Pro-dynoprhin is the pro-peptide responsible for the synthesis of dynorphin. Studies detail ing the developmental expression profi le of either the pro-peptide or the actual peptide i n pain related areas of the CNS are very sparse and no clear consensus or opinion has been reached (McDowell and Kitchen, 1987 ). Developmental expression It is apparent from the current literature that investigations int o the normal development of the endogenous opioid system have slowed considerably from the late 1980s and early 1990s. Th ere is little data regarding the presence of endogenous opio id peptides in the pri- mary aff erent fi bres through postnatal development. Th ere is lim - ited evidence for the presence of opioid peptides in some aff erent fi bres in the prenat al environment (Haynes et al., 1982 ), but this weak immunoreactivity disappeared postnatal ly and was restricted to white matter tracts. Fiber staining within the spinal DH was absen t. Th is study pre-dated a full description of the composition of the family of opioid pept ides and concentrated only on the expres- sion of -endorphin. More recent studies regard ing the distribution of these peptides in the peripheral nervous system has focussed on the adult rat. In these studies (Marviz n et al., 2009 ), although enkephalin was never obs erved to be co-localized with primary aff erent terminals in the spinal DH, there was some co-localiza- tion of dynorphin with calcitonin gene-related peptide (CGRP), a marker of pep tidergic C fi bres. A thorough study through postnatal development has not yet been perform ed. Role of opioid peptides in normal development Do opioid peptides have a role in normal deve lopment? Reviewing the available literature suggests that opioid peptides act as inhibi- to ry growth factors at a cellular level, with antagonism of opioid receptors in vivo resultin g in increased dendritic length and spine CHAPTER 44 developmental pharmacology of opioids 451 formation in neurons in the cerebellum (Hauser et al., 1987 , 1989 ). However, alterations in the anatomical development of the nerv- ous system by opioids are not restricted to neur ons. Astrocyte numbers have decreased in mouse cortical cultures exposed to met-enkephalin by a receptor-dependent mechanism (Stiene- Martin and Hauser, 1990 ). More recently this ef f ect of opioids on the development of non-neuronal cells in the nervous system has been co nfi rmed. To model eff ects of prolonged opiate exposure of off spring of opiate-dependent mothers, Sanchez et al. ( 2008 ) implanted pregnant rat dams with osmotic mini-pumps that c on- tinuously delivered buprenorphine from day 7 of gestation until postnatal day 21, ensur ing that pups were exposed prenatally to the drug via the placenta and postnatally via mate rnal milk. In these pups it was shown that lower doses of buprenorphine increased the expre ssion of myelin basic protein whilst higher doses signifi cantly decreased levels of this p rotein, particularly when animals were examined at postnatal day 12. Anatomical examination of the cor- pus callosum showed that in rats exposed to buprenorphine there were signifi c antly more large caliber myelinated fi bres but the same distributions of non-myelinated fi bres compared to controls. Th is increase in caliber was also associated with a thinner my elin sheath surrounding these axons suggesting an altered balance between axon growth and m yelin thickness. Th us, exposure to high levels of opioid for relatively prolonged periods during prenatal develop- ment can alter CNS structure. Further studies are required to eval u- ate the impact of diff erent doses and durations of exposure during postnatal developmen t. ontogeny of adult endogenous opioid networks in the CNS, how- ever the application of quant itative genomic and post-genomic techniques would signifi cantly add to this work. Equally, utilizing immunohistochemical techniques to uncover the distribution of these peptides and the co-transmitters utilized in diff erent neuro- nal populations through the postnatal pe riod would be valuable. Many of the supraspinal structures involved in pain modulation are anatomically small and the analyzes detailed earlier were too crude to detect subtle variat ions in expression levels within refi ned brain nuclei. Even with today \200\231s state-of- the art RNA amplifi cation meth- ods accurately and repeatedly dissecting these areas from postmor- tem tissue is technically challenging. Opioid receptors Opioid peptides mediate their eff ects via interactions with a family of o pioid receptors, which are metabotropic, membrane-expressed receptors that act through seco nd messengers (i.e. in contrast to ionotropic receptors that form an ion channel pore). Th ere are three main subclasses of opioid receptor named mu ( ), delta ( ), and kappa ( ), as well as a less well recognized and investigated receptor, ORL-1 (opioid-like rece ptor). Receptors are composed of seven hydrophobic membrane-spanning domains, with extra- c ellular components that are involved in specifying interactions with ligands, and intracell ular domains that interact with intracel- lular signaling mediators. Opioid receptors media te their actions via second messenger G-proteins (see later). In the adult nervous systems activation of opioid receptors engage intracellular signal- ling systems that are in large part inhibitory, and act to decrease neuronal excitability by increasing inwardly rectifyin g potassium (K + ) conductances, decreasing voltage-dependent calcium (Ca 2+ ) channels, an d by decreasing the production of cyclic adenosine monophosphate (cAMP). More recently data from our laboratory has demonstrated a specifi c trophic role for endoge nous opioids in the maturation of supraspinal centers responsible for the descending contro l of spinal pain processing (Hathway et al., 2012 ). In this study we implanted subcutaneou s mini-pumps containing naloxone HCl at P14, P21, or P28, and continuously delivered the dr ug for 7-day periods. Animals were then allowed to mature to P40. In the group that had dru g delivered between P21 to P28, the RVM failed to mature properly, and descending inhibitio n of spinal pain processing could not be evoked in adulthood. Th ese data showed that the a ction of endogenous opioids are central to the normal maturation of supraspinal pain contro l systems, but even more interestingly that this occurs relatively late in postnatal develo pment, at a period when rats would be judged to be adolescent. Th is is a true \200\230cri tical period \200\231 as the same manipulation one week before or aft er this does not prev ent or eff ect normal maturation. In an extension of this study we implanted neonatal, 7-da y-old, rats with mini-pumps that delivered morphine for 7 days and showed that at developme ntal stages where the RVM is normally immature and unable to provide descending inhibitory control of spinal processing, that neonatal morphine treatment had accelerated the maturati on of the RVM. In these animals adult like stimulus response profi les from the RVM were ev oked. Th is work demonstrates the importance of the opioidergic system for the normal postn atal development of pain processing in the nervous system. Peripheral nervous system Th e peripheral nervous system detects stimuli and conveys sensor y information to the CNS. Peripheral sensory aff erent neurons are subdivided into three br oad categories according to their physi- ological and anatomical properties: \231 A fi bres are large, myelinated fi bres that usually convey non- noxious information to the CNS. \231 A fi bres are small myelinated fi bres and convey both noxious and non-noxious information. \231 C fi bres are small unmyelinated fi bres that convey noxious infor- mation to the CN S, that are further subdivided into peptider- gic or non-peptidergic, depending on their ne urotransmitter phenotype. All of these primary aff erent fi bres innervate target tissues such as skin, muscle, and v iscera and their cell bodies are to be found in the dorsal root ganglia (DRG) which run par allel to the spinal cord. Th ese neurons then have spinal projections which enter the DH of the spinal cord. Each primary aff erent subtype has diff erent central termination pattern s within the DH. Th ese terminations are imma- ture at birth and refi ne to their adult sta te via activity dependent processes (see Walker and Baccei, Chapter 6, this volume). It is intriguing to speculate on the results of studies using more recent quantitative and non-quantitative methods to further evalu- ate the changing expression of opioid peptides i n the developing nervous system. Th e majority of studies listed above utilized the most se nsitive assays available at the time, such as radioimmu- noassay and in situ hybridization. Th e value of these studies remains high and they are still our best source of information regarding the A detailed study of the expression pattern of -opioid receptors in the DRG over postnata l development has shown considerable changes in early life (Nandi et al., 2004 ). Receptor expression in SECTION 6 pharmacological interventions 452 adulthood was confi ned to mainly small and medium sized neu- rons (presumably C and A f i bres). However, in the neonatal rat, there were signifi cantly more -opioid receptors expressed on large-diameter myelinated fi bres (Beland and Fitzgerald, 2001 ). Th is increa sed expression was mirrored when Ca 2+ imaging dem- onstrated increased fl uorescence in re sponse to activation of the -opioid receptor with the receptor agonist DAMGO, in a great er proportion of DRG neurons than in the adult (Nandi et al., 2004 ). Th is suggests that n ot only are more receptors present, but they are also functional and can be activated by ag onists, as confi rmed by an enhanced response or left ward shift in the dose \200\223respon se relation- ship for systemic morphine in younger rats. Th e mechanisms that regulate the expression of the -opioid receptor in primary aff erent neurons are not known. Although age-dependent alterations in the central terminals of primary aff erent neurons in the DH a re related to changes in levels of neural activity (Beggs et al., 2002 ; Bremner and Fitzge rald, 2008 ), it is not known if opioid receptor expression is similarly regulated. Both th e and subtypes are expressed on the terminals of adult primary aff erent neurons, but developmental changes have not been evaluated. -, -, and -opioid receptor expression in the spinal cord, with intense staining to all three seen in the superfi cial layers in adult rats (Gray et al., 2006 ). Positive cel ls were generally ovoid in shape and aligned to follow the rostrocaudal axis of the spinal cord itself. staining was most intense in lamina II and was localized to the cell membra ne of cell bodies and dendrites, whereas immuno- reactivity was restricted to cell bodie s. staining was particularly intense in lamina I due to the presence of heavily stained fi bres, but cell body staining was also prominent here. Immunohistochemistry uses antibodi es to selectively label diff erent proteins and provides increased spatial resolution, as p rotein expression can be directly detected at a subcellular level. Th is is a distinct adva ntage over other techniques, such as radioligand binding, which requires tissue to be expos ed to irradiated drug and resultant images on X-ray or similar fi lms to then be analyzed. Improvements in immunohistochemi- cal technologies, from antibody design and production, th rough to signal amplifi cation and advances in microscopy, have meant that immunohistochemi stry is now more routinely employed in labora- tory studies, but to date there has been lim ited immunohistochemi- cal investigation of the postnatal expression of opioid receptors in the spinal cord. Th e presence of opioid receptors on the terminals of primary aff erent neurons suggests a role in inhibiting primary aff erent fi ring in response to noxious stimuli. In postnatal r ats, electrical stimu- lation of incoming fi bres in the dorsal root produces excitatory po stsynaptic potentials in the DH; but these were reduced by the -opioid receptor agonist DAMGO via inhibition of both A and C fi bres. DAMGO was also more effi cacious and poten t at inhibiting C fi bres than A fi bres, but identical intracellular processes medi- at ed the inhibition in both fi bre types. Tissue from 17- to 25-day- old rat pups, which is t oward the limit of technical feasibility of the techniques employed were used, but these ar e not fully mature animals (Heinke et al., 2011 ). It was not reported whether stimulat- in g the dorsal root at A strengths was also sensitive to DAMGO application in young animal s, as would be predicted on the basis of developmental studies (Nandi et al., 2004 ). Autoradiography has been used to map postnatal changes in opioid receptor biding in the spi nal cord (Rahman et al., 1998 ). At birth, binding is relatively diff use, increases ove r the fi rst postna- tal week, and then decreases until P21. A progressive pattern and refi nement of binding could be seen with increasing age, so that binding sites became progr essively more restricted to the super- fi cial laminae of the DH as seen in the adult. b inding followed a similar pattern to in that diff use binding was present at birth. Tota l binding increased over the fi rst postnatal week and then decreased so that at P14 the re was approximately 50% of the bind- ing seen at P7. At P21 and P56 (adult) greater prefer ence for bind- ing in the superfi cial DH was observed. opioid receptor binding was n ot present at birth, was fi rst observed at P7, but was diff use and remained like this unt il at least P56. Spinal cord Th e spinal cord is the fi rst site of integration of sensory informa- tion wit hin the CNS. Primary aff erent sensory neurons enter the spinal cord via the dorsal root an d synapse with second-order neurons. Th ese second-order neurons are central to the percep- tion of pain; being intrinsically involved in both the transmission of nociception-related information to supraspinal centers, and also in receiving converging descending informatio n from other supraspinal sites that can act to increase or decrease their excit- ability. A utoradiographic and immunohistochemical studies have shown that the majority of opioid rece ptors are located either in laminae I and II of the DH and to a lesser extent in deeper lay ers (Besse et al., 1990 ; Rahman et al., 1998 ). Th is refl ects termination of primary aff erents (see earlier), but approximately 30% (Besse et al., 1990 ) of receptors are located postsynaptically and may relate to descending projections. Brainstem Analysis of the published literature shows that, as with other areas of the nervo us system, a detailed account of the postnatal develop- ment of opioid receptors is elusive . For the purposes of this chapter, changes in opioid receptor expression that take place i n areas of the brain that are recognized as being central to the modulation of spinal cord excitability, namely the PAG and the RVM, have been highlighted ( Figure 44.1 ). Immunohist ochemical studies (Gray et al., 2006 ) have shown intense immunoreactivity for all three op ioid receptors within the PAG; with considerable co-localization of and receptors in the medial and lateral parts of this midbrain structure. Light to moderate staining was seen and positive cells were closely apposed to those that expressed opioid receptors . immunoreactivity was also most noticeable within the caudal and venterolateral parts; the latter is of particular interest as it is this part of the PAG that projects to the RVM . Within the nucleus raphe magnus of the RVM strong staining was seen in dense cell bodi es whereas immunoreactivity was associated with fi bres and was more diff use. As in the PAG there was some co-localization of both and receptors. Small round cells which we re positive for immunoreactivity were noted to be \200\230intermingled \200\231 with positive cell bodies. Th e locus coeruleus (LC) is the source of noradrenaline Studies in the 1990s using in situ hybridization illustrated that mRNA for at least the - and -opioid receptors can be found within the DH (Maekawa et al., 1994 ). Th e highest density of mRNA for the -opioid receptor could be found in laminae I, II, and VIII whil st was densest in laminae I and II. Recent immu- nohistochemical studies examined the an atomical distribution of CHAPTER 44 developmental pharmacology of opioids 453 receptors may follow diff erent trajectories in diff erent regions of the brain. Again the application of modern post-genomic approaches to the study of the development of opioid rec eptor expression and their pharmacology is required. CORTEX HYPOTHAL- AMUS AMYGDALA Idiosyncrasies of age and receptor function It is tempting to think of neonates, children, or young animals as smaller versions of adults. Compensations are clearly made in terms of the dosing requirements based on weight and pharmacokinetic data, but what of the fundament al action of the drug at its recep- tor? Previously we have shown that the action of midazo lam at the GABA A receptor is signifi cantly altered in neonatal rats as it is pro- nocicep tive and fails to produce sedation (Koch et al., 2008 ). Could the result of opioid recepto r agonism also undergo age-dependent changes? As discussed previously, there seem to be no major diff er- ences in the basic receptor-agonist interaction with postnatal age as assess ed by radio-ligand binding in whole brain homogenates. It must be stressed that the availab le literature confi rms that opioid mediated analgesia can be evoked at all ages. Th is is true via which- ever route the drugs are administered; be that systemic (Abbott and Guy, 19 95 ; Nandi et al., 2004 ; Th ornton et al., 1998 ), epidural (Marsh et al., 1999 a, 1999 b) , intrathecal (Barr et al., 1992 ; Westin et al., 2010 ) or peripherally (Barr, 1999 ). PAG RYM + \200\223 DORSAL HORN Figure 44.1 Supraspinal modulation of dorsal horn excitability. Th e ability of supraspinal sites to control the excitability of neurons within the dorsal horn of the spinal cord is one of the major mechanisms that underpin opioid analgesia. Forebrain regions commonly inne rvate the periaqueductal gray (PAG) which in turns innervates the rostroventral medical med ulla (RVM). Via spinally projecting neurons this structure directly controls pain processin g in the spinal cord. Th e RVM bi-directionally controls spinal excitability it is able to both facilitate (+) or inhibit ( \210\222) the spinal cord depending on the nature of the i nput to the RVM itself. Th e RVM is therefore central to opioid analgesia mediating these e \200 ects via descending inhibitory pathways and in the maintenance of chronic pain state s via facilitatory pathways. Assumptions seem to have been made in the literature that -opioid receptor activity \200 \223function relationships in one part of the CNS refl ect the situation in the entire CNS. Recently we have shown that this is not the case. Microinjection of the -opioid recepto r agonist DAMGO into the RVM of juvenile (P21) rats is able to dose-dependently facilitate spinal nociception, that is it increases pain, which is in contrast to adult ( > P40) rats where the same inter- vention results in dose-dependent analgesia (Hathway et al., 2012 ). Th is alteration in response to receptor agonism is not associated with an age-related alte ration in the level or pattern of expression of -opioid receptors within the RVM, but se ems to be a fundamental shift in the signaling pathways activated by the receptor following its activation. It is therefore apparent that although the same recep- tor can be expresse d within the same animal in multiple tissues at any point during postnatal development ther e can be subtle, tissue specifi c alterations in the receptor that radically alter the cons e- quences of its activation. for the entire central nervous system and also has descending pro- jections which powerfull y control spinal excitability. Intense stain- ing was found within cell bodies of the LC in adult rats; staining although above background levels was weak; and a few -positi ve cells were present (Gray et al., 2006 ). Multiple radioligand binding studies have quantifi ed opioid receptor binding at diff erent postnatal ages in brain tissue. In gen- eral, it appears that the expression of opioid rec eptor binding sites within the brain alters with age and that the diff erent opioid recep- tors follow diff erent trajectories (Kitchen et al., 1990 ; McDowell and Kitchen, 1986 , 19 87 ; Spain et al., 1985 ; Volterra et al., 1986 ,). Despite the diffi culty in making compa risons between studies it appears that receptors are present at birth and are able to bi nd ligands with the same affi nity (dissociation constant, Kd) as in the adult. Binding of selective ligands increase from birth and reach adult levels around the third postnatal week. receptors seem to be absent at birth, but reliable binding is detectable from the second postnatal week, and then increases with age. binding is detectable at P5, increas es to high levels by P10, then decreases to adult levels by P25. Binding characteristics of the and receptor are the same in neonates as they are in adults. Whilst informative, these studies provide no data regarding the expression of opioid receptors in spe- cifi c brain nuclei. Most use whole brain homogenates which have limited sensitivity, and Spain et al. ( 1985 ) use rostral and caudal brain portions. It is not unreasonable to hypothesize that as well as following diff erent paths between subtypes, the maturation of opioid Analysis of the literature reveals some interesting developmen- tal changes. Th ere are age -related increases in the total -opioid expression within the brainstem of the developin g rat whilst there is a concomitant decrease in expression (Kivell et al., 2004 ). It wa s also been noted that MOR in brainstem tissue existed as two diff erent isoforms, both a 5 0 kD protein and a 70 kD protein, and that levels of the heavier isoform increased with pos tnatal age whilst the lighter isoform decreased. No alteration in the expres- sion pattern of -opioid receptor was seen, in agreement with our later study (Hathway et al., 2012 ). Th is age-related alteration in receptor mass may represent alterations in: the splicing o f mRNA for the receptor; post-translational glycosylation; use of alterna- tive promoters f or the OPRM1 gene that encodes the receptor; or oligomerization of the receptor. Several st udies have noted region specifi c distribution of -opioid receptor splice variants acros s the adult rodent CNS (Abbadie et al., 2000 a, 2000 b; Pan et al., 1999 ; Schulz et al., 1 998 ), yet this study was the fi rst to detail an appar- ent change in receptor isoform exp ression that correlated with postnatal age.