent mean f S.E.M. of labelled neurons in a single 25-pm slice in the lumbar segments L,-L, expressed as n/s. The suppressive effect of morphine (10 mg/kg) was reversed by intravenous naloxone (1 mg/kg, open circle; 10 mg/kg, filled circle; investigated at time points of maximal expression). Note the halved scaling of the y-axis for neurons of deep laminae. The additional table shows the results (F-values and corresponding significances) for a two-way analysis of variance (ANOVA) performed for c-Fos, Fos B and Krox-24 neurons in laminae I-II and laminae III-VII and X. The considered factors were drug-treatment (0 vs lOmg/kg morphine) and time (basal expression = 0, 2, 4, 8 h).

Table 1). The absolute number of neurons in dDH was exceptionally low for c-Jun with 3 + 1 n/s. In the dDH the general time course of maximal expression was comparable to the sDH, but at a lower level (Figs 5, 6; note the halved scaling of the y-axis).

The number of neurons immunoreactive for Fos B and Jun D increased continuously in sDH and dDH for the entire observation period of 8 h (Figs 5 and 6; P-values of Duncan-tests in Table 1). Jun D demonstrated a delayed onset of expression. In contrast to

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Fig. 6. Time-course of expression of c-Jun, Jun B and Jun D proteins in laminae I-II and laminae III-VII and X neurons in the ipsilateral rat spinal cord following noxious thermal stimulation of one hind foot (basal expression = 0, 2,4, 8 h; closed line) and modulation by prior application of intravenous morphine (10 mg/kg i.v.; dashed line). Values given represent mean k S.E.M. of labelled neurons in a single 25-pm slice in the lumbar segments L4-LS expressed as n/s. The suppressive effect of morphine (10 mg/kg) was reversed by intravenous naloxone (1 mg/kg, open circle, 10 mglkg, filled circle; investigated at time points of maximal expression). Note the halved scaling of they-axis for neurons of deep laminae. The additional table shows the results (F-values and corresponding significances) for a two-way analysis of variance (ANOVA) performed for c-Jun, Jun B and Jun D neurons in laminae I-II and laminae III-VII and X. The considered factors were drug-treatment (0 vs 10 mg/kg morphine) and time (basal expression = 0, 2,

4, 8 h).

all other IEGs Jun D expression 2 h following stimulation did not significantly differ from basal levels. The first enhanced expression of Jun D in individual neurons-as evidenced by an intensified staining+ompared to the observed background

constitutive expression appeared only after 2-4 h following termination of noxious stimulation (Fig 6; Table 1). After 4 h 34 +211/s in the sDH and 24 _t 3 n/s in the dDH were labelled; at 8 h these values had significantly increased to 45 f 3 n/s in the

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(Duncan-tests,

P < 0.01).

On the contralateral side after 8 h about 10 cells with an enhanced staining for Jun D appeared in both the sDH and dDH. Noteworthy, the other IEGs were observed only in a few neurons contralateral to the side of stimulation and in no case did their number exceed 5-10% of the initial number of neurons observed on the ipsilateral side in all animals studied (not shown). Dose-related

effects

of morphine

At analgesic doses, morphine (10 mg/kg i.v.) did not influence the basal IEG expression (Table 1; Figs 14). One-way analysis of variance showed a significant main effect of morphine for all IEGs investigated (F4,r, = 50.1 for c-Fos; F4,,4 = 85.2 for Fos B; F4,,8 = 9.3 for Krox-24; F4,,2 = 20.4 for c-Jun; P’4,15= 34.3 for Jun B; P’4,,3= 27.5 for Jun D; for all IEGs signifcance of F < 0.001). Following noxious stimulation and application of 5 mg/kg morphine the overall mean number of stained neurons in the dorsal horn was significantly reduced for all IEGs (Duncantests, P < 0.01) and ranged between 45 and 65% of control levels (Fig. 7). With the higher dose (10 mg/kg iv.) of morphine the level of reduction ranged between 2247% of control expression. The differences between 5 mg/kg and 10 mg/kg of morphine were also significant for all IEGs (Duncan-tests, P < 0.01 for c-Fos, Fos B and Jun D; P < 0.05 for Krox-24 and Jun B), except c-Jun. Both doses of morphine showed a more pronounced suppression of IEG expression in neurons in deep rather than in superficial laminae. Descriptive statistics revealed that with the higher dose the level of reduction ranged between 30 and 60% of control levels in sDH and between 5 and 30% in dDH. This effect was consistent for all IEGs investigated: IEGs : sDH,dDH; c-Fos: 36%, 12%; Fos B: 44%, 25%; Krox-24: 60%, 16%; Jun B: 42%, 28%. The only exception was c-Jun: 31%, 29% and Jun D, where the differential suppression of sDH vs dDH was not evident with morphine at either 5 m&kg: 69 rf: 10%; 55 f 5% or lOmg/kg: 48 rf: 3%; 40 & 3%. In addition to the decreased number of stained neurons, the intensity of the immunostaining in morphine-treated animals was occasionally weaker than in control animals. Combined administration of naloxone (1 or 10 mg/kg iv.) prior to morphine reversed the suppressive effect of morphine (10 mg/kg). There was neither a significant difference in the number of IEG-positive neurons compared to control levels nor was there any significant difference between the naloxone groups. c-Fos was the only IEG that reproducibly showed an increased number of neurons following application of 10 mg/kg naloxone (Fig. 7). However, although this difference was in the range of 117 + 4% of control, this was statistically neither significant for values of the whole dorsal horn (Fig. 7) nor for the sDH or dDH taken alone (Fig. 5).

Time-related

e#kcts of morphine

Two-way analysis of variance, performed for the anatomical regions sDH and dDH, showed a significant main effect of drug (0 vs lOmg/kg morphine) and time for all IEGs investigated and a significant drug x time interaction for most IEGs (F-values and corresponding significances in Figs 5 and 6). Application of morphine (10 mg/kg) decreased the noxious stimuli evoked expressions of almost all IEGs examined in sDH and dDH of the spinal cord at almost all time points after the cessation of noxious stimulation (Figs 14). The 2-h values for c-Fos, Krox-24. c-Jun and Jun B and the 8-h values for Fos B and Jun D have been described above (see dose-related effects of morphine). At 4 h the number of neurons expressing the various IEGs showed a significant reduction in sDH and dDH, except for Jun D in the dDH (P-values of Duncan-tests in Table 1). Descriptive statistics revealed that c-Fos and Jun B-positive neurons were homogeneously decreased to 2&30% of control in sDH and dDH of the spinal cord. Krox-24 showed a level of reduction to 20% in sDH and was reduced to only 4% of control levels in dDH. Fos B was reduced to 53% in the sDH and 4% in the dDH. Jun D, expressed above basal expression in untreated animals at this time-point, was now also reduced by morphine to 75% of control animals in sDH and dDH (Table 1). At 8 h morphine had depressed c-Fos, Krox-24 and Jun B to basal levels in all laminae investigated, while the corresponding control animals still demonstrated considerable expressions (Figs 5, 6; Table 1). The number of c-Fos, Krox-24, c-Jun and Jun B neurons in morphine-treated animals at 8 h following noxious stimulation was not significantly different from basal expression (Duncan-tests, P = n.s.). The only IEGs that were not reduced to basal expression by morphine by the end of the 8 h observation period were Fos B and Jun D. Fos B was present in 12 k 2 n/s in superficial laminae (Fig. 2; Table 1) and Jun D was expressed in 23 i 3 n/s in superficial and 13 + 1 n/s in deep laminae (Fig. 4h, Table 1). These values represented 30% levels of remaining activity for Fos B in sDH and 40-50% levels for Jun D in sDH and dDH and were significantly above basal expression seen in unstimulated animals for Fos B in the sDH and for Jun D in the sDH and dDH (Duncan-tests, P < 0.01).

DISCUSSION

The present data show that peripheral noxious heat stimulation triggers the expression of several transcription factors with a distinct temporospatial pattern. The observed cascade of dynamic alterations in the expression of members of the Fos family (c-Fos, Fos B), the Jun family (c-Jun, Jun B and Jun D) and Krox-24 is prone to a differential

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Fig. 7. Expression of IEG positive neurons in laminae I-VII and X of the rat spinal cord following noxious thermal stimulation and pre-treatment with increasing doses of intravenous morphine (5 and 10 mg/kg). As a control for morphine effects animals were intravenously injected with naloxone (1 or 10mgjkg) before application of morphine (lOmg/kg). The effects of morphine and naloxone were investigated at time points where the various IEGs showed maximal expression following termination of noxious stimulation (2 h for c-Fos, Krox-24, cJun and Jun B; 8 h for Fos B and Jun D). Values given represent mean + S.E.M. of neurons per section relative to the corresponding control values. Asterisks indicate statistical significances (*P < 0.05; **P < 0.01) in Duncan post hoc tests following a one-way ANOVA. Significance for 5 mg/kg morphine is expressed as compared to control, and for 10mg/kg morphine is expressed as compared to 5 mgikg morphine.

pharmacological modulation by systemic application of morphine prior to the noxious stimulation. Temporospatial pattern of immediate-early gene expression c-Fos, c-Jun, Jun B and Krox-24 proteins show maximal staining in the sDH after 2 h. This is in agreement with the initial finding of Hunt et al.” with regard to c-Fos and in line with an increase in nerve growth factorl-A, nerve growth factorl-B, c-fos, SRF and c-jun mRNAs following a single noxious

heat stimulus reported previouslyE as well as with a specific spatial and temporal pattern of Jun B, Jun D and Fos B immunoreactivity following repetitive noxious heat stimuli or C-fibre stimulation.27,29 Our data extend the concept of complex transcriptional operations in the spinal cord following noxious stimulation by adding the time- dependent expression of the zinc-finger DNA binding protein Krox-24.4s,5’ Temporospatial analysis reveals considerable qualitative and quantitative differences in the expression characteristics of the members of the

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various IEG families with an exceptional incongruency being observed in the dDH. In this compartment c-Jun, Jun B and Fos B are almost absent, whereas c-Fos and Jun D are expressed in numerous nuclei. In the sDH several IEGs show overlapping temporal and spatial pattern. The time-course of Krox-24 and c-Fos for instance in the sDH together with in vitro observations of a strikingly similar induction kinetic’j favours the idea of co-expression of various IEGs at least in subsets of spinal neurons. Up to now, however, only co-localization of jun B mRNA and c-Fos protein has been described.57 Besides the possibility of co-expression in models of noxious stimulation, experiments such as long-term potentiation and axotomy suggest a differential induction of various IEGs”.*~.*~ depending on the experimental conditions. With regard to the IEG c-J&, one hypothesis is that the amount of IEG protein produced by dorsal horn neurons is proportional to the degree of synaptic excitation and that the expression of c-fos depends upon the neurochemistry of synaptic transmission and/or upon the postsynaptic propensity for its induction or both.34,82 Assuming that this hypothesis is correct, the diversity of chemical neuroanatomy for neurotransmitters and neuromodulators in the dorsal horn (for reference, see Ref 89) alone might well explain the varying IEG pattern in various laminae of the spinal cord. In addition functional organization evidenced in electrophysiological studies has demonstrated that nociceptive-specific and wide-dynamic range nociceptive neurons are located predominantly in the superficial dorsal horn (laminae I-II) and in the neck of the dorsal horn (laminae V) (for review, see Ref. 7). Many of these neurons contribute to ascending pathways, 50.56show distinct firing patterns or respond to stimulation of peripheral fibres at C-fibre strength with prolonged excitations.” Tracer studies have shown that c-Fos positive ascending tract neurons constitute only a small percentage of all spinal neurons that express c-Fos in response to noxious stimulation5’ suggesting that most of the c-Fos labelled neurons may be interneurons or propriospinal neurons. The observed temporospatial pattern of IEG expression in superficial and deep laminae may reflect the different discharge activity of functionally distinct sets of neurons in the dorsal horn driven mono- or polysynaptically through segmental or supraspinal loop~.‘~~‘~~~~~*’ A slow onset of synaptically evoked excitation could account for the delayed appearance and/or longer lasting persistance of the IEG signal.27~29~81 The weak induction of cJun compared to Jun B following noxious stimulation may be due in part to the relatively low affinity of the specific c-Jun antibody in immunohistochemical experiments. Immunocytochemical considerations aside, transsynaptic stimulation does not seem to be very effective in inducing cJun protein as has been described in in vitro and in uivo experiments.4s28 However, in

contrast to trdnssynaptic stimulation, transection 01 peripheral and central nerve fibres is a highly effective stimulus for induction of c-Jun.26.3h The high basal level of Jun D and its delayed inducibility agrees with recent observations that jun D mRNA is constitutively expressed and shows a delayed onset of expression following stimulation in citrf1.‘2.40.4’.41~J’ Therefore it seems likely that the late onset of Jun D results from a delay in transcription rather than a delay in translation. The slow increase and the long persistance of the Fos B proteins is also consistent with in vitro findings of a prolonged mRNA expression and more stable protein compared to c-Fos.~~~~~Wisden et aLE4were not able to detect changes of jun B or jun B mRNA following single (20 s) noxious heat stimulation. The differences to the present data probably arise to some extent from the rather short interval between the end of a single noxious stimulation and in situ detection of IEG mRNA in this study. This time-interval and the low stimulus rate would be expected to be critical with regard to the levels of the slowly transcribed ,jun D. Other data indicate that the results of any study employing c-Fos as a marker of neuronal activity may be affected by the duration of the excitation stimulus. Noxious heat stimulation of 5 sx2 or electrical nerve stimulation of 3 s duration’* have been shown to induce c-Fos expression in a limited number of neurons in the superficial dorsal horn, whereas continuous stimulation over a period of hours produced many more labelled neurons in all laminae of the spinal cord. ‘* Taken together, beside the neurochemistry of transmission and IEG-specific intracellular kinetics for their induction and translation. some differences in temporospatial patterns may also emerge from the amount of excitation. In the present study almost all induced immunoreactivity was restricted to the ipsilateral dorsal horn. Although we observed the same change in the spatial pattern with time, e.g. for c-Fos from superficial to deeper laminae” we were unable, like others,“,‘” to observe a “second wave” of noxious heat-induced Fos-positive neurons with a marked contralateral component reported previously.*’ The different kinds and depths of anaesthesia used in the various other studies’2.29.81and in the present study may account for differences in the pattern of ipsi and contralateral expression. The only IEG that was found in our study 8 h after stimulation on the contralateral side in a considerable number of neurons was Jun D. Morphine and immediate early gene expression Opioid peptide containing neurons and all known subtypes of opioid ligand binding sites have been documented in the spinal cord. Systemic administration of morphine suppresses neuronal activity at the spinal cord leve186 by a direct action or indirectly, by activation of monaminergic inhibitory control systems originating in the brainstem.‘,3’.42 Both spinal and supraspinal sites probably interact

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morphine or a distinct propensity of neurons located to achieve the analgesic action of opioids (for review, in deeper laminae&2s76to express IEGs is the reason see Ref. 89). why they are more readily affected by opioids. The It has been reported that systemic application of comparatively low sensitivity of Fos B and Jun D the p-agonist morphine and the rc-agonist U-50488 expression to morphine could result from distinct supressed c-Fos induction in the spinal cord followintracellular pathways that initiate or control the ing acute noxious heat stimulation or inflamstart and the persistence of expression of these two mation.25~64~76 Both drugs produced a dose-dependent IEGs. Triggered by the same combination of neuroinhibition of pain behaviour and a dose-dependent transmitters released from small calibre primary suppression of the number of Fos-positive neurons afferents, different metabolic pathways may provide in the spinal corda as well as in more rostra1 variable links to the various members of the activator structures.25 The authors conclude that the proprotein 1 binding family. This assumption is in duction of complete anti-nociception is not associaccordance with observations on IEG expression and ated with a total suppression of Fos-positive long-term potentiation in the hippocampus as well as neurons.24,25@’ Alternatively, behavioural analgesia IEG expression in the spinal cord following synaptic might be achieved without complete anti-nociception stimulation. These studies revealed a selective inducat least on the level of the spinal cord. Similar tion of IEGs as a consequence of distinct receptor to systemic application of morphine, electrical activation.‘7.39.‘8 stimulation in the nucleus raphe magnus and intracerebroventricular administration of [D-Ala*, Functional aspects: immediate early genes and target NMe-Phe4, Gly-o15]enkephalin (DAMGO) reduced gene control the number of dorsal horn neurons that expressed Experimental pain alters the release6’ and levels of c-Fos in response to various peripheral noxious stimseveral neurotransmitters in the spinal cord. Among uli.24.27This effect was not demonstrable after midthothe best studied examples is the increase of opioid racic lesions. Neurons in laminae I and II of the rat synthesis in spinal neurons during experimental spinal dorsal horn which express c-Fos in response to polyarthritis’4,‘3 and localized hind limb inflaminflammatory stimulation receive a dense projection mation.35.55,67 This enhanced opioid synthesis is from enkephalin and serotonin immunoreactive preceded by an ipsilateral increase in c-Fos exvaricosities.65 Experiments in spinally transsected anipression.2”,58 Furthermore, c-Fos, prodynorphin and malsZ4 revealed little contribution of descending pathproenkephalin mRNA have been demonstrated to ways on the pattern of IEG expression following be co-localized in some of these neurons.586o The noxious stimulation.” consecutive appearance of both genes in the same Pretreatment with morphine substantially reduced the expression of all IEG proteins measured in a neurons and the demonstration of AP-I sequences in the prodynorphin gene promoter region are further naloxone-reversible manner. However, at any given evidence in favour of a functional link.47,58A recent time point there was considerable potential for transtudy in transgenic mice demonstrated that the upregscriptional activity left over time in a considerable ulation of proenkephalin synthesis in the spinal number of neurons throughout the whole spinal cord cord in response to pain acts through a 193 base lumbar enlargement. Only at 8 h with morphine pair promoter region in the prodynorphin gene.75 pretreatment did the IEGs c-Fos, Krox-24, c-Jun and Jun B return to basal levels, while Fos B and Jun D Although a causal relationship between peripheral stimulation, IEG induction and long-term changes in were still expressed in 12-23 neurons per 25-pm the expression of target genes is still lacking, circumsection and still available to constitute various and stantial evidence indicates that at least some longvarying transcription forming complexes. The depressant effect of opioid agonists,?4,25.64,76 term effects triggered by peripheral injury in central neurons may be initiated by transactivation of IEGs. antiepileptic drugs” and alpha-2-adrenoceptor agoCompared to the numerous-partly coinduced”nists18,63on IEG expression may bc attributed to their IEG labelled neurons observed following chronic well-documented depressant action on neuronal discharge activity (for review see Refs 86, 89, 90).*’ noxious stimulation’s74 only a limited number of neurons, mostly in the substantia gelatinosa change There is evidence from extra- and intracellular studies mRNA levels for target genes, e.g. genes coding that morphine preferentially supresses C-fibre evoked for opioid peptide precursors.‘7,59sw This obvious disactivity whereas early components of the response crepancy in the number of IEGs and target gene resulting from the activation of large myelinated expressing neurons favours the idea that a highly fibres are affected only slightly, if at all, even by high developed overlap of various transcription factors doses of morphine.‘,‘9,89 Neuronal activity evoked by has to be fulfilled in a single neuron to achieve sustained synaptic bombardment and/or excitation transactivation of a specific secondary target gene derived from low-threshold afferents activated during or, alternatively, only genes with a low level of noxious heat stimulation are probably responsible expression, such as Fos B, c-Jun or Jun D have for the residual expression of IEGs still detected under morphine. It remains to be shown whether a the capacity to induce long-term changes. The actual affinities, specificities and activities of these more pronounced liability to the depressant effect of

318

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transcriptionally active complexes depend upon subunit composition, post-translational modifications, competing ligands, flanking nucleotide sequences and overlapping binding sites.6,‘8.48,49.54.66,68 The level of complexity is extended even further by the finding that IEGs act as negative regulators of their own expression.“,69,83 The functional relevance of these multi-combinatorial transcriptional complexes for DNA binding will depend upon the competition of the various ligands for DNA binding in the nucleus of a given cell. These complexes may uniquely regulate subsets of target genes containing a particular variant of the AP-1 site, or may have different effects upon the same set of regulatory elements.7’,72 The highest binding affinity to DNA is demonstrated for Fos B and c-Jun followed by Jun D containing complexes. ” These IEGs were found in neurons located in regions associated with long-term changes in neuronal excitability in the spinal cord dorsal horn. The high transcriptional activity of Fos B and Jun D could thus trigger transcriptional operations and neuronal plasticity even under protection of a

high dosage stimulation.

of morphine

applied

before

noxious

CONCLUSION

Most of the currently available data on IEG expression by noxious stimulation are based on the IEG c-Fos and experiments employing acute or subacute stimuli. Conclusions drawn from studies such as these, to explain mechanisms that may contribute to states of pain in man such as hyperalgesia, phantomlimb pain or trigeminal neuralgia,‘6,2’.22,70 should be treated with caution, as different mechanisms may operate in states of acute and chronic noxious stimulation. The present experimental observations suggest that, provided the prevention of transcriptional operations is needed in order to avoid central a more elaborate pharmacological sensitization, treatment is required. AcknoM?ledgrments-The authors wish to thank Dr Richard A. Hughes for his critical reading of the manuscript and Dr Wolfgang Wiilwer for assistance with the statistical analysis.

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