Magdalena Kostrzewa 1 , Mateusz Kucharczyk 1 , Katarzyna Starowicz 1 , Alessandra Luchetti 3 , Francesca d’Amato 3 , Flaminia Pavone 3 , Barbara Przewlocka 2 1 Laboratory of Pain Patophysiology, Department of Pain Pharmacology, Polish Academy of Sciences, Krakow, Poland; 2 Department of Pain Pharmacology, Polish Academy of Sciences, Krakow, Poland; 3 Istituto di Biologia Cellulare e Neurobiologia, CNR, Roma, Italy Neurochemical changes in opioid systems activity in adult mice as consequences of neonatal painful stimulation METHODS Pain experienced in the infancy influence the pain sensitivity in adult life and neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. Additionally, the relation with mother in infancy and social behavior later are also changed, as was shown by our previous behawioral study (Pavone et al. own results). An increased focus on the neurobiology of developing pain pathways attests the awareness of the importance of pain experience in infancy. The impact of neonatal pain on developing nociceptive circuitry, nociceptive processing and the subsequent perception of pain has attracted our attention. The opioid system is highly implicated in pain perception and in emotional behaviors (Kieffer and Gaveriaux-Ruff 2002). Each of the three opioid family derives from a distinct precursor protein encoded by three separate genes and has a characteristic anatomical distribution. Proopiomelanocortin (POMC), proenkephalin and prodynorphin are prohormons for endorphins, enkephalins and dynorphins, respectively. Endogenous opioid peptides are ligands for numerous types of opioid receptors. The opioid receptors belong to the large superfamily of G-protein-coupled receptors. Three main types of these receptors mu-, delta-, and kappa- (MOR, DOR and KOR, respectively) are expressed in several brain areas along nociceptive pathways that modulate pain transmission. Intriguingly, the biological signal(s) generated by POMC may ultimately be mediated by a family of peptides which are in complex functional interaction with each other. RESULTS BACKGROUND Early life manipulation Acute neonatal inflammation was induced by unilateral injection of carrageenan (CARR) into the plantar surface of the right hindpaw of albino NMRI mice within 12h of birth. Control animals received intraplantar sterile saline. AIM OF WORK The objective of the present study was to determine if neonatal inflammatory insult (post- natal carrageenan) influence the developing nervous system of the pups and change the activity of endogenous opioid systems. 1) Neonatal inflammatory insult increases maternal behavior both immediately after pups’ neonatal painfull manipulations as well as during the first week after their birth as was shown by our previous study. 2) Carrageenan (CARR) administration causes the differences in the spinal cord between males and females in POMC, PENK and all opioid receptors 3) As a consequence of neonatal treatment the POMC system in adult females seems to be more affected than in males, a simultaneous decrease of both, peptides and MOR receptor level in spinal cord was observed 4) Opposite regulation is observed in the PAG, where level of POMC is increased in female mice. 5) The pain stimulus in post-natal day 1 seems to change the functioning of the descending antinociceptive system by lowering opioidergic tone especially in female mice. 6) Neonatal treatment which can be considered as chronic stress affected animals’ cognitive function in adulthood 7) Important contribution of the POMC system in the thalamus in modulating affective motivational components of pain may be suggested . CONCLUSION 0 2 4 6 8 10 12 14 16 18 nursig posture grooming pups Maternal behaviour during the first hour after pups treatment CARR SAL 0 20 40 60 80 100 120 nursig posture grooming pups Maternal behaviour during the first week after pups treatment CARR SAL Time spent in nursing position (hours) 0 20 40 60 object interaction partner interaction object interaction partner interaction Permanence time (sec) MALE FEMALE * CARR SAL 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 POMC PENK PDYN PNOC Fold change (Hprt1 normalised) PAG: OPIOID PRECURSORS MALE FEMALE 0 0,5 1 1,5 2 2,5 3 3,5 MOR DOR KOR NOR MC4R Fold change (Hprt1 normalised) PAG: OPIOID RECEPTORS MALE FEMALE 0 0,5 1 1,5 2 2,5 POMC MOR MC4R Fold change (Hprt1 normalised) Thalamus MALE FEMALE Acknowledgements. Supported by grant MAESTRO NCN2012/06/A/NZ4/00028 and statutory funds and The Joint Research Project between PAS-Poland /CNR-Italy POMC-neurons release multiple peptide transmitters including α-melanocyte-stimulating hormone (α-MSH) and the endogenous opioid β- endorphin that modulate aspects such as motivation, food intake analgesia and mood. The presence of POMC-derived peptides in supraspinal and spinal central nervous system pathways as well as in the pituitary gland, involved in stress-related phenomena, suggests that POMC may serve as a link between stress response systems in the body and endogenous pain control (Roberts et al. 1993; Przewłocki and Przewłocka 2001; Przewłocki 2002, van Meeteren et al. 1997; Akil et al. 1986). It is likely that both melanocortin and opioid receptors that bind POMC-derived peptides may mediate opposite biological functions. Carr/Sal Treatment Social approach avoidance Animal Sacrifice Maternal behaviour 12 h Quanitative PCR Samples were formalin-fixed and design not to be analysed by quantitative real-time polymerase chain reaction (RT-qPCR) we were forced to conduct special protocol, that let us reverse cross- linkage of nucleic acids with proteins caused by formalin perfusion. Based on Körbler et all paper we homogenised tissue samples in buffer B (10 mMNaCl, 500 mM Tris (pH 7.6), 20 mM EDTA, 1% SDS) by Tissue Lyser II apparatus (Life Tech.). Afterwards proteinase K (500 μg/mL, A&A Biotech.) was added and incubated overnight in 45°C with shaking (500 rpm). From obtained lysate we isolate total RNA with TRIzol reagent (Ambion) followed by Chomczynski et all method. After isolation RNA quality was checked by chip-based capillary electrophoresis Agilent RNA 6000 Nano Kit and Agilent Bioanalyser 2100 (Agilent) according to the manufacturer’s instructions. RNA was rewritted in transcription reaction using iScript Reverse Transcription Supermix (BioRad). RT-qPCR reactions were carried out by iTaq Universal Probes Supermix (BioRad) with 100ng of CDNA and primer sets (TaqMan Gene Expression Assay, Life Tech.) according to the manufacturer’s protocol. Expression levels were assessed against housekeeping gene – HPRT1. C cycle threshold values were calculated automatically by the iCycler IQ 3.0 software. RNA abundance was calculated as 2 -(threshold cycle) CONSEQUENCES OF NEONATAL PAINFUL STIMULATION ON MATERNAL BEHAVIOR EFFCET OF CARRAGEENAN (CARR) TREATMENT ON OPIOID SYSTEM LOCATED IN PAIN MODULATION CENTERS: SPINAL CORD AND PAG NEONATAL STRESS AFFECTS SOCIAL BEHAVIOR IN ADULT ANIMALS POMC SYSTEM IS IMPORTANT FOR AFFECTIVE-MOTIVATIONAL COMPONENT OF PAIN The analgesic function of opioids is subject to modulation by a range of non-opioid systems, i.e. melanocortin system Time spent in nursing position (hours) POMC -endorphin endogenous opioid peptide Analgesia -MSH endogenous non-opioid peptide Hyperalgesia 0 0,5 1 1,5 2 2,5 3 POMC PENK PDYN PNOC Fold change (Hprt1 normalised) SPINAL CORD: OPIOID PRECURSORS MALE FEMALE 0 0,5 1 1,5 2 MOR DOR KOR NOR Fold change (Hprt1 normalised) SPINAL CORD: OPIOID RECEPTORS MALE FEMALE POMC