- Email: [email protected]
Mu- and delta-opioid receptors are downregulated in the largest diameter primary sensory neurons during postnatal development in rats
Pain 90 (2001) 143±150
www.elsevier.nl/locate/pain
Mu- and delta-opioid receptors are downregulated in the largest diameter primary sensory neurons during postnatal development in rats Barbara Beland 1, Maria Fitzgerald* Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Received 18 January 2000; received in revised form 20 July 2000; accepted 2 August 2000
Abstract The aim of this study was to investigate the postnatal development of m-(MOR) and d-opioid receptor (DOR) immunoreactivity in rat dorsal root ganglia. Lumbar dorsal root ganglia (DRG) from postnatal day (P) 0, 3, 7 and 21 rat pups were immunostained for MOR and DOR. Proportions of MOR 1ve and DOR 1ve cells were calculated from pro®le counts. Diameters of MOR 1ve and DOR 1ve cells were measured and compared to 2ve cells. The coexpression of MOR and neuro®lament (NF200) in DRG over this postnatal period was also investigated. A greater proportion of cells were immunoreactive for MOR and DOR in neonatal rat DRG at P0, P3 and P7 compared to P21. At P3, 39.5 ^ 1.7% of cells were MOR 1ve and 30.3 ^ 1.5% were DOR 1ve, whereas at P21, the values were 30.1 ^ 1.7% and 21.8 ^ 1.6% (mean ^ SEM), respectively. During the ®rst postnatal week both opioid receptors were expressed in cells across the whole diameter range but by 3 weeks of age, expression was restricted to small and medium diameter cells. Furthermore, a signi®cantly higher proportion of NF200 1ve cells expressed MOR in new-born compared to P21 rats. The results show that MOR and DOR expression is downregulated in the largest diameter, NF200 1ve primary sensory neurons postnatally. Since these neurons are mainly non-nociceptive, this may explain previous reports of opioid agonists affecting re¯ex responses to both innocuous and noxious stimuli in rat pups. The results highlight an important difference between opioid function in the immature and adult nervous system. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Opioid receptor; Dorsal root ganglion; Primary sensory neuron; Neonate; Morphine; Paediatric pain
1. Introduction Despite the fact that opioids are now widely used in neonates and infants (Purcell-Jones et al., 1987; De Lima et al., 1996) there is still a lack of knowledge on their speci®c effects in this population of patients (Marsh et al., 1997). Differences in opioid pharmacology compared to the adult may arise from changes in drug metabolism and transport, receptor expression and function and also from differences in information processing due to immature neuronal connections (Fitzgerald, 1999). Of importance also is the development of other transmitter systems in¯uencing opioid effects, such as NMDA and cholecystokinin (Dickenson, 1994; Fitzgerald, 1997). The analgesic effectiveness of opioid agonists is likely to be different in neonates compared to adults although this has not been directly studied in humans and animal data varies between labora* Corresponding author. Fax: 144-171-387-0905. E-mail address: m.®[email protected] (M. Fitzgerald). 1 Present address: Klinik und Poliklinik fuer Anaesthesiologie und operative Intensivmedizin, Westfaelische Wilhems-Universitaet Muenster, Albert-Schweitzer-Strasse 33, 49149, Muenster, Germany.
tories (Zhang and Pasternak, 1981; McLaughlin and Dewey, 1994; Windh and Kuhn, 1995; Rahman et al., 1998; Thornton et al., 1998; Rahman and Dickenson, 1999). In addition, there is increasing evidence from animal studies that opioids have qualitatively different effects in the immature compared to the mature nervous system. While opioid agonists speci®cally depress nociceptive C and Ad inputs in the adult (Dickenson et al., 1987), studies in vitro and in vivo suggest that non-nociceptive Ab-mediated stimuli are also depressed in neonatal rats (Faber et al., 1997; Marsh et al., 1999). One possible mechanism for this lack of opioid selectivity in the young rat spinal cord could be a different pattern of expression of opioid receptors which is regulated over the postnatal period. The ontogeny of opioid binding, receptor protein and gene expression and function in the central nervous system has been studied in animals and humans (Szucs and Coscia, 1990; Kar and Quirion, 1995; Windh and Kuhn, 1995; Rahman et al., 1998; Zhu et al., 1998; Ray and Wadhwa, 1999) but information on the development of opioid receptor expression in primary afferent neurons is limited (Zhu et al., 1998). In the spinal cord, most opioid receptors (OR) are
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(00)00397-3
144
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
located presynaptically on central terminals of primary afferents (Besse et al., 1990) and so a study of their expression in developing dorsal root ganglia (DRG) is clearly necessary if we are to understand the ontogeny of opioid systems and to use opioids effectively in the management of infant pain. The aim of this study, therefore, was to investigate the postnatal development of m- (MOR) and d-opioid receptor (DOR) expression in dorsal root ganglia of neonatal and adolescent rats. 2. Methods Sprague±Dawley rats were obtained from UCL Biological Services. All experiments were performed under personal and project licences in accordance with the UK Animals (Scienti®c Procedures) Act, 1986. L4/5 dorsal root ganglia (DRG) were investigated from rat pups of both sexes at postnatal day (P) 0, 3, 7 and 21. Six animals of each age group were used for MOR and DOR diaminobenzidine immunostaining and four animals of each group were used for MOR/neuro®lament (NF200) double¯uorescence immunohistochemistry. 2.1. Immunohistochemistry Following perfusion ®xation with 4% paraformaldehyde/ 0.04% picric acid in 0.1 M phosphate-buffered saline (PBS), L4/5 DRG were dissected out, post®xed overnight and cryoprotected for 24 h in 30% sucrose in PBS. Cryostat sections (10 mm) were collected on aminosilane-coated slides (Sigma), air-dried overnight and stored frozen for up to 2 months. For each DRG, four sections were sampled which were equally distributed within the ganglion. The ®rst section was sampled randomly. For staining, sections were brought back to room temperature and rehydrated in PBS for 15 min. A blocking solution of 20% goat serum (v/v) (Vector Laboratories) containing 0.4% Triton (v/v) (T-x, Sigma) was applied for 1 h. All antibodies were diluted in PBS containing 2% goat serum and 0.4% T-x. Incubation with primary antibodies was done for 48 h at 48C; the secondary antibody solution was left to incubate for 3 h at room temperature. Slides were washed three times for 10 min each in PBS after each incubation. For diaminobenzidine (DAB) staining the secondary antibody (biotinylated anti-rabbit, 1:200, Vector) was followed by ABC solution (ABC elite, Vector) and incubated for 3 h. Slides were then washed followed by a nickel enhanced DAB reaction (Llewellyn-Smith et al., 1992). They were counterstained in Toluidine Blue, dehydrated in a series of alcohols and cleared in Histo-Clear (National Diagnostics). Slides were coverslipped using DPX (Fisher Scienti®c) and air-dried. Primary antibodies for MOR and DOR were a generous gift from R. Elde. The antibody speci®c to MOR is raised in rabbits against a peptide corresponding to the predicted
carboxy terminus of cloned rat MOR1 (Arvidsson et al., 1995b) and was used in 1:4000 dilution for DAB staining. Rabbit anti-DOR is raised against a peptide corresponding to aminoacids 3±17 of the DOR1 sequence (Arvidsson et al., 1995a). It was used in 1:2000 dilution for DAB staining. To investigate coexpression of MOR and NF200, DRG were double-immunostained with anti-MOR in combination with anti-NF200 (clone 52). This antibody of the mouse clone N52 (Sigma) against phosphorylated and unphosphorylated high molecular weight neuro®lament (NF200) can be used to label large cells in neonatal and adolescent rats (unpublished data). The basic protocol for double-¯uorescence immunohistochemistry was as described above. The primary antibody solution contained both anti-MOR (1:1000) and anti-NF200 (1:500). Alexa ¯uor 594 conjugated anti-rabbit and Alexa ¯uor 488 conjugated antimouse obtained from Molecular Probes were used as secondary antibodies (1:200). Red emitting Alexa ¯uor 594 can be used as an alternative to Texas Red dye while the green emitting Alexa ¯uor 488 is an alternative to ¯uorescein. Slides were coverslipped using Prolong anti-fade mounting medium (Molecular Probes) and air-dried. In each immunostaining run, sections from animals of each age group were processed simultaneously. 2.2. Analysis To calculate the proportions of the OR immunoreactive cells, light microscope images of DAB-stained DRG sections were grabbed with a colour CCD camera (Sony) using Leica Imaging Systems Ltd software. All cell pro®les (both labelled and unlabelled) with a visible nucleus were counted in four equally distributed sections within each DRG and the proportions of labelled cells per DRG were calculated. Cell diameters of all cell pro®les (both labelled and unlabelled) with a visible nucleus were measured in DAB-stained DRG sections using the Leica software. At least 200 cell diameters were measured in each age group (P0, P3, P7, P21). To quantify MOR/NF200 coexpression, photographs of ¯uorescent double-stained sections were taken using a Nikon G-2A ®lter for Alexa 594 ¯uorescence and ®lter B2A for Alexa 488. The two corresponding photographs of each section were scanned into a computer and overlapped using image processing software. Cell pro®les were counted from images of four equally distributed sections/DRG that were 1ve for MOR or NF200 only or both markers. Proportions of labelled cells from the different age groups were analyzed by non-parametrical analysis of variance (Kruskal±Wallis test) followed by Dunn's test for multiple comparisons using a standard statistical software program. Cell size distributions of OR 1ve and OR 2ve cell populations were compared by parametrical analysis of variance followed by the Bonferroni test for multiple comparisons. A P value of 0.05 was chosen as a level for statistical signi®cance.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
3. Results 3.1. General aspects of OR staining in neonatal and adolescent rats Both MOR and DOR immunoreactivity (Figs. 1 and 2) were detected in DRG cell bodies of neonatal rats as early as P0. Large as well as small cells were positive (1ve) for MOR and DOR in younger animals while expression was con®ned mainly to small and medium cells in P21 rats. In P0 and P3 rats OR immunoreactivity was rarely seen in ®bres. 3.2. MOR and DOR immunoreactive cell proportions in neonatal and adolescent rats Proportions of MOR and DOR 1ve cell pro®les were estimated in P0, P3, P7 and P21 rats (n 6 for each age group). In immature pups at age P3 and P7 the proportion of MOR and DOR 1ve DRG cells was signi®cantly higher compared to mature P21 rats. Proportions of MOR 1ve pro®les were 36.9 ^ 2.3% at P0, 39.5 ^ 1.7% at P3, 39.5 ^ 1.0% at P7 and 30.1 ^ 1.7% at P21 (mean ^ SEM, Fig. 3). A smaller proportion of cells expressed DOR, but a similar pattern of postnatal downregulation was observed: 28.5 ^ 1.9% at P0, 30.3 ^ 1.5% at P3, 31.2 ^ 1.5% at P7 and 21.8 ^ 1.6% at P21 (mean ^ SEM, Fig. 3). 3.3. Size distribution of MOR and DOR immunoreactive DRG cells in neonatal and adolescent rats Both MOR and DOR were expressed in cells across all diameters at P0, P3 and P7. In these neonatal rats, the mean diameter of MOR 1ve and MOR 2ve cells was the same, illustrating their equal size distribution (MOR 1ve at P0: 15.3 ^ 3.7 mm, MOR 2ve at P0: 15.1 ^ 3.4 mm; MOR 1ve at P3: 15.4 ^ 4.0 mm, MOR 2ve at P3: 15.5 ^ 4.6 mm; MOR 1ve at P7: 15.3 ^ 5.0 mm, MOR 2ve at P7: 15.8 ^ 5.6 mm; mean ^ SD, Fig. 4). The mean diameter of DOR 1ve and 2ve cell populations also did not differ at P0 and P7 (DOR 1ve at P0: 12.1 ^ 4.3 mm, DOR 2ve at P0: 12.7 ^ 2.9 mm; DOR 1ve at P7: 16.0 ^ 5.5 mm, DOR 2ve at P7: 15.3 ^ 5.0 mm; mean ^ SD, Fig. 5) while at P3, DOR 1ve cell pro®les were signi®cantly larger than the DOR 2ve population (DOR 1ve at P3: 15.4 ^ 5.0 mm, DOR 2ve at P3: 12.7 ^ 3.8 mm; mean ^ SD, Fig. 5). In contrast, at P21 very few large DRG cells showed MOR and DOR immunoreactivity, which was restricted to the small to medium sized population. The mean cell diameter of OR 1ve pro®les was signi®cantly less than that of OR 2ve pro®les (MOR 1ve: 18.5 ^ 5.7 mm, MOR 2ve: 24.3 ^ 8.3 mm; mean ^ SD, Fig. 4; DOR 1ve: 17.8 ^ 4.2 mm, DOR 2ve: 23.6 ^ 8.4 mm; mean ^ SD, Fig. 5).
145
3.4. Postnatal development of MOR/NF coexpression in DRG cells Since the largest diameter cells in the ®rst postnatal week are considerably smaller than at P21, it is important to establish whether there is a genuine postnatal downregulation of MOR expression in the largest DRG cells or simply that the population of large cells is not apparent until P21. To determine this, coexpression of MOR with NF200, a marker for large diameter cells was investigated in P0, P3, P7 and P21 rats (n 4 for each age group, Fig. 7). In new-born P0 pups a signi®cantly higher proportion of cells coexpressed MOR and NF200 (Fig. 6). At P0, 26.3 ^ 0.6% of MOR 1ve pro®les also expressed NF200 compared to 14.3 ^ 1.5% at P21 (mean ^ SEM, Fig. 6A). At P0, 25 ^ 2.8% of NF200 1ve cells also expressed MOR compared to 13.3 ^ 1.6% at P21 (mean ^ SEM, Fig. 6B).
4. Discussion The results presented here show that both MOR and DOR are downregulated in DRG neurons during postnatal development in the rat. These neurons, being NF200 1ve represent the largest diameter, non-nociceptive population of sensory afferents (Lawson and Waddell, 1991). In the ®rst postnatal week, signi®cantly more DRG cells express MOR and DOR distributed across all diameters. By 3 weeks of age, fewer cells express the receptors and the distribution is con®ned to small to medium-sized cells. This anatomical observation provides an explanation for the effects of m- and d-opioid agonists on non-nociceptive Ab-mediated stimuli observed in neonatal rats (Faber et al., 1997; Marsh et al., 1999). The expression of OR mRNA has been previously investigated in embryonic mice DRG. DOR mRNA is detected ®rst at embryonic day (E) 12.5 followed by MOR at E13.5. Expression of k receptors (KOR) mRNA is seen much later at E17.5 (Zhu et al., 1998). Thus, MOR and DOR are transcribed at a time point during embryonic development when large DRG neurons are generated (Lawson et al., 1974). MOR binding was also recently reported in P1 rat DRG (Ray and Wadhwa, 1999). The results of the present study provide the ®rst information about the postnatal developmental regulation of OR expression in different populations of DRG cells. The proportions of cells expressing OR found here are consistent with previous reports. MOR immunoreactivity has been reported in 17% (Zhang et al., 1998a) and 21% (Ji et al., 1995) of adult rat DRG cells although high level MOR mRNA expression has been found in 55% (Minami et al., 1995). In two studies, DOR immunoreactivity has been observed in 14% of adult rat DRG cells (Zhang et al., 1998a; Ji et al., 1995) although another study reports a higher 40% (Zhang et al., 1998b). High level DOR mRNA expression occurs in 19% of rat DRG cells (Minami et al., 1995). Since the proportions in adult DRG are slightly lower
146
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Fig. 1. m-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. Scale bars represent 50 mm.
Fig. 2. d-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. Scale bars represent 50 mm.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
147
Fig. 3. (A) Proportions of m-opioid receptor immunoreactive cells in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. n 6 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons). (B) Proportions of d-opioid receptor immunoreactive cells in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. n 6 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons).
than those found at P21 in this study, a further downregulation of MOR and DOR expression may occur during later development. Differences may also arise from immunostaining and cell counting methods. The results of the present and previously reported studies in adult animals are based on pro®le counts and the proportions of the different subpopulations can only be estimates. There is an ongoing discussion about the bene®ts of different methods of DRG cell counting (Guillery and Herrup, 1997; West, 1999). Previous studies have shown m-opioid agonist binding in rat spinal cord at birth but delayed d-agonist binding for the ®rst two postnatal weeks (Attali et al., 1990; McDowell and Kitchen, 1987; Rahman et al., 1998). DOR immunoreactivity in rat DRG was clearly seen as early as P0 in this study and DOR mRNA expression appears in early embryonic mice DRG (Zhu et al., 1998). This suggests that anterograde transport of DOR binding sites from the DRG to central terminals in the spinal cord is delayed in the immature animal. Both MOR and DOR were found to be expressed in cells
of all sizes in immature neonatal DRG but restricted to small and medium DRG neurons in mature P21 rats. Since the overall size of the whole DRG cell population increases over this period, it is important that the study of MOR and NF200 coexpression con®rmed the downregulation of OR expression in large DRG neurons during the second and third postnatal week. We have recently shown that two populations of DRG cells, large and small to medium, can be distinguished by NF200 (clone N52) immunostaining as early as P0 (unpublished data). These ®ndings may explain the postnatal changes in m-opioid agonist binding within the spinal cord. In the adult animal, m-opioid binding is restricted to the super®cial dorsal horn, the site of C-®bre termination (Besse et al., 1990), but in the neonatal spinal cord it is also seen in deep dorsal horn where A-®bres terminate (Kar and Quirion, 1995; Rahman et al., 1998). These anatomical results have functional implications. The majority of larger DRG neurons are the cell bodies of non-nociceptive Ab mechanoreceptors and proprioceptors (Lawson and Waddell, 1991) and there is evidence that
Fig. 4. Cell size distribution of m-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21.
148
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Fig. 5. Cell size distribution of d-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21.
opioids can depress Ab-®bre-mediated responses in the neonatal spinal cord. The neonatal withdrawal re¯ex to low intensity mechanical stimuli is thought to have an Ab-mediated component (Fitzgerald and Jennings, 1999) and epidural opioid agonists increase the low baseline mechanical threshold of these re¯exes in neonatal rats (Marsh et al., 1999). In addition, morphine depresses both
Ab- and C-®bre-mediated ventral root re¯exes in the isolated neonatal spinal cord (Faber et al., 1997). This is in contrast to older animals where m- and d-opioid agonists have been shown to selectively depress C- and Ad-®bre but not Ab-®bre input to the dorsal horn (Dickenson et al., 1987; Rahman and Dickenson, 1999) and opioid agonists inhibit membrane currents in small but not in large adult
Fig. 6. Double-¯uorescence immunohistochemistry for m-opioid receptor (A) and neuro®lament (B) in L4/5 dorsal root ganglia of rat pups at postnatal day 0, and double-¯uorescence immunohistochemistry for m-opioid receptor (C) and neuro®lament (D) in L4/5 dorsal root ganglia of rat pups at postnatal day 21. Arrows indicate cell pro®les coexpressing both m-opioid receptor and neuro®lament. Scale bars represent 50 mm.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
149
Fig. 7. (A) Proportions of m-opioid receptor immunoreactive cells coexpressing neuro®lament 200 (NF200) in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21 as seen in double-¯uorescence immunohistochemistry. n 4 for each age group. Mean ^ SEM. **P , 0:01 versus P21 (Dunn's test for multiple comparisons). (B) Proportions of NF200 immunoreactive cells coexpressing m-opioid receptor in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21 as seen in double-¯uorescence immunohistochemistry. n 4 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons).
DRG neurons in culture (Taddese et al., 1995; Abdulla and Smith, 1998). The study presented here provides a mechanism to explain the qualitatively different effect of opioids on sensory processing in the immature animal affecting both nociceptive and non-nociceptive input. The wider depression of sensory inputs by opioids may be advantageous to protect the central nervous system from inputs that, while not necessarily noxious, may still be stressful. The effects of opioids in the immature animal and human will be in¯uenced by many different mechanisms apart from the pattern of OR expression. In the adult, opioid analgesia is mediated via presynaptic receptors reducing transmitter release, postsynaptic receptors hyperpolarizing the cell and effects on descending inhibitory pathways (Dickenson, 1994). The delayed maturation of descending inhibition of spinal cord activity over the ®rst three postnatal weeks will no doubt affect analgesic pathways (Fitzgerald and Koltzenburg, 1986). Altered opioid effects may also arise from differences in receptor dynamics and second messenger systems. G-protein coupling and activity are known to change during postnatal development in rats (Szucs and Coscia, 1990; Windh and Kuhn, 1995). Nothing is known of the developmental regulation of opioid receptor heterodimerization which leads to altered binding and function (Jordan and Devi, 1999) and which would not be detected by immunohistochemistry. The transient postnatal expression of m- and d-opioid receptors in larger NF200 1ve cells of the DRG is an example of how neonatal and adult opioid systems differ. Studies such as these have important implications for the safety and effectiveness of opioid administration in neonates and infants.
Acknowledgements We are grateful to Robert Elde, Department of Pharmacology, University of Minnesota, Minneapolis for the generous gift of MOR and DOR antibodies. We also wish to
acknowledge J. Meredith-Middleton and A. Allchorne for excellent technical assistance. This study was supported by grants from the IMF and DFG, Germany to B.B. and the MRC, UK to M.F.
References Abdulla FA, Smith PA. Axotomy reduces the effect of analgesic opioids yet increases the effect of nociception on dorsal root ganglion neurons. J Neurosci 1998;18:9685±9694. Arvidsson U, Dado RJ, Riedl M, Lee JH, Law PY, Loh HH, Elde R, Wessendorf MW. Delta-opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 1995a;15:1215±1235. Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 1995b;15:3328±3341. Attali B, Saya D, Vogel Z. Pre- and postnatal development of opiate receptor subtypes in rat spinal cord. Brain Res Dev Brain Res 1990;53:97± 102. Besse D, Lombard MC, Zajac JM, Roques BP, Besson JM. Pre- and postsynaptic distribution of mu, delta and kappa opioid receptors in the super®cial layers of the cervical dorsal horn of the rat spinal cord. Brain Res 1990;521:15±22. De Lima J, Lloyd-Thomas AR, Howard RF, Sumner E, Quinn TM. Infant and neonatal pain: anaesthetists' perceptions and prescribing pattern. Br Med J 1996;313:787. Dickenson AH. Where and how do opioids act? In: Gebhardt GF, Hammond DL, Jensen TS, editors. Progress in pain research and management, 2. Seattle, WA: International Association for the Study of Pain, 1994. pp. 525±552. Dickenson AH, Sullivan AF, Knox R, Zajac JM, Roques BP. Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with mand d-opioid receptor agonists in the control of nociception. Brain Res 1987;413:36±44. Faber ESL, Chambers JP, Brugger F, Evans RH. Depression of A and C ®bre-evoked segmental re¯exes by morphine and clonidine in the in vitro spinal cord of the neonatal rat. Br J Pharmacol 1997;120:1390± 1396. Fitzgerald M. Neonatal pharmacology of pain. In: Besson J-M, Dickenson AH, editors. Handbook of experimental pharmacology, Heidelberg: Springer-Verlag, 1997. pp. 447±465. Fitzgerald M. The developmental neurobiology of pain. In: Wall PD, editor.
150
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Textbook of pain, Edinburgh: Churchill-Livingstone, 1999. pp. 153± 170. Fitzgerald M, Jennings E. The postnatal development of spinal sensory processing. Proc Natl Acad Sci USA 1999;96:7719±7722. Fitzgerald M, Koltzenburg M. The functional development of descending inhibitory pathways in the dorsolateral funiculus of the newborn rat spinal cord. Dev Brain Res 1986;24:261±270. Guillery RW, Herrup K. Quanti®cation without ponti®cation: choosing a method for counting objects in sectioned tissues. J Comp Neurol 1997;386:2±7. Ji R-R, Zhang Q, Law P-Y, Low HH, Elde R, Hokfelt T. Expression of mu-, delta-, and kappa-opioid receptor like immunoreactivities in rat dorsal root ganglia after carrageenan-induced in¯ammation. J Neurosci 1995;15:8156±8166. Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature 1999;399:697±700. Kar S, Quirion R. Neuropeptide receptors in developing and adult rat spinal cord: an in vitro quantitative audioradiography study of calcitonin generelated peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors. J Comp Neurol 1995;354:253±281. Lawson SN, Waddell PJ. Soma neuro®lament immunoreactivity is related to cell size and ®ber conduction velocity in rat primary neurones. J Physiol (Lond) 1991;435:41±63. Lawson SN, Caddy KWT, Biscoe TJ. Development of rat dorsal root ganglion neurones. Cell Tissue Res 1974;153:399±413. Llewellyn-Smith IJ, Pilowsky P, Minson JB. Retrograde tracers for light and electron microscopy. In: Bolam JP, editor. Experimental neuroanatomy, Oxford: Oxford University Press, 1992. pp. 37±38. Marsh DF, Hatch DJ, Fitzgerald M. Opioid systems and the newborn. Br J Anaesth 1997;79:787±795. Marsh D, Dickenson A, Hatch D, Fitzgerald M. Epidural opioid analgesia in infant rats I: mechanical and heat responses. Pain 1999;82:23±32. McDowell J, Kitchen I. Development of opioid systems: peptides, receptors and pharmacology. Brain Res 1987;434:397±421. McLaughlin CR, Dewey WL. A comparison of the antinociceptive effects of opioid agonists in neonatal and adult rats in phasic and tonic nociceptive tests. Pharmacol Biochem Behav 1994;49:1017±1023. Minami M, Maekawa K, Yabuuchi K, Satoh M. Double in situ hybridiza-
tion study on coexistence of mu-, delta- and kappa-opioid receptor mRNAs with preprotachkinin A mRNA in the rat dorsal root ganglia. Mol Brain Res 1995;30:203±210. Purcell-Jones G, Dormon F, Sumner E. The use of opioids in neonates. A retrospective study of 933 cases. Anaesthesiology 1987;42:1316±1320. Rahman W, Dickenson AH. Electrophysiological studies on the postnatal development of the spinal antinociceptive effects of the delta opioid receptor agonist DPDPE in the rat. Br J Pharmacol 1999;126:1115± 1122. Rahman W, Dashwood MR, Fitzgerald M, Aynsley-Green A, Dickenson AH. Postnatal development of multiple opioid receptors in the spinal cord and development of spinal morphine analgesia. Dev Brain Res 1998;108:239±254. Ray SB, Wadhwa S. Mu opioid receptors in developing human spinal cord. J Anat 1999;195:11±18. Szucs M, Coscia CJ. Evidence for delta-opioid binding and GTP-regulatory proteins in 5-day-old rat brain membranes. J Neurochem 1990;54:1419±1425. Taddese A, Nah S-Y, McCleskey AW. Selective opioid inhibition of small nociceptive neurons. Science 1995;270:1366±1369. Thornton SR, Compton DR, Smith FL. Ontogeny of mu opioid agonist antinociception in postnatal rats. Dev Brain Res 1998;105:269±276. West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci 1999;22:51±61. Windh RT, Kuhn CM. Increased sensitivity to mu opiate antinociception in the neonatal rat despite weaker receptor-guanyl nucleotide binding protein coupling. J Pharmacol Exp Ther 1995;273:1353±1360. Zhang AZ, Pasternak GW. Ontogeny of pharmacology and receptors: high and low af®nity site differences. Eur J Pharmacol 1981;73:29±40. Zhang Q, Schaffer M, Elde R, Stein C. Effects of neurotoxins and hindpaw in¯ammation on opioid receptor immunoreactivities in dorsal root ganglia. Neuroscience 1998a;85:281±291. Zhang X, Bao L, Arvidsson U, Elde R, Hokfelt T. Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles. Neuroscience 1998b;82:1225±1242. Zhu Y, Hsu M-S, Pintar JE. Developmental expression of the mu, kappa, and delta opioid receptor mRNAs in mouse. J Neurosci 1998;18:2538±2549.
www.elsevier.nl/locate/pain
Mu- and delta-opioid receptors are downregulated in the largest diameter primary sensory neurons during postnatal development in rats Barbara Beland 1, Maria Fitzgerald* Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Received 18 January 2000; received in revised form 20 July 2000; accepted 2 August 2000
Abstract The aim of this study was to investigate the postnatal development of m-(MOR) and d-opioid receptor (DOR) immunoreactivity in rat dorsal root ganglia. Lumbar dorsal root ganglia (DRG) from postnatal day (P) 0, 3, 7 and 21 rat pups were immunostained for MOR and DOR. Proportions of MOR 1ve and DOR 1ve cells were calculated from pro®le counts. Diameters of MOR 1ve and DOR 1ve cells were measured and compared to 2ve cells. The coexpression of MOR and neuro®lament (NF200) in DRG over this postnatal period was also investigated. A greater proportion of cells were immunoreactive for MOR and DOR in neonatal rat DRG at P0, P3 and P7 compared to P21. At P3, 39.5 ^ 1.7% of cells were MOR 1ve and 30.3 ^ 1.5% were DOR 1ve, whereas at P21, the values were 30.1 ^ 1.7% and 21.8 ^ 1.6% (mean ^ SEM), respectively. During the ®rst postnatal week both opioid receptors were expressed in cells across the whole diameter range but by 3 weeks of age, expression was restricted to small and medium diameter cells. Furthermore, a signi®cantly higher proportion of NF200 1ve cells expressed MOR in new-born compared to P21 rats. The results show that MOR and DOR expression is downregulated in the largest diameter, NF200 1ve primary sensory neurons postnatally. Since these neurons are mainly non-nociceptive, this may explain previous reports of opioid agonists affecting re¯ex responses to both innocuous and noxious stimuli in rat pups. The results highlight an important difference between opioid function in the immature and adult nervous system. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Opioid receptor; Dorsal root ganglion; Primary sensory neuron; Neonate; Morphine; Paediatric pain
1. Introduction Despite the fact that opioids are now widely used in neonates and infants (Purcell-Jones et al., 1987; De Lima et al., 1996) there is still a lack of knowledge on their speci®c effects in this population of patients (Marsh et al., 1997). Differences in opioid pharmacology compared to the adult may arise from changes in drug metabolism and transport, receptor expression and function and also from differences in information processing due to immature neuronal connections (Fitzgerald, 1999). Of importance also is the development of other transmitter systems in¯uencing opioid effects, such as NMDA and cholecystokinin (Dickenson, 1994; Fitzgerald, 1997). The analgesic effectiveness of opioid agonists is likely to be different in neonates compared to adults although this has not been directly studied in humans and animal data varies between labora* Corresponding author. Fax: 144-171-387-0905. E-mail address: m.®[email protected] (M. Fitzgerald). 1 Present address: Klinik und Poliklinik fuer Anaesthesiologie und operative Intensivmedizin, Westfaelische Wilhems-Universitaet Muenster, Albert-Schweitzer-Strasse 33, 49149, Muenster, Germany.
tories (Zhang and Pasternak, 1981; McLaughlin and Dewey, 1994; Windh and Kuhn, 1995; Rahman et al., 1998; Thornton et al., 1998; Rahman and Dickenson, 1999). In addition, there is increasing evidence from animal studies that opioids have qualitatively different effects in the immature compared to the mature nervous system. While opioid agonists speci®cally depress nociceptive C and Ad inputs in the adult (Dickenson et al., 1987), studies in vitro and in vivo suggest that non-nociceptive Ab-mediated stimuli are also depressed in neonatal rats (Faber et al., 1997; Marsh et al., 1999). One possible mechanism for this lack of opioid selectivity in the young rat spinal cord could be a different pattern of expression of opioid receptors which is regulated over the postnatal period. The ontogeny of opioid binding, receptor protein and gene expression and function in the central nervous system has been studied in animals and humans (Szucs and Coscia, 1990; Kar and Quirion, 1995; Windh and Kuhn, 1995; Rahman et al., 1998; Zhu et al., 1998; Ray and Wadhwa, 1999) but information on the development of opioid receptor expression in primary afferent neurons is limited (Zhu et al., 1998). In the spinal cord, most opioid receptors (OR) are
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(00)00397-3
144
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
located presynaptically on central terminals of primary afferents (Besse et al., 1990) and so a study of their expression in developing dorsal root ganglia (DRG) is clearly necessary if we are to understand the ontogeny of opioid systems and to use opioids effectively in the management of infant pain. The aim of this study, therefore, was to investigate the postnatal development of m- (MOR) and d-opioid receptor (DOR) expression in dorsal root ganglia of neonatal and adolescent rats. 2. Methods Sprague±Dawley rats were obtained from UCL Biological Services. All experiments were performed under personal and project licences in accordance with the UK Animals (Scienti®c Procedures) Act, 1986. L4/5 dorsal root ganglia (DRG) were investigated from rat pups of both sexes at postnatal day (P) 0, 3, 7 and 21. Six animals of each age group were used for MOR and DOR diaminobenzidine immunostaining and four animals of each group were used for MOR/neuro®lament (NF200) double¯uorescence immunohistochemistry. 2.1. Immunohistochemistry Following perfusion ®xation with 4% paraformaldehyde/ 0.04% picric acid in 0.1 M phosphate-buffered saline (PBS), L4/5 DRG were dissected out, post®xed overnight and cryoprotected for 24 h in 30% sucrose in PBS. Cryostat sections (10 mm) were collected on aminosilane-coated slides (Sigma), air-dried overnight and stored frozen for up to 2 months. For each DRG, four sections were sampled which were equally distributed within the ganglion. The ®rst section was sampled randomly. For staining, sections were brought back to room temperature and rehydrated in PBS for 15 min. A blocking solution of 20% goat serum (v/v) (Vector Laboratories) containing 0.4% Triton (v/v) (T-x, Sigma) was applied for 1 h. All antibodies were diluted in PBS containing 2% goat serum and 0.4% T-x. Incubation with primary antibodies was done for 48 h at 48C; the secondary antibody solution was left to incubate for 3 h at room temperature. Slides were washed three times for 10 min each in PBS after each incubation. For diaminobenzidine (DAB) staining the secondary antibody (biotinylated anti-rabbit, 1:200, Vector) was followed by ABC solution (ABC elite, Vector) and incubated for 3 h. Slides were then washed followed by a nickel enhanced DAB reaction (Llewellyn-Smith et al., 1992). They were counterstained in Toluidine Blue, dehydrated in a series of alcohols and cleared in Histo-Clear (National Diagnostics). Slides were coverslipped using DPX (Fisher Scienti®c) and air-dried. Primary antibodies for MOR and DOR were a generous gift from R. Elde. The antibody speci®c to MOR is raised in rabbits against a peptide corresponding to the predicted
carboxy terminus of cloned rat MOR1 (Arvidsson et al., 1995b) and was used in 1:4000 dilution for DAB staining. Rabbit anti-DOR is raised against a peptide corresponding to aminoacids 3±17 of the DOR1 sequence (Arvidsson et al., 1995a). It was used in 1:2000 dilution for DAB staining. To investigate coexpression of MOR and NF200, DRG were double-immunostained with anti-MOR in combination with anti-NF200 (clone 52). This antibody of the mouse clone N52 (Sigma) against phosphorylated and unphosphorylated high molecular weight neuro®lament (NF200) can be used to label large cells in neonatal and adolescent rats (unpublished data). The basic protocol for double-¯uorescence immunohistochemistry was as described above. The primary antibody solution contained both anti-MOR (1:1000) and anti-NF200 (1:500). Alexa ¯uor 594 conjugated anti-rabbit and Alexa ¯uor 488 conjugated antimouse obtained from Molecular Probes were used as secondary antibodies (1:200). Red emitting Alexa ¯uor 594 can be used as an alternative to Texas Red dye while the green emitting Alexa ¯uor 488 is an alternative to ¯uorescein. Slides were coverslipped using Prolong anti-fade mounting medium (Molecular Probes) and air-dried. In each immunostaining run, sections from animals of each age group were processed simultaneously. 2.2. Analysis To calculate the proportions of the OR immunoreactive cells, light microscope images of DAB-stained DRG sections were grabbed with a colour CCD camera (Sony) using Leica Imaging Systems Ltd software. All cell pro®les (both labelled and unlabelled) with a visible nucleus were counted in four equally distributed sections within each DRG and the proportions of labelled cells per DRG were calculated. Cell diameters of all cell pro®les (both labelled and unlabelled) with a visible nucleus were measured in DAB-stained DRG sections using the Leica software. At least 200 cell diameters were measured in each age group (P0, P3, P7, P21). To quantify MOR/NF200 coexpression, photographs of ¯uorescent double-stained sections were taken using a Nikon G-2A ®lter for Alexa 594 ¯uorescence and ®lter B2A for Alexa 488. The two corresponding photographs of each section were scanned into a computer and overlapped using image processing software. Cell pro®les were counted from images of four equally distributed sections/DRG that were 1ve for MOR or NF200 only or both markers. Proportions of labelled cells from the different age groups were analyzed by non-parametrical analysis of variance (Kruskal±Wallis test) followed by Dunn's test for multiple comparisons using a standard statistical software program. Cell size distributions of OR 1ve and OR 2ve cell populations were compared by parametrical analysis of variance followed by the Bonferroni test for multiple comparisons. A P value of 0.05 was chosen as a level for statistical signi®cance.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
3. Results 3.1. General aspects of OR staining in neonatal and adolescent rats Both MOR and DOR immunoreactivity (Figs. 1 and 2) were detected in DRG cell bodies of neonatal rats as early as P0. Large as well as small cells were positive (1ve) for MOR and DOR in younger animals while expression was con®ned mainly to small and medium cells in P21 rats. In P0 and P3 rats OR immunoreactivity was rarely seen in ®bres. 3.2. MOR and DOR immunoreactive cell proportions in neonatal and adolescent rats Proportions of MOR and DOR 1ve cell pro®les were estimated in P0, P3, P7 and P21 rats (n 6 for each age group). In immature pups at age P3 and P7 the proportion of MOR and DOR 1ve DRG cells was signi®cantly higher compared to mature P21 rats. Proportions of MOR 1ve pro®les were 36.9 ^ 2.3% at P0, 39.5 ^ 1.7% at P3, 39.5 ^ 1.0% at P7 and 30.1 ^ 1.7% at P21 (mean ^ SEM, Fig. 3). A smaller proportion of cells expressed DOR, but a similar pattern of postnatal downregulation was observed: 28.5 ^ 1.9% at P0, 30.3 ^ 1.5% at P3, 31.2 ^ 1.5% at P7 and 21.8 ^ 1.6% at P21 (mean ^ SEM, Fig. 3). 3.3. Size distribution of MOR and DOR immunoreactive DRG cells in neonatal and adolescent rats Both MOR and DOR were expressed in cells across all diameters at P0, P3 and P7. In these neonatal rats, the mean diameter of MOR 1ve and MOR 2ve cells was the same, illustrating their equal size distribution (MOR 1ve at P0: 15.3 ^ 3.7 mm, MOR 2ve at P0: 15.1 ^ 3.4 mm; MOR 1ve at P3: 15.4 ^ 4.0 mm, MOR 2ve at P3: 15.5 ^ 4.6 mm; MOR 1ve at P7: 15.3 ^ 5.0 mm, MOR 2ve at P7: 15.8 ^ 5.6 mm; mean ^ SD, Fig. 4). The mean diameter of DOR 1ve and 2ve cell populations also did not differ at P0 and P7 (DOR 1ve at P0: 12.1 ^ 4.3 mm, DOR 2ve at P0: 12.7 ^ 2.9 mm; DOR 1ve at P7: 16.0 ^ 5.5 mm, DOR 2ve at P7: 15.3 ^ 5.0 mm; mean ^ SD, Fig. 5) while at P3, DOR 1ve cell pro®les were signi®cantly larger than the DOR 2ve population (DOR 1ve at P3: 15.4 ^ 5.0 mm, DOR 2ve at P3: 12.7 ^ 3.8 mm; mean ^ SD, Fig. 5). In contrast, at P21 very few large DRG cells showed MOR and DOR immunoreactivity, which was restricted to the small to medium sized population. The mean cell diameter of OR 1ve pro®les was signi®cantly less than that of OR 2ve pro®les (MOR 1ve: 18.5 ^ 5.7 mm, MOR 2ve: 24.3 ^ 8.3 mm; mean ^ SD, Fig. 4; DOR 1ve: 17.8 ^ 4.2 mm, DOR 2ve: 23.6 ^ 8.4 mm; mean ^ SD, Fig. 5).
145
3.4. Postnatal development of MOR/NF coexpression in DRG cells Since the largest diameter cells in the ®rst postnatal week are considerably smaller than at P21, it is important to establish whether there is a genuine postnatal downregulation of MOR expression in the largest DRG cells or simply that the population of large cells is not apparent until P21. To determine this, coexpression of MOR with NF200, a marker for large diameter cells was investigated in P0, P3, P7 and P21 rats (n 4 for each age group, Fig. 7). In new-born P0 pups a signi®cantly higher proportion of cells coexpressed MOR and NF200 (Fig. 6). At P0, 26.3 ^ 0.6% of MOR 1ve pro®les also expressed NF200 compared to 14.3 ^ 1.5% at P21 (mean ^ SEM, Fig. 6A). At P0, 25 ^ 2.8% of NF200 1ve cells also expressed MOR compared to 13.3 ^ 1.6% at P21 (mean ^ SEM, Fig. 6B).
4. Discussion The results presented here show that both MOR and DOR are downregulated in DRG neurons during postnatal development in the rat. These neurons, being NF200 1ve represent the largest diameter, non-nociceptive population of sensory afferents (Lawson and Waddell, 1991). In the ®rst postnatal week, signi®cantly more DRG cells express MOR and DOR distributed across all diameters. By 3 weeks of age, fewer cells express the receptors and the distribution is con®ned to small to medium-sized cells. This anatomical observation provides an explanation for the effects of m- and d-opioid agonists on non-nociceptive Ab-mediated stimuli observed in neonatal rats (Faber et al., 1997; Marsh et al., 1999). The expression of OR mRNA has been previously investigated in embryonic mice DRG. DOR mRNA is detected ®rst at embryonic day (E) 12.5 followed by MOR at E13.5. Expression of k receptors (KOR) mRNA is seen much later at E17.5 (Zhu et al., 1998). Thus, MOR and DOR are transcribed at a time point during embryonic development when large DRG neurons are generated (Lawson et al., 1974). MOR binding was also recently reported in P1 rat DRG (Ray and Wadhwa, 1999). The results of the present study provide the ®rst information about the postnatal developmental regulation of OR expression in different populations of DRG cells. The proportions of cells expressing OR found here are consistent with previous reports. MOR immunoreactivity has been reported in 17% (Zhang et al., 1998a) and 21% (Ji et al., 1995) of adult rat DRG cells although high level MOR mRNA expression has been found in 55% (Minami et al., 1995). In two studies, DOR immunoreactivity has been observed in 14% of adult rat DRG cells (Zhang et al., 1998a; Ji et al., 1995) although another study reports a higher 40% (Zhang et al., 1998b). High level DOR mRNA expression occurs in 19% of rat DRG cells (Minami et al., 1995). Since the proportions in adult DRG are slightly lower
146
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Fig. 1. m-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. Scale bars represent 50 mm.
Fig. 2. d-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. Scale bars represent 50 mm.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
147
Fig. 3. (A) Proportions of m-opioid receptor immunoreactive cells in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. n 6 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons). (B) Proportions of d-opioid receptor immunoreactive cells in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21. n 6 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons).
than those found at P21 in this study, a further downregulation of MOR and DOR expression may occur during later development. Differences may also arise from immunostaining and cell counting methods. The results of the present and previously reported studies in adult animals are based on pro®le counts and the proportions of the different subpopulations can only be estimates. There is an ongoing discussion about the bene®ts of different methods of DRG cell counting (Guillery and Herrup, 1997; West, 1999). Previous studies have shown m-opioid agonist binding in rat spinal cord at birth but delayed d-agonist binding for the ®rst two postnatal weeks (Attali et al., 1990; McDowell and Kitchen, 1987; Rahman et al., 1998). DOR immunoreactivity in rat DRG was clearly seen as early as P0 in this study and DOR mRNA expression appears in early embryonic mice DRG (Zhu et al., 1998). This suggests that anterograde transport of DOR binding sites from the DRG to central terminals in the spinal cord is delayed in the immature animal. Both MOR and DOR were found to be expressed in cells
of all sizes in immature neonatal DRG but restricted to small and medium DRG neurons in mature P21 rats. Since the overall size of the whole DRG cell population increases over this period, it is important that the study of MOR and NF200 coexpression con®rmed the downregulation of OR expression in large DRG neurons during the second and third postnatal week. We have recently shown that two populations of DRG cells, large and small to medium, can be distinguished by NF200 (clone N52) immunostaining as early as P0 (unpublished data). These ®ndings may explain the postnatal changes in m-opioid agonist binding within the spinal cord. In the adult animal, m-opioid binding is restricted to the super®cial dorsal horn, the site of C-®bre termination (Besse et al., 1990), but in the neonatal spinal cord it is also seen in deep dorsal horn where A-®bres terminate (Kar and Quirion, 1995; Rahman et al., 1998). These anatomical results have functional implications. The majority of larger DRG neurons are the cell bodies of non-nociceptive Ab mechanoreceptors and proprioceptors (Lawson and Waddell, 1991) and there is evidence that
Fig. 4. Cell size distribution of m-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21.
148
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Fig. 5. Cell size distribution of d-opioid receptor immunoreactivity in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21.
opioids can depress Ab-®bre-mediated responses in the neonatal spinal cord. The neonatal withdrawal re¯ex to low intensity mechanical stimuli is thought to have an Ab-mediated component (Fitzgerald and Jennings, 1999) and epidural opioid agonists increase the low baseline mechanical threshold of these re¯exes in neonatal rats (Marsh et al., 1999). In addition, morphine depresses both
Ab- and C-®bre-mediated ventral root re¯exes in the isolated neonatal spinal cord (Faber et al., 1997). This is in contrast to older animals where m- and d-opioid agonists have been shown to selectively depress C- and Ad-®bre but not Ab-®bre input to the dorsal horn (Dickenson et al., 1987; Rahman and Dickenson, 1999) and opioid agonists inhibit membrane currents in small but not in large adult
Fig. 6. Double-¯uorescence immunohistochemistry for m-opioid receptor (A) and neuro®lament (B) in L4/5 dorsal root ganglia of rat pups at postnatal day 0, and double-¯uorescence immunohistochemistry for m-opioid receptor (C) and neuro®lament (D) in L4/5 dorsal root ganglia of rat pups at postnatal day 21. Arrows indicate cell pro®les coexpressing both m-opioid receptor and neuro®lament. Scale bars represent 50 mm.
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
149
Fig. 7. (A) Proportions of m-opioid receptor immunoreactive cells coexpressing neuro®lament 200 (NF200) in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21 as seen in double-¯uorescence immunohistochemistry. n 4 for each age group. Mean ^ SEM. **P , 0:01 versus P21 (Dunn's test for multiple comparisons). (B) Proportions of NF200 immunoreactive cells coexpressing m-opioid receptor in L4/5 dorsal root ganglia of rat pups at postnatal day (P) 0, 3, 7 and 21 as seen in double-¯uorescence immunohistochemistry. n 4 for each age group. Mean ^ SEM. *P , 0:05 versus P21 (Dunn's test for multiple comparisons).
DRG neurons in culture (Taddese et al., 1995; Abdulla and Smith, 1998). The study presented here provides a mechanism to explain the qualitatively different effect of opioids on sensory processing in the immature animal affecting both nociceptive and non-nociceptive input. The wider depression of sensory inputs by opioids may be advantageous to protect the central nervous system from inputs that, while not necessarily noxious, may still be stressful. The effects of opioids in the immature animal and human will be in¯uenced by many different mechanisms apart from the pattern of OR expression. In the adult, opioid analgesia is mediated via presynaptic receptors reducing transmitter release, postsynaptic receptors hyperpolarizing the cell and effects on descending inhibitory pathways (Dickenson, 1994). The delayed maturation of descending inhibition of spinal cord activity over the ®rst three postnatal weeks will no doubt affect analgesic pathways (Fitzgerald and Koltzenburg, 1986). Altered opioid effects may also arise from differences in receptor dynamics and second messenger systems. G-protein coupling and activity are known to change during postnatal development in rats (Szucs and Coscia, 1990; Windh and Kuhn, 1995). Nothing is known of the developmental regulation of opioid receptor heterodimerization which leads to altered binding and function (Jordan and Devi, 1999) and which would not be detected by immunohistochemistry. The transient postnatal expression of m- and d-opioid receptors in larger NF200 1ve cells of the DRG is an example of how neonatal and adult opioid systems differ. Studies such as these have important implications for the safety and effectiveness of opioid administration in neonates and infants.
Acknowledgements We are grateful to Robert Elde, Department of Pharmacology, University of Minnesota, Minneapolis for the generous gift of MOR and DOR antibodies. We also wish to
acknowledge J. Meredith-Middleton and A. Allchorne for excellent technical assistance. This study was supported by grants from the IMF and DFG, Germany to B.B. and the MRC, UK to M.F.
References Abdulla FA, Smith PA. Axotomy reduces the effect of analgesic opioids yet increases the effect of nociception on dorsal root ganglion neurons. J Neurosci 1998;18:9685±9694. Arvidsson U, Dado RJ, Riedl M, Lee JH, Law PY, Loh HH, Elde R, Wessendorf MW. Delta-opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 1995a;15:1215±1235. Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 1995b;15:3328±3341. Attali B, Saya D, Vogel Z. Pre- and postnatal development of opiate receptor subtypes in rat spinal cord. Brain Res Dev Brain Res 1990;53:97± 102. Besse D, Lombard MC, Zajac JM, Roques BP, Besson JM. Pre- and postsynaptic distribution of mu, delta and kappa opioid receptors in the super®cial layers of the cervical dorsal horn of the rat spinal cord. Brain Res 1990;521:15±22. De Lima J, Lloyd-Thomas AR, Howard RF, Sumner E, Quinn TM. Infant and neonatal pain: anaesthetists' perceptions and prescribing pattern. Br Med J 1996;313:787. Dickenson AH. Where and how do opioids act? In: Gebhardt GF, Hammond DL, Jensen TS, editors. Progress in pain research and management, 2. Seattle, WA: International Association for the Study of Pain, 1994. pp. 525±552. Dickenson AH, Sullivan AF, Knox R, Zajac JM, Roques BP. Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with mand d-opioid receptor agonists in the control of nociception. Brain Res 1987;413:36±44. Faber ESL, Chambers JP, Brugger F, Evans RH. Depression of A and C ®bre-evoked segmental re¯exes by morphine and clonidine in the in vitro spinal cord of the neonatal rat. Br J Pharmacol 1997;120:1390± 1396. Fitzgerald M. Neonatal pharmacology of pain. In: Besson J-M, Dickenson AH, editors. Handbook of experimental pharmacology, Heidelberg: Springer-Verlag, 1997. pp. 447±465. Fitzgerald M. The developmental neurobiology of pain. In: Wall PD, editor.
150
B. Beland, M. Fitzgerald / Pain 90 (2001) 143±150
Textbook of pain, Edinburgh: Churchill-Livingstone, 1999. pp. 153± 170. Fitzgerald M, Jennings E. The postnatal development of spinal sensory processing. Proc Natl Acad Sci USA 1999;96:7719±7722. Fitzgerald M, Koltzenburg M. The functional development of descending inhibitory pathways in the dorsolateral funiculus of the newborn rat spinal cord. Dev Brain Res 1986;24:261±270. Guillery RW, Herrup K. Quanti®cation without ponti®cation: choosing a method for counting objects in sectioned tissues. J Comp Neurol 1997;386:2±7. Ji R-R, Zhang Q, Law P-Y, Low HH, Elde R, Hokfelt T. Expression of mu-, delta-, and kappa-opioid receptor like immunoreactivities in rat dorsal root ganglia after carrageenan-induced in¯ammation. J Neurosci 1995;15:8156±8166. Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature 1999;399:697±700. Kar S, Quirion R. Neuropeptide receptors in developing and adult rat spinal cord: an in vitro quantitative audioradiography study of calcitonin generelated peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors. J Comp Neurol 1995;354:253±281. Lawson SN, Waddell PJ. Soma neuro®lament immunoreactivity is related to cell size and ®ber conduction velocity in rat primary neurones. J Physiol (Lond) 1991;435:41±63. Lawson SN, Caddy KWT, Biscoe TJ. Development of rat dorsal root ganglion neurones. Cell Tissue Res 1974;153:399±413. Llewellyn-Smith IJ, Pilowsky P, Minson JB. Retrograde tracers for light and electron microscopy. In: Bolam JP, editor. Experimental neuroanatomy, Oxford: Oxford University Press, 1992. pp. 37±38. Marsh DF, Hatch DJ, Fitzgerald M. Opioid systems and the newborn. Br J Anaesth 1997;79:787±795. Marsh D, Dickenson A, Hatch D, Fitzgerald M. Epidural opioid analgesia in infant rats I: mechanical and heat responses. Pain 1999;82:23±32. McDowell J, Kitchen I. Development of opioid systems: peptides, receptors and pharmacology. Brain Res 1987;434:397±421. McLaughlin CR, Dewey WL. A comparison of the antinociceptive effects of opioid agonists in neonatal and adult rats in phasic and tonic nociceptive tests. Pharmacol Biochem Behav 1994;49:1017±1023. Minami M, Maekawa K, Yabuuchi K, Satoh M. Double in situ hybridiza-
tion study on coexistence of mu-, delta- and kappa-opioid receptor mRNAs with preprotachkinin A mRNA in the rat dorsal root ganglia. Mol Brain Res 1995;30:203±210. Purcell-Jones G, Dormon F, Sumner E. The use of opioids in neonates. A retrospective study of 933 cases. Anaesthesiology 1987;42:1316±1320. Rahman W, Dickenson AH. Electrophysiological studies on the postnatal development of the spinal antinociceptive effects of the delta opioid receptor agonist DPDPE in the rat. Br J Pharmacol 1999;126:1115± 1122. Rahman W, Dashwood MR, Fitzgerald M, Aynsley-Green A, Dickenson AH. Postnatal development of multiple opioid receptors in the spinal cord and development of spinal morphine analgesia. Dev Brain Res 1998;108:239±254. Ray SB, Wadhwa S. Mu opioid receptors in developing human spinal cord. J Anat 1999;195:11±18. Szucs M, Coscia CJ. Evidence for delta-opioid binding and GTP-regulatory proteins in 5-day-old rat brain membranes. J Neurochem 1990;54:1419±1425. Taddese A, Nah S-Y, McCleskey AW. Selective opioid inhibition of small nociceptive neurons. Science 1995;270:1366±1369. Thornton SR, Compton DR, Smith FL. Ontogeny of mu opioid agonist antinociception in postnatal rats. Dev Brain Res 1998;105:269±276. West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci 1999;22:51±61. Windh RT, Kuhn CM. Increased sensitivity to mu opiate antinociception in the neonatal rat despite weaker receptor-guanyl nucleotide binding protein coupling. J Pharmacol Exp Ther 1995;273:1353±1360. Zhang AZ, Pasternak GW. Ontogeny of pharmacology and receptors: high and low af®nity site differences. Eur J Pharmacol 1981;73:29±40. Zhang Q, Schaffer M, Elde R, Stein C. Effects of neurotoxins and hindpaw in¯ammation on opioid receptor immunoreactivities in dorsal root ganglia. Neuroscience 1998a;85:281±291. Zhang X, Bao L, Arvidsson U, Elde R, Hokfelt T. Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles. Neuroscience 1998b;82:1225±1242. Zhu Y, Hsu M-S, Pintar JE. Developmental expression of the mu, kappa, and delta opioid receptor mRNAs in mouse. J Neurosci 1998;18:2538±2549.