Psychoneuroendocrinology (2014) 41, 1—12

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Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge Ihssane Zouikr a, Melissa A. Tadros b, Javad Barouei a, Kenneth W. Beagley c, Vicki L. Clifton d, Robert J. Callister b, Deborah M. Hodgson a,* a

Laboratory of Neuroimmunology, School of Psychology, University of Newcastle, Newcastle, New South Wales, Australia b School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, New South Wales, Australia c Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia d Robinson Institute, University of Adelaide, Adelaide, South Australia, Australia Received 29 August 2013; received in revised form 18 November 2013; accepted 18 November 2013

KEYWORDS LPS; HPA-axis; Hypothalamus; SDH neurons; Formalin test; Pain; Corticosterone

Summary The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking ( p < .01), and at PND 22, increased flinching in response to formalin injection ( p < .05). LPSchallenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 ( p < .001)

* Corresponding author at: Laboratory of Neuroimmunology, School of Psychology, University of Newcastle, University Drive, Callaghan, Newcastle, New South Wales 2308, Australia. Tel.: +61 0249216701; fax: +61 0249216980. E-mail address: [email protected] (D.M. Hodgson). 0306-4530/$ — see front matter. Crown Copyright # 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psyneuen.2013.11.016

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I. Zouikr et al. but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts ( p < .05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis. Crown Copyright # 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction It is now well accepted that the immune, endocrine and nervous systems communicate in a tightly regulated manner (Czura and Tracey, 2005; Hodyl et al., 2007; Sternberg, 2006). The hypothalamic-pituitary-adrenal (HPA) axis is a key component in this communication and its activation results in altered plasma corticosterone levels and altered pain responses, most notably in response to a painful inflammatory insult, such as the formalin test (Aloisi et al., 1998; Sorg et al., 2001). Elevated plasma corticosterone levels are observed in both neonatal (Butkevich et al., 2013) and preadolescent (Butkevich et al., 2009a) rats after formalin injection into the hindpaw, suggesting inflammatory pain results in rapid activation of the HPA axis. Interestingly, HPA axis function in adults can be modulated by early life events (for review see Matthews, 2002). One example of this ‘‘programming’’ of the HPA axis by early life events is potentiation of stress responses and reduced natural killer cell activity in adult rats after neonatal exposure to lipopolysaccharide (LPS) (Hodgson et al., 2001). Neonatal exposure to LPS also increases basal corticosterone secretion and lymphocyte sensitivity in adult animals (Shanks et al., 2000). Furthermore, rats exposed to LPS during early postnatal development display thermal and mechanical hyperalgesia and allodynia when tested as adults (Boisse et al., 2005). These data suggest neonatal LPS exposure alters neuroimmunoendocrine communication and nociceptive behaviours. While numerous studies have examined the effects of neonatal LPS exposure on responses to noxious thermal and mechanical stimuli in adults, few have examined the effects of neonatal LPS on responses to inflammatory pain stimuli, such as the formalin test, during the neonatal and preadolescent period. This is surprising as gestational stress enhances nociceptive behaviours and elevates plasma corticosterone levels in response to the formalin test in neonatal (PND 7) rats (Butkevich et al., 2013), suggesting early life events programme the HPA axis and inflammatory pain responses at least in the first week after birth. Responses to the formalin test also appear to vary during early postnatal development, with infant rats being susceptible to much lower concentrations of formalin than adults (Gagliese and Melzack, 1999; Zouikr et al., 2013). Thus, the effect of neonatal exposure to LPS on the development of inflammatory pain response appears to be complex and is not well understood.

Like components of the HPA axis, the development of spinal cord dorsal horn circuits is also known to be vulnerable to early life events. For example, substantial changes in synaptic function are observed in adult superficial dorsal horn (SDH) neurons following neonatal injury (Baccei, 2010). It appears the neonatal period represents a critical time point when major perturbations can disrupt the maturation of SDH neurons and their connections (Baccei and Fitzgerald, 2005; Walsh et al., 2009). Surprisingly, there have been no investigations of the long-term effects of neonatal exposure to LPS on both the HPA axis and dorsal horn activity even though we know formalin injection in adult animals increases metabolic activity in the spinal cord (Aloisi et al., 1993), and alters dorsal horn signalling (Dickenson and Sullivan, 1987; McCall et al., 1996). Clearly early life events are important for programming both the HPA axis and nociceptive pathways. In order to test how neonatal inflammation impacts the development of components of the HPA axis and inflammatory pain responses we exposed neonatal rats to LPS and assessed the effects of a neonatal immune challenge on behavioural, endocrine and neural responses to inflammatory pain stimuli at three time points during the first three weeks after birth. Our data suggest neonatal LPS exposure results in developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuronal properties following noxious stimulation later in life.

2. Materials and methods 2.1. Experimental procedures Eight experimentally naı¨ve female Wistar rats were obtained from the University of Newcastle Animal House and allowed one week acclimatization prior to mating in a vivarium. Mating resulted in 86 offspring for this study. The male was removed from the harem after two weeks and dams were housed individually in custom designed polycarbonateperspex home boxes (43.5 cm  28 cm  12.5 cm; Mascot Wire Works, Sydney, Australia). At PND 3 and 5 (PND 1 as day of birth), pups were briefly removed from their home boxes, weighed and injected (IP) with either LPS (Salmonella enterica, serotype enteritidis; Sigma—Aldrich Chemical Co., USA, dissolved in sterile pyrogen-free saline, 0.05 mg/kg) or an equal volume of saline (Livingstone International, Australia).

Plasticity following a neonatal immune challenge Following neonatal drug administration, pups were left undisturbed with their mothers until testing, when they were exposed to the formalin test as described below. Firstly, rats were randomly assigned testing days based on the three ages examined, PND 7, 13 and 22, with a maximum of 3 pups per litter being assigned to each group. After undergoing the formalin test, trunk (PND 7) or cardiac (PND 13 and 22) blood was collected and subsequently used for assessment of plasma corticosterone levels. The hypothalamus was isolated for quantification of glucocorticoid and mineralocorticoid receptor mRNA expression (GR and MR, respectively), and the lumbosacral enlargement of the spinal cord was removed for measurement of intrinsic electrophysiological properties of superficial dorsal horn (SDH) neurons. Animals were distributed evenly from all litters used per treatment to avoid potential litter bias. Until their testing day, rats were maintained in a temperature (21  1 8C) and humidity (60%) controlled environment, under a 12 h/12 h light—dark cycle (light on 06:00 h) with food and water available ad libitum. All experiments were carried out in accordance with the 2004 National Health and Medical Research Council of Australia Code of Practice for the care and use of animals for scientific use. All procedures were reviewed and approved by the Ethics committee of the University of Newcastle.

2.2. Formalin behavioural testing Pups were randomly assigned to the three age groups. At PND 7, rats were injected with 0.5% formalin into the plantar surface of the left hindpaw. At PND 13, rats were injected with 0.8% formalin into the plantar surface of the right hindpaw. At PND 22, rats were injected with 1.1% formalin into the plantar surface of the left hindpaw. The choice of formalin concentrations and injection volume (10 ml for each age group) was based on previous studies (Guy and Abbott, 1992; Zouikr et al., 2013) and the site of injection was alternated at each developmental stage. Injections were made using a 31 gage needle. The testing apparatus as well as testing conditions for the ages examined have been described elsewhere in detail by our group (Zouikr et al., 2013). No saline-injected rats were included since it has been previously shown that rats subjected to a subcutaneous injection of saline into the hindpaw do not display flinching or licking behaviour when tested during the first three postnatal weeks or in adulthood (Butkevich and Vershinina, 2001; Guy and Abbott, 1992; Okuda et al., 2001).

2.3. Behavioural analysis Flinching and licking responses were scored based on the method of Wheeler-Aceto and Cowan (Wheeler-Aceto and Cowan, 1991). The one hour recording was divided into an early phase (the first 5 min) and a late phase (10—60 min) during which the frequency of flinches and the duration (in seconds) spent licking the injected paw was scored. Two methods were applied to analyze the behavioural data: during the early phase (the first 5 min), an independent samples t-test was used where flinching and licking at 5 min were both analyzed as outcome variables with treatment as a fixed factor. During the late phase, the Area Under

3 the Curve (AUC) for both flinching and licking was calculated as the sum of responses from 10 to 60 min and analyzed as an outcome variable. Treatment and sex were considered fixed factors. Plots of the mean levels of flinching and licking, and a histogram displaying the AUC, were generated. Initially, groups were divided by sex and when no sex differences were observed, data from males and females were combined.

2.4. Blood collection and radioimmunoassay procedures A total of 85 rats were used for assessment of plasma corticosterone levels 1 h after the completion of the formalin test (PND 7 = 28; PND 13 = 32; PND 22 = 25; evenly distributed between saline- and LPS-treated and sexes in all three age groups). Animals were left undisturbed in their individual testing boxes during this period. At PND 7, rats were rapidly decapitated and trunk blood collected into EDTA-coated tubes (Livingstone International, Australia). At PND 13 and 22, rats were overdosed with Lethabarb1 (2 ml/kg IP.; Virbac, Pty. Ltd., Milperra, Australia) and cardiac blood was collected in EDTA-coated tubes. All blood samples were centrifuged at 1000  g at 4 8C for 20 min and plasma was stored at 20 8C until assayed. Plasma corticosterone concentrations were determined using a rat corticosterone 125I radioimmunoassay kit (MP Biomedicals, CA, USA). The recovery of free corticosterone was 100% with a mean inter- and intra-assay variability of 4.4% and 6.5%, respectively.

2.5. RNA extraction and qRT-PCR 23 rats exposed to the formalin test at PND 22 (evenly distributed between saline- and LPS-challenged and sexes) were used in order to assess the expression of mRNA levels of glucocorticoid (GR) and mineralocorticoid (MR) receptors in the hypothalamus. One hour post formalin behavioural assessment, animals were overdosed with Lethabarb1, their brains were rapidly removed and the hypothalamus isolated. Samples were then immersed in RNAlater1 solution (Ambion, Austin, TX, USA), stored at 4 8C overnight and then kept at 80 8C until further analysis. Total RNA was extracted from the hypothalamus using RNeasy1 Lipid Tissue Mini kit (Qiagen Inc., Valencia, CA, USA). Samples were disrupted and homogenized by adding 5 mm beads (Qiagen, Hilden, Germany) into 1 ml QIAzol Lysis Reagent and using a Tissue Lyser LT (Qiagen, Hilden, Germany) for 4 min at 50 Hz. The supernatant was allowed to sit for 5 min prior to being passed through a genomic DNA eliminator spin column. Chloroform (200 ml) was added and the lysate was then centrifuged for 15 min (12,000  g at 4 8C). Ethanol (500 ml of 70%) was added and precipitates were mixed thoroughly. A sample (700 ml) was then transferred to an RNeasy Mini spin column and centrifuged for 15 s at 8000  g (at room temperature). Contaminants were cleared using Buffer RW1 and RPE. RNA was finally eluted in 30 ml of RNase free water. RNA concentrations were determined by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). 260/280 and 260/230 ratios were

4 examined to test the purity of RNA, values ranged between 1.98 and 2.09 for the former and between 1.97 and 2.27 for latter indicating that the RNA was pure and free from protein, phenol and other contaminants. First-strand cDNA was synthesized from 2 mg of total RNA using a Super-Script1 VILOTM cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA, USA). A combination of 4 ml 5X VILOTM Reaction Mix, 2 ml 10X SuperScript1 Enzyme Mix, 2.5 mg RNA and up to 20 ml of DEPC-treated water was gently mixed and incubated at 25 8C for 10 min. Tubes were then transferred to MyCyclerTM thermal cycler (Bio-Rad, Hercules, CA, USA) and heated at 42 8C for one hour. The reaction was stopped at 85 8C for 5 min. The resulting cDNA was then stored at 20 8C until use. Quantitative real-time PCR (qRT-PCR) was performed on an Applied Biosystems 7500 Fast System instrument (Applied Biosystems, Foster City, CA, USA). qRT-PCR was conducted using TaqMan1 Gene Expression Master Mix and TaqMan1 Gene Expression primer/probe Assay (Applied Biosystems, Foster City, CA, USA) on each sample for the target genes: Nr3c1 (GR) and Nr3c2 (MR) (Assay ID’s Rn00561369_m1 and Rn00565562_m1, respectively). The total volume of 20 ml per single PCR reaction mix consisted of 1 ml 20x TaqMan1 Gene Expression Assay, 10 ml 2 TaqMan1 Gene Expression Master Mix, 4 ml cDNA (5 ng/ml), and 5 ml RNase-free water. All samples were assayed in duplicate and standardized using the endogenous control gene Hprt1 (Assay ID Rn01527840_m1). Amplification and detection were carried out using the following thermal conditions: 50 8C for 2 min, 95 8C for 10 min, and 40 cycles at 95 8C for 15 s and 1 min at 60 8C. In order to determine the relative quantitation of GR and MR mRNA, a Quantitation-Comparative Ct (DDCt) method was applied using the equation 2 DDCt which expresses the fold change in the target gene (Schmittgen and Livak, 2008).

2.6. Electrophysiological recordings Following the formalin test, spinal cords from 58 rats (PND 7 = 15; PND 13 = 18; PND 22 = 25; equally distributed between treatment groups and sexes) were dissected out for subsequent electrophysiological recording from superficial dorsal horn (SDH) neurons in laminae I-II. Rats were anaesthetized with Ketamine (100 mg/kg) and decapitated. The vertebral column and posterior thoracic wall were isolated and rapidly immersed in ice cold sucrose substituted artificial cerebrospinal fluid (sACSF) containing (in mM): 250 sucrose, 25 NaHCO2, 10 glucose, 2.5 KCl, 1 NaH2PO4, 1 MgCl2 and 2.5 CaCl2. The sACSF was continually bubbled with 95% O2—5% CO2 (pH of 7.3—7.4; Tadros et al., 2012). The lumbosacral enlargement of the spinal cord was removed, and marked to allow identification of ipsi- and contralateral dorsal horns. Transverse slices from L3-5 spinal segments (300 mm thick) were prepared using a vibratome (Leica VT1200s, Leica Microsystems, Wetzlar, Germany). Slices were then transferred to an interface storage chamber containing ACSF (118 mM NaCl substituted for sucrose in sACSF) and allowed to equilibrate for 1 h at room temperature (22—24 8C) prior to recording. Slices were transferred to a recording chamber (volume 0.4 ml) and continually superfused (4—6 bath volumes/minute) with ACSF. Recording temperature was maintained at 22—24 8C using an in-line temperature control unit (TC-324B,

I. Zouikr et al. Warner Instruments, Hamden, CT). Whole cell patch clamp recordings were obtained from SDH neurons, visualized with infrared differential contrast optics and an infrared-sensitive camera (Rolera-XR, Olympus, NJ), using patch pipettes (3— 4 MV resistance) prepared from thin walled borosilicate glass (PG150T-15, Harvard Apparatus, Kent, UK). Patch pipettes were filled with a potassium-based internal containing (in mM): 135 KCH3SO4, 6 NaCl, 2 MgCl2, 10 HEPES, 0.1 EGTA, 2 MgATP, 0.3 NaGTP, pH 7.3 (with 1 M KOH). Whole cell patch clamp recordings were made using a Multiclamp 700B Amplifier (Molecular Devices, Sunnyvale, CA). The whole cell recording configuration was first established in voltage clamp mode (holding potential — 60 mV). Series resistance was measured from the averaged response (five trials) to a 5 mV (10 ms long) hyperpolarizing pulse. This was measured at the beginning and end of each recording session and data were rejected if values changed by >20%. All measurements were subsequently made after switching into the current clamp recording mode. The membrane potential observed 15 s after this switch was designated resting membrane potential (RMP). In order to obtain a rheobase action potential (AP), a series of depolarising steps (20 pA increments, 800 ms duration) were injected from RMP. A series of measurements were made to allow comparison of the intrinsic properties of SDH neurons from saline- and LPStreated rats. Input resistance was obtained by calculating the chord conductance across a minimum of four responses to hyperpolarizing current injections (from 70 mV; 10 pA increments in current clamp recording mode). Individual APs were captured using the derivative threshold method, using the optimum threshold for each age group examined (PND 7, dv/dt = 10; PND 13, dv/dt = 15; PND 22, dv/dt = 20). AP threshold was measured at the inflection point during the rising phase of the AP. Rheobase current was defined as the smallest step-current that elicited at least one AP. The amplitude of each AP was measured as the difference between its threshold and maximum positive peak. AP half-width was calculated at 50% of AP amplitude. AP afterhyperpolarization (AHP) amplitude was measured as the difference between AP threshold and its maximum negative peak.

2.7. Data analysis Electrophysiological data were subjected to pair-wise comparisons (using Student’s t-test). Data that failed Levene’s test of homogeneity of variance were compared using the non-parametric Mann—Whitney test. Statistical significance was set at p < 0.05 and all data are presented as means  SEM. For all other data, an analysis of covariance (ANCOVA) was applied. Litter size and male-to-female ratio were not significant covariates. Planned comparisons were performed using the Least Significant Differences (LSD) and a was set to 0.05.

3. Results 3.1. Inflammatory pain behaviour A total of 86 rats underwent the formalin test, (PND 7 = 28; PND 13 = 32; PND 22 = 26; equal numbers of males and females). At each time point examined, no differences were

Plasticity following a neonatal immune challenge observed between male and female data. Therefore, data from both sexes were combined to produce plots of mean flinching and licking behaviours over the one-hour recording period. 3.1.1. PND 7 rats At this time-point, the pattern of flinching and licking the injected (ipsilateral) paw was similar between saline(n = 14; female n = 7, male n = 7) and LPS-challenged rats (n = 14; female n = 7, male n = 7) (Fig. 1). At this age, we also observed flinching of the contralateral paw (Fig. 1C), but to a lesser extent than the ipsilateral paw. This contralateral flinching also followed the response profile observed in the ipsilateral paw. Calculation of the AUC for contralateral flinching revealed no significant differences between saline and LPS-challenged rats (Fig. 1D). 3.1.2. PND 13 rats At this age, the characteristic biphasic pattern was observed in both licking and flinching responses (Fig. 2A and B). During the early phase, no significant differences were found in

5 flinching or licking the ipsilateral paw between the two groups. Analysis of the AUC in the licking response during the late phase revealed a significant main effect of treatment [F(1,122) = 11.77, p < .01] with LPS-challenged rats spending significantly more time licking the ipsilateral paw (M = 21.79 sec, SEM = 3.59, n = 16) compared to the salinechallenged group (M = 4.06 sec, SEM = 3.71, n = 16; Fig. 2C). Although some brief contralateral flinching and licking was observed in PND 13 rats, it was only present for very short periods. Therefore, no further analysis was conducted for the contralateral responses at PND 13. 3.1.3. Preadolescent rats (PND 22) At PND 22, a clear biphasic pattern was noticed in both flinching and licking responses (Fig. 3). Although no significant differences were observed during the early phase in either flinching or licking, analysis of the AUC in flinching the ipsilateral paw during the late phase revealed a significant main effect of treatment [F(1,20) = 4.88, p < .05; Fig. 3B] with LPS-treated rats displaying significantly more flinching of the ipsilateral paw (M = 114.08, SEM = 20.52, n = 14)

Figure 1 Neonatal LPS exposure does not alter formalin-induced nociception in PND 7 rats. Time course of flinching and licking the ipsilateral (A and B) and contralateral (C) paw in PND 7 rats after injection of 0.5% formalin (mean  SEM). (D) The Area Under the Curve (AUC) for flinching the contralateral paw during the late phase in PND 7 rats. Ipsi: ipsilateral. Contra: contralateral to formalin injection. n.s.: non significant.

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I. Zouikr et al. pattern of these responses did not differ between the two groups (Fig. 3D and E).

3.2. Plasma corticosterone levels Plasma corticosterone levels were measured one hour after formalin injection in 85 rats (PND 7 = 28; PND 13 = 32; PND 22 = 25; equally distributed between males and females). Since no significant differences between the sexes were observed, data from males and females were combined. At PND 7, a significant effect of treatment [F(1,22) = 25.64, p < .001] was observed, with LPS-challenged rats displaying increased levels of plasma corticosterone than salinechallenged rats (Fig. 4A). Although no significant differences in plasma corticosterone were observed at PND 13 (Fig. 4B), LPS-challenged rats showed slightly elevated plasma corticosterone. In contrast, at PND 22 there was a significant effect of treatment [F(1,19) = 17.70, p < .01] with LPS-challenged rats exhibiting increased levels of plasma corticosterone one hour post formalin injection compared to saline-treated rats (Fig. 4C). These data show that the activation of the HPA axis in response to neonatal exposure to LPS is developmentally regulated.

3.3. Gene expression at PND 22 Comparison of hypothalamic GR and MR mRNA levels between males and females revealed no differences, so data from both sexes were combined. No significant differences were observed between saline (n = 11) and LPS-challenged rats (n = 12) for both GR and MR mRNA levels in the hypothalamus (Fig. 5). However, there was a slight increase in mRNA levels of MR in the hypothalamus of LPS-treated rats after exposure to the formalin test ( p = .077). These data imply that the balance between these two types of corticoid receptors was slightly disturbed as a consequence of neonatal LPS exposure and a subsequent formalin test at PND 22.

3.4. Intrinsic properties of SDH neurons

Figure 2 Neonatal LPS exposure induces increased licking responses during the late phase in PND 13 rats. Time course of flinching and licking the ipsilateral paw (ipsi) in PND 13 rats (A and B) after injection of 0.8% formalin (mean  SEM). (C) The Area Under the Curve (AUC) of licking the ipsilateral paw during the late phase in PND 13 rats.**p < .01.

compared to saline-challenged rats (M = 45.98, SEM = 22.22, n = 12). Licking of the ipsilateral paw did not differ between the two groups during the late phase of the behavioural response (Fig. 3C). Interestingly, we observed contralateral flinching and licking of the injected paw at PND 22, but the

A total of 228 recordings (PND 7 = 65; PND 13 = 77; PND 22 = 86) were obtained from SDH neurons from 58 rats (PND 7 = 15; PND 13 = 18; PND 22 = 25; equally distributed between treatment groups and sexes, Table 1). Recordings were made from neurons classified as either ipsi- or contralateral according to the site of injection (i.e., left or right hindpaw). Comparisons were made between ipsi- or contralateral SDH neurons from saline- or LPS-challenged rats within each age group. At PND 7, intrinsic properties of SDH neurons were remarkably similar between saline- and LPS-challenged rats, for both ipsi- and contralateral sides of the spinal cord. At PND 13, SDH neurons from LPS-challenged rats showed more depolarised RMPs on the contralateral spinal cord compared to the ipsilateral ( p = 0.005). Input resistance and AP properties were unchanged between saline- and LPS-challenged rats at PND 13. In contrast, at PND 22 the input resistance of ipsilateral SDH neurons in LPS-challenged rats was significantly lower than both the ipsilateral SDH neurons from saline-challenged rats ( p = 0.05) and the contralateral SDH neurons from LPS-challenged rats ( p = 0.03). In addition, AP

Plasticity following a neonatal immune challenge

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Figure 3 Neonatal LPS exposure produces increased flinching responses during the late phase in PND 22 rats. (A) and (C) Time-course of flinching and licking (respectively) the ipsilateral paw following the injection of 1.1% formalin (mean  SEM). (B) The Area Under the Curve (AUC) of flinching the ipsilateral (ipsi) paw during the late phase. (D) and (E) the AUC of flinching and licking (respectively) the contralateral paw during the late phase. n.s.: non significant. *p < .05.

amplitude was decreased in ipsilateral SDH neurons from LPSchallenged rats compared to ipsilateral SDH neurons from saline-challenged rats ( p = 0.006). Overall, these data show neonatal exposure to LPS results in subtle changes in the intrinsic properties of SDH neurons. These changes have the potential to alter the excitability of SDH neurons well after the initial immune response to the LPS-challenge has cleared.

PND 22. There were also changes in the intrinsic properties of SDH neurons in PND 22 rats after neonatal LPS exposure. These data provide the first evidence that neonatal immune challenge produces developmentally regulated changes in formalin-induced nociception, HPA axis function and SDH neuronal properties.

4. Discussion

4.1. The impact of neonatal immune challenge on formalin-induced nociception is agedependent.

Our study demonstrates that an immune challenge during the neonatal period exerts long-term effects on inflammatory pain responses in a developmentally regulated manner. Enhanced susceptibility (i.e., hyperalgesia) to formalininduced licking (at PND 13) and flinching (at PND 22) responses was observed in response to neonatal LPS exposure. This behavioural hyperalgesia was associated with increased plasma corticosterone levels at PND 22, but not PND 13, and a shift in the balance of glucocorticoid and mineralocorticoid receptor mRNA in the hypothalamus at

Although PND 7 rats exhibited flinching and licking responses, the profile of the response was unaltered by neonatal LPS exposure. In contrast, LPS-challenged PND 13 and PND 22 rats displayed increased licking or flinching behaviours, respectively. These data suggest distinct changes in formalininduced pain behaviours occur with both age and response type. The rhythmic nature of flinching suggests this behaviour is spinally modulated, whereas the complex movements involved in licking would require supraspinal modulation. Indeed, intrathecal injection of Lidocaine into the lumbar

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Figure 4 Neonatal exposure to LPS alters corticosterone response in a developmentally regulated way after subsequent challenge with the formalin test. Corticosterone plasma levels (ng/ml) one hour post formalin injection in PND 7 (A), PND 13 (B), and PND 22 (C) rats. Data are presented as mean  SEM. n.s.: non significant. ***p < .001.

spinal cord prior to formalin injection abolished flinching during the late phase of the response (Coderre et al., 1994). In contrast, complete transection of the spinal cord at the mid-thoracic level did not affect formalin-induced flinching (Coderre et al., 1994), but completely abolished formalininduced licking behaviours (Wheeler-Aceto and Cowan, 1991). Taken together, these data confirm that distinct neural

I. Zouikr et al. pathways are responsible for generating flinching or licking behaviours. Therefore, adverse events occurring during the neonatal period can modulate the development of either spinal or supraspinal neural circuits and alter the profile of formalin-induced pain behaviours later in life. Previous studies examining the emergence of formalininduced licking and flinching behaviours also suggest distinct developmental pathways exist for the two responses. For example, Butkevich and colleagues observed flinching, but no licking, in PND 7 rats and flinching and licking in PND 10 rats exposed to 2.5% formalin (Butkevich et al., 2009b). Lower doses of formalin (0.5%) could elicit both licking and flinching in PND 7 rats, and a dose-dependent increase in flinching response was observed at PND 13 (Zouikr et al., 2013). Additionally, prenatal exposure to restraint stress results in increased flinching at PND 7 and PND 25, but not PND 10, with no differences in licking behaviours at PND 25 (Butkevich et al., 2009b; Butkevich and Vershinina, 2001). This suggests that fine-tuning of flinching and licking occurs throughout the first month of postnatal life, and that immune challenge during this time can result in long-term effects on either behaviour. We observed contralateral formalin-induced pain behaviours at PND 7 and 22 (Figs. 1 and 3), which was rarely observed at PND 13 (1/32 rats). Contralateral formalininduced pain behaviours have been previously documented in adult rats (Aloisi et al., 1993), however, we report for the first time that contralateral behaviours also occur during early postnatal development. It has been proposed that contralateral pain responses result from peripherally circulating hormonal factors or by centrally mediated neuronal changes (Koltzenburg et al., 1999). Due to the specificity of the contralateral pain behaviours, as well as their rapid onset (within 5 min at PND 7; Fig. 1, and 10 min at PND 22; data not shown), it is likely that the contralateral flinching we observed is modulated by neuronal mechanisms. Indeed, functional activity of dorsal horn neurons in the spinal cord increases bilaterally within one hour of unilateral formalin injection in adult rats (Aloisi et al., 1993). Interestingly, the contralateral hyperalgesia we observed in infant and preadolescent rats was not affected by neonatal LPS-challenge, indicating the neural pathway responsible for generating contralateral responses is less susceptible to long-term effects of neonatal immune challenge. One possible mechanism for the bilateral effect of formalin injection is the existence of commissural fibres between opposing dorsal horns of the spinal cord. For example, Golgi staining in adult cats and dogs has revealed decussating fibres at all levels of the spinal cord, with synaptic contacts between laminae I-II neurons of both sides of the dorsal horn (Szentagothai, 1964). The development, and susceptibility to neonatal insult, of these commissural fibres in rodents is presently unknown, but our data suggest they are present and functional during the early stages of postnatal development.

4.2. Neonatal immune challenge alters HPA axis function in preadolescent rats following an inflammatory noxious challenge. The immune and nervous system communicate closely via the action of glucocorticoids (i.e., HPA axis) which constitutes a powerful modulator of immune function (Besedovsky and del

Plasticity following a neonatal immune challenge

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Figure 5 Expression of GR and MR mRNA levels in the hypothalamus of PND 22 rats following neonatal LPS-challenge. GR and MR mRNA levels in the hypothalamus represented as fold change relative to the endogenous gene, Hprt1 in PND 22 rats. (A) The level of glucocorticoid receptors mRNA levels in the hypothalamus of PND 22 male and female rats combined one hour post formalin injection into the left hindpaw. (B) The level of mineralocorticoid receptors mRNA levels in the hypothalamus of PND 22 one hour post formalin injection into the left hindpaw. Data are presented as mean  SEM. n.s.: non significant.

Rey, 1992). Pain is an aversive and stressful experience and therefore capable of activating the HPA axis (BlackburnMunro, 2004). Previous studies have documented that PND 7 and 10 rats displayed enhanced flinching responses 30 min after formalin injection that was parallel with an increase of corticosterone levels compared to its basal level (Butkevich et al., 2013; Mikhailenko et al., 2013). In the present study, neonatal LPS exposure enhanced post formalin levels of plasma corticosterone at PND 7 and 22 but not PND 13. The combination of increased formalin-induced behavioural responses and plasma corticosterone levels was only observed at PND 22. The HPA axis goes through a ‘‘stress hyporesponsive period’’ (SHRP) from PND 4 to PND 14 during which adrenal sensitivity to stressors is diminished and corticosterone concentrations are maintained at low levels (Levine, 1994). Given that PND 13 falls inside this critical window of HPA axis development, it is possible we did not see changes in plasma corticosterone levels at this age due to the SHRP. We did, however, observe an increase in plasma corticosterone at PND 7, suggesting that the SHRP is sensitive to an immune challenge alone or in combination with an inflammatory challenge during the first postnatal week. Experimental and clinical evidence have previously documented sex differences in pain and HPA axis function in infant (Butkevich et al., 2007, 2011) and pre-pubertal (Butkevich and Vershinina, 2001) periods. PND 7—8 male rats injected with formalin exhibit higher flinching in response to prenatal stress compared to females. In addition, formalin injection at PND 25 evoked higher flinching and plasma corticosterone levels in females (Butkevich et al., 2009a). We did not observe sex differences in any measured parameters in our study. This discrepancy may be attributed to the difference in the timing (gestational vs. neonatal) and nature (restraint stress vs. immune challenge) of the stressor, which can differently modulate formalin-induced behaviours. In addition, the difference in formalin concentrations should be

considered (2.5% vs. 1.1%). In summary, it is clear that early life programming of sex-specific responses to formalin induced pain behaviours is highly dependent on the timing and nature of the stressor. We also demonstrated that neonatally LPS-challenged rats displayed decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus at PND 22 compared to controls (Fig. 5). Whilst these differences were not significant, they suggest a shift in the relative balance of these two receptors in preadolescent rats following neonatal LPS-challenge. GRs are activated by high levels of plasma corticosterone, and aid in recovery from stress, whereas MRs respond to low levels and are thought to set the threshold for stress responsiveness. The balance between activation of these two receptors is thought to set an individual’s response to stressors, and any disturbance in this balance can dysregulate the stress system (for review see Groeneweg et al., 2012). Although we cannot confirm whether MR or GR mRNA was translated into functional protein, we speculate that an increase in MRs would reduce the threshold for stress responsiveness, and that a decrease in GRs would result in poorer recovery from stress. Combined with the increased plasma corticosterone levels observed at PND 22, preadolescent rats neonatally challenged with LPS are likely to be more susceptible to stress-related disorders than their saline-challenged counterparts. MRs and GRs show a distinctive ontogenetic pattern in the rat hypothalamus, with GR mRNA detectable at gestational day 16 in the hypothalamus (Yi et al., 1994), whereas MR mRNA is not detected until PND 2 (van Eekelen et al., 1991). Since we exposed rats to LPS at PNDs 3 and 5, it is possible that this timing had a greater affect on MR ontogeny than GR. This may result in altered MR density in the hypothalamus later in life and lead to less efficient negative-feed back during stress. Although speculative, this assumption is consistent with the finding that daily handling from day 2 to 8 resulted in down-regulation of hypothalamic

23.4  1.2

4.3. Neonatal LPS-challenge produces subtle, but potentially important, long-term effects on SDH neurons

1.6  0.1

26.0  3.0 18.6  2.9 21.8  2.2 1.4  0.1 1.8  0.3 1.5  0.2

corticotrophin releasing hormone mRNA levels in PND 23 rats (Avishai-Eliner et al., 2001). To the best of our knowledge, no study has focused on the impact of neonatal immune challenge on hypothalamic GR and MR mRNA in preadolescent rats following formalin injection.

86  15

LPS

Contra (n = 28)

indicate significant difference (details in brackets). *

49.1  1.9

27.9  1.8

62.5  2.3 50.7  5.3 47.6  4.2* (vs. Saline Ipsi, p = 0.006) 52.3  3.2 34.3  1.3 32.0  2.1 32.0  1.4 60  27 75  15 119  12 51.5  3.6 50.1  2.2 50.2  2.0 Ipsi (n = 15) Contra (n = 23) Ipsi (n = 20) PND 22

LPS

Saline

216  31 219  20 155  14* (vs. LPS Contra, p = 0.03, vs. Saline Ipsi, p = 0.05) 222  19

19.2  1.6 19.9  1.8 19.0  0.8 22.8  2.0 2.0  0.2 1.5  0.1 1.9  0.1 1.6  0.1 52.0  3.3 57.5  3.4 53.4  1.9 54.7  3.8 34.1  1.5 34.0  1.1 33.0  0.8 32.4  1.5 66  15 67  16 76  12 54  8 52.1  2.1 48.1  3.3 53.2  1.7 42.8  2.3* (vs. LPS Ipsi, p = 0.005) Ipsi (n = 17) Contra (n = 11) Ipsi (n = 39) Contra (n = 10) PND 13

LPS

Saline

286  30 262  37 275  21 270  21

2.7  0.3 2.5  0.2 2.5  0.3 2.6  0.2 40.8  3.4 38.5  3.1 47.8  4.5 43.1  3.7 31.8  1.2 31.8  1.0 34.0  1.6 28.6  1.7 71  16 69  12 40  11 75  15 43.1  3.0 47.0  2.5 43.1  3.5 48.4  2.1 Ipsi (n = 16) Contra (n = 17) Ipsi (n = 8) Contra (n = 24) PND 7

Saline

345  35 398  48 355  77 376  39

AP half-width (ms) AP amplitude (mV) AP threshold (mV) Rheobase (pA) Resting membrane potential (mV) Input resistance (MV) Sight of recording Treatment Group

The intrinsic properties of superficial dorsal horn (SDH) neurons in PND 7, 13, and 22 rats following neonatal LPS exposure. Table 1

19.3  2.0 19.0  2.5 14.2  1.6 16.2  1.8

I. Zouikr et al. AHP amplitude (mV)

10

SDH neurons (laminae I & II) are the first component of the central nervous system to receive incoming sensory information regarding noxious stimuli, and their output is determined by a combination of their synaptic inputs and intrinsic neuronal properties (Hille, 2001). We observed remarkably similar intrinsic properties in SDH neurons from saline and LPS-challenged rats at PND 7 and 13 (Table 1). In contrast, SDH neurons in preadolescent LPS-challenged rats had lower input resistances compared to their saline-challenged counterparts. Importantly, this change was only observed in SDH neurons ipsilateral to the injected hindpaw, suggesting the decreased input resistance occurred in response to the formalin injection, not the neonatal exposure to LPS. Hindpaw injection of formalin is associated with an increase in the release of numerous substances in the spinal cord dorsal horn, including prostaglandin E2 (Malmberg and Yaksh, 1995). Bath application of prostaglandin E2 results in changes of intrinsic properties of dorsal horn neurons, including a decrease in input resistance (Baba et al., 2001). Since this change was only observed in LPS-challenged rats, it is possible that the neonatal exposure to LPS results in either an increased release of pro-inflammatory substances into the dorsal horn, or an increase in SDH neuron susceptibility to these substances. Changes in formalin-induced pain behaviours were not always accompanied by changes in the electrophysiological properties of SDH neurons. However, it is still possible that the overall excitability of the dorsal horn circuitry has been altered in response to neonatal exposure to LPS. For example, chronic stress in adult rats was associated with impaired function of the hippocampal network without any significant differences in intrinsic properties of hippocampal neurons (Schnell et al., 2012). Moreover, increased levels of BDNF in the spinal cord results in increased excitatory drive within the dorsal horn, but no change in intrinsic properties of SDH neurons (Lu et al., 2009). There are also supraspinal networks involved in generating formalin-induced pain behaviours that may be affected by neonatal exposure to LPS. Intracerebroventricular, but not intrathecal, administration of acetaminophen reduced LPS-induced hyperalgesia during the formalin test in adult mice, suggesting that systemic LPS plays a prominent role in supraspinal modulation (Seo et al., 2008). Therefore, it is possible that neonatal exposure to LPS can affect both spinal and supraspinal neural networks.

4.4. Conclusions Previous studies have documented the importance of the neonatal experience to subsequent responses to a variety of stressors in adulthood (Hodyl et al., 2007; Beggs et al., 2012; Hodgson et al., 2001). Here, we report several long-term

Plasticity following a neonatal immune challenge effects of neonatal LPS exposure that include neural, endocrine and behavioural responses to inflammatory pain induced by formalin injection. Interestingly, these changes appear to be developmentally regulated, with distinct changes observed at PND 7, 13 and 22.

Contributors Kenneth W. Beagley and Vicki L. Clifton contributed intellectually and provided critical input. Ihssane Zouikr; Melissa A. Tadros; Robert J. Callister; Deborah M. Hodgson conceived and designed the experiments. Ihssane Zouikr; Melissa A. Tadros; Javad Barouei performed the experiments. Ihssane Zouikr; Melissa A. Tadros analyzed the data. Ihssane Zouikr; Melissa A. Tadros; Robert J. Callister; Deborah M. Hodgson wrote the paper.

Role of the funding source This work was supported by a grant from the Australian Research Council (ARC DP 09787599). ARC had no further role in the study design, running experiments, data collection, analysis and interpretation of data, in the writing of the manuscript; and in the decision to submit the paper for publication.

Conflict of interest The authors declare no competing financial or other relationships that might lead to a conflict of interest.

Acknowledgments We would like to thank Donna Catford and all conjoint BSAF staff for their assistance in animal husbandry. We also acknowledge Kim Colyvas for his assistance with statistical analysis.

References Aloisi, A.M., Ceccarelli, I., Lupo, C., 1998. Behavioural and hormonal effects of restraint stress and formalin test in male and female rats. Brain Res. Bull. 47, 57—62. Aloisi, A.M., Porro, C.A., Cavazzuti, M., Baraldi, P., Carli, G., 1993. ‘Mirror pain’ in the formalin test: behavioral and 2-deoxyglucose studies. Pain 55, 267—273. Avishai-Eliner, S., Eghbal-Ahmadi, M., Tabachnik, E., Brunson, K.L., Baram, T.Z., 2001. Down-regulation of hypothalamic corticotropin-releasing hormone messenger ribonucleic acid (mRNA) precedes early-life experience-induced changes in hippocampal glucocorticoid receptor mRNA. Endocrinology 142, 89—97. Baba, H., Kohno, T., Moore, K.A., Woolf, C.J., 2001. Direct activation of rat spinal dorsal horn neurons by prostaglandin E2. J. Neurosci. 21, 1750—1756. Baccei, M.L., 2010. Modulation of developing dorsal horn synapses by tissue injury. Ann. N. Y. Acad. Sci. 1198, 159—167. Baccei, M.L., Fitzgerald, M., 2005. Intrinsic firing properties of developing rat superficial dorsal horn neurons. Neuroreport 16, 1325—1328. Beggs, S., Currie, G., Salter, M.W., Fitzgerald, M., Walker, S.M., 2012. Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activity. Brain 135, 404— 417.

11 Besedovsky, H.O., del Rey, A., 1992. Immune-neuroendocrine circuits: integrative role of cytokines. Front. Neuroendocrinol. 13, 61—94. Blackburn-Munro, G., 2004. Hypothalamo-pituitary-adrenal axis dysfunction as a contributory factor to chronic pain and depression. Curr. Pain Headache Rep. 8, 116—124. Boisse, L., Spencer, S.J., Mouihate, A., Vergnolle, N., Pittman, Q.J., 2005. Neonatal immune challenge alters nociception in the adult rat. Pain 119, 133—141. Butkevich, I.P., Barr, G.A., Vershinina, E.A., 2007. Sex differences in formalin-induced pain in prenatally stressed infant rats. Eur. J. Pain 11, 888—894. Butkevich, I.P., Mikhailenko, V.A., Bagaeva, T.R., Makukhina, G.V., 2009a. Persistent pain responses in inflammation and corticosterone levels in juvenile rats born to adrenalectomized dams. Neurosci. Behav. Physiol. 39, 297—300. Butkevich, I.P., Mikhailenko, V.A., Bagaeva, T.R., Vershinina, E.A., Aloisi, A.M., Otellin, V.A., 2013. Inflammatory pain and corticosterone response in infant rats: effect of 5-HT1A agonist buspirone prior to gestational stress. Mediators Inflamm. 2013, 915189. Butkevich, I.P., Mikhailenko, V.A., Vershinina, E.A., Otellin, V.A., Aloisi, A.M., 2011. Buspirone before prenatal stress protects against adverse effects of stress on emotional and inflammatory pain-related behaviors in infant rats: age and sex differences. Brain Res. 1419, 76—84. Butkevich, I.P., Mikhailenko, V.A., Vershinina, E.A., Semionov, P.O., Otellin, V.A., Aloisi, A.M., 2009b. Heterogeneity of the infant stage of rat development: inflammatory pain response, depression-related behavior, and effects of prenatal stress. Brain Res. 1286, 53—59. Butkevich, I.P., Vershinina, E.A., 2001. Prenatal stress alters time characteristics and intensity of formalin-induced pain responses in juvenile rats. Brain Res. 915, 88—93. Coderre, T.J., Yashpal, K., Henry, J.L., 1994. Specific contribution of lumbar spinal mechanisms to persistent nociceptive responses in the formalin test. Neuroreport 5, 1337—1340. Czura, C.J., Tracey, K.J., 2005. Autonomic neural regulation of immunity. J. Intern. Med. 257, 156—166. Dickenson, A.H., Sullivan, A.F., 1987. Subcutaneous formalin-induced activity of dorsal horn neurones in the rat: differential response to an intrathecal opiate administered pre or post formalin. Pain 30, 349—360. Gagliese, L., Melzack, R., 1999. Age differences in the response to the formalin test in rats. Neurobiol. Aging 20, 699—707. Groeneweg, F.L., Karst, H., de Kloet, E.R., Joels, M., 2012. Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signalling. Mol. Cell. Endocrinol. 350, 299—309. Guy, E.R., Abbott, F.V., 1992. The behavioral response to formalin in preweanling rats. Pain 51, 81—90. Hille, B., 2001. Ionic Channels of Excitable Membranes,, 3rd ed. Sinauer Associates, Inc., Sunderland, MA. Hodgson, D.M., Knott, B., Walker, F.R., 2001. Neonatal endotoxin exposure influences HPA responsivity and impairs tumor immunity in Fischer 344 rats in adulthood. Pediatr. Res. 50, 750—755. Hodyl, N.A., Krivanek, K.M., Lawrence, E., Clifton, V.L., Hodgson, D.M., 2007. Prenatal exposure to a pro-inflammatory stimulus causes delays in the development of the innate immune response to LPS in the offspring. J. Neuroimmunol. 190, 61—71. Koltzenburg, M., Wall, P.D., McMahon, S.B., 1999. Does the right side know what the left is doing? Trends Neurosci. 22, 122—127. Levine, S., 1994. The ontogeny of the hypothalamic-pituitary-adrenal axis. The influence of maternal factors. Ann. N. Y. Acad. Sci. 746, 275—288 (discussion 289-293). Lu, V.B., Biggs, J.E., Stebbing, M.J., Balasubramanyan, S., Todd, K.G., Lai, A.Y., Colmers, W.F., Dawbarn, D., Ballanyi, K., Smith, P.A., 2009. Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve injury. J. Physiol. 587, 1013—1032.

12 Malmberg, A.B., Yaksh, T.L., 1995. The effect of morphine on formalin-evoked behaviour and spinal release of excitatory amino acids and prostaglandin E2 using microdialysis in conscious rats. Br. J. Pharmacol. 114, 1069—1075. Matthews, S.G., 2002. Early programming of the hypothalamo-pituitary-adrenal axis. Trends Endocrinol. Metab. 13, 373—380. McCall, W.D., Tanner, K.D., Levine, J.D., 1996. Formalin induces biphasic activity in C-fibers in the rat. Neurosci. Lett. 208, 45—48. Mikhailenko, V.A., Butkevich, I.P., Lavrova, Y.A., Bagaeva, T.R., Otellin, V.A., 2013. Effect of tonic pain on the corticosterone level in rat pups of various ages subjected to prenatal stress and opportunities for correction of stress-induced impairments. Dokl. Biol. Sci. 450, 134—138. Okuda, K., Sakurada, C., Takahashi, M., Yamada, T., Sakurada, T., 2001. Characterization of nociceptive responses and spinal releases of nitric oxide metabolites and glutamate evoked by different concentrations of formalin in rats. Pain 92, 107—115. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101—1108. Schnell, C., Janc, O.A., Kempkes, B., Callis, C.A., Flugge, G., Hulsmann, S., Muller, M., 2012. Restraint stress intensifies interstitial K(+) accumulation during severe hypoxia. Front. Pharmacol. 3, 53. Seo, Y.J., Kwon, M.S., Choi, H.W., Choi, S.M., Nam, J.S., Lee, J.K., Jung, J.S., Park, S.H., Suh, H.W., 2008. The differential effects of acetaminophen on lipopolysaccharide induced hyperalgesia in various mouse pain models. Pharmacol. Biochem. Behav. 91, 121—127. Shanks, N., Windle, R.J., Perks, P.A., Harbuz, M.S., Jessop, D.S., Ingram, C.D., Lightman, S.L., 2000. Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal function and predisposition to inflammation. Proc. Natl. Acad. Sci. U. S. A. 97, 5645— 5650.

I. Zouikr et al. Sorg, B.A., Bailie, T.M., Tschirgi, M.L., Li, N., Wu, W.R., 2001. Exposure to repeated low-level formaldehyde in rats increases basal corticosterone levels and enhances the corticosterone response to subsequent formaldehyde. Brain Res. 898, 314—320. Sternberg, E.M., 2006. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat. Rev. Immunol. 6, 318—328. Szentagothai, J., 1964. Neuronal and synaptic arrangement in the Substantia gelatinosa rolandi. J. Comp. Neurol. 122, 219—239. Tadros, M.A., Harris, B.M., Anderson, W.B., Brichta, A.M., Graham, B.A., Callister, R.J., 2012. Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord. J. Physiol. 590, 2409—2425. van Eekelen, J.A., Bohn, M.C., de Kloet, E.R., 1991. Postnatal ontogeny of mineralocorticoid and glucocorticoid receptor gene expression in regions of the rat tel- and diencephalon. Brain Res. Dev. Brain Res. 61, 33—43. Walsh, M.A., Graham, B.A., Brichta, A.M., Callister, R.J., 2009. Evidence for a critical period in the development of excitability and potassium currents in mouse lumbar superficial dorsal horn neurons. J. Neurophysiol. 101, 1800—1812. Wheeler-Aceto, H., Cowan, A., 1991. Standardization of the rat paw formalin test for the evaluation of analgesics. Psychopharmacology (Berl.) 104, 35—44. Yi, S.J., Masters, J.N., Baram, T.Z., 1994. Glucocorticoid receptor mRNA ontogeny in the fetal and postnatal rat forebrain. Mol. Cell. Neurosci. 5, 385—393. Zouikr, I., Tadros, M.A., Clifton, V.L., Beagley, K.W., Hodgson, D.M., 2013. Low formalin concentrations induce fine-tuned responses that are sex and age-dependent: a developmental study. PLoS ONE 8, e53384.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge.

The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation...
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