Excitatory synapses in the rat superficial dorsal horn are strengthened following peripheral inflammation during early postnatal development

PAINÒ 143 (2009) 56–64

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Excitatory synapses in the rat superficial dorsal horn are strengthened following peripheral inflammation during early postnatal development Jie Li, Mark L. Baccei * Pain Research Center, Department of Anesthesiology, University of Cincinnati Medical Center, 231 Albert Sabin Way, Cincinnati, OH 45267, USA

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Article history: Received 29 September 2008 Received in revised form 30 December 2008 Accepted 22 January 2009

Keywords: Dorsal horn Neonatal Inflammation Patch-clamp Development Glutamate

a b s t r a c t Peripheral inflammation can cause prolonged changes in pain sensitivity if it occurs during a critical period of early postnatal development, suggesting that neonatal pain circuits respond to tissue damage in a fundamentally different manner. However, the effects of early tissue injury on the function of emergent central pain networks have yet to be characterized at the synaptic level. Here, we investigated whether inflammation at different postnatal ages has distinct consequences for synaptic function in the superficial dorsal horn (SDH) of the rat spinal cord using in vitro patch-clamp techniques. Subcutaneous injections of carrageenan (CARR) into the hindpaw at postnatal day (P) 2 significantly increased the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) in P3–4 SDH neurons compared to naïve controls, while no changes were observed at inhibitory synapses onto the same neurons. The paired-pulse ratio of evoked EPSCs was unaltered by CARR at P2, suggesting that the elevation in mEPSC frequency following inflammation does not reflect an enhanced probability of glutamate release within the SDH. The potentiation of glutamatergic synapses did not persist beyond the duration of the injury, as the observed difference in mEPSC frequency had resolved by P10–11. In contrast, peripheral inflammation at P9 or P17 failed to significantly alter excitatory or inhibitory synaptic efficacy in the SDH. Meanwhile, carrageenan decreased the inward rectification of AMPAR-mediated currents throughout the first three postnatal weeks. These data suggest that distinct synaptic mechanisms may underlie central sensitization in the immature and mature spinal cords under pathological conditions. Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction Previous work has demonstrated that exposure to inflammatory mediators results in a significant increase in the excitability of immature primary afferent neurons [32]. This appears sufficient to induce central sensitization in the neonatal spinal cord, as hyperalgesia and allodynia are observed in both human infants and rat pups after tissue injury or inflammation [17,22,39,40,49]. Although both the neonate and the adult show heightened pain sensitivity in response to peripheral inflammation, it is still unknown if the potential underlying mechanisms of central sensitization are similar at various stages of postnatal development. Mounting evidence suggests that central sensitization following tissue injury in the adult is driven by significant alterations in synaptic function within the superficial dorsal horn (SDH) of the spinal cord, which may share similar mechanisms with the long-term potentiation (LTP) of synaptic strength in the cortex [27]. As a result, it is interesting to note that the cellular mechanisms underlying hippocampal LTP appear to be developmentally regulated [57]. This highlights the possibility that the nature of the synaptic mod* Corresponding author. Tel.: +1 513 558 5037; fax: +1 513 558 0995. E-mail address: [email protected] (M.L. Baccei).

ifications that produce spinal hyperexcitability under pathological conditions also varies during postnatal development. Age-dependent changes in synaptic efficacy within central pain networks such as the SDH could explain why tissue damage during the early postnatal period is uniquely capable of triggering persistent changes in pain sensitivity [12,35,48]. Novel effects of neonatal tissue injury on synaptic transmission within the dorsal horn might in fact be expected, given the distinct organization of the SDH synaptic network at early postnatal ages. For example, newborn rat lamina II neurons are distinguished by their relatively weak C-fiber inputs [18] and an absence of glycinergic inhibition [5]. In addition, a subset of neonatal SDH cells are depolarized in response to GABAAR activation [5], which likely reflects a reduced expression of the K+/Cl co-transporter KCC2 [8,50]. Subsequent work has demonstrated that a fully mature Cl extrusion capacity is not apparent in SDH neurons until the third postnatal week [14]. GABAergic signaling in the immature SDH is also strongly modulated by a tonic production of 5a-reduced neurosteroids, which does not occur in the adult spinal cord under normal conditions [30]. These ongoing developmental changes in synaptic function during the early postnatal period could affect how the SDH network responds to aberrant sensory input under pathological conditions. Unfortunately, little is currently

0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2009.01.023

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known about how tissue injury modulates synaptic transmission in the neonatal SDH. The present results demonstrate that peripheral inflammation during the first postnatal week transiently increases the efficacy of excitatory synapses in the immature SDH. Although the exact mechanisms underlying this potentiation of glutamatergic signaling remain unclear, the data suggest that it is unlikely to be explained by alterations in the probability of glutamate release in the SDH following tissue damage. Meanwhile, carrageenan treatment evoked similar changes in the properties of postsynaptic AMPARs in SDH neurons throughout the postnatal period. Collectively, the results indicate that the precise complement of synaptic changes evoked by tissue injury will vary according to the age at which the injury occurs. 2. Materials and methods All experiments adhered to animal welfare guidelines established by the University of Cincinnati Institutional Animal Care and Use Committee and the Committee for Research and Ethical Issues of the IASP. 2.1. Induction of hindpaw inflammation with carrageenan Neonatal Sprague–Dawley rat pups (postnatal days 2–17) were anesthetized with isoflurane (2–3%) and placed on a heating pad maintained at 37 °C. k-Carrageenan (2% in sterile saline; Fisher Scientific, Florence, KY) was injected into the left hindpaw at 0.7 ll/g of body weight using a microsyringe with a 30 gauge needle. The degree of hindpaw edema following carrageenan treatment was quantified by measuring the hindpaw diameter (from the dorsal to ventral surface) using a calibrated caliper, and expressed as the ratio of ipsilateral (i.e. injected) paw diameter over contralateral paw diameter as a function of time after the injection (see Fig. 1A). Naïve littermates were used as controls. 2.2. Preparation of spinal cord slices for in vitro recording Pups (P3–P20) were deeply anesthetized with sodium pentobarbital (30 mg/kg) and then perfused transcardially with ice-cold dissection solution consisting of (in mM) 250 sucrose, 2.5 KCl, 25 NaHCO3, 1.0 NaH2PO4, 6 MgCl2, 0.5 CaCl2, and 25 glucose continuously bubbled with 95% O2/5% CO2. The lumbar spinal cord was isolated, immersed in low-melting-point agarose (3% in above solution; Invitrogen, Carlsbad, CA), and parasagittal slices (350– 400 lm) were cut from the ipsilateral side using a Vibroslice tissue slicer (HA-752; Campden Instruments, Lafayette, IN). The slices were placed in a chamber filled with oxygenated dissection solution for 30 min then allowed to recover in an oxygenated artificial CSF (aCSF) solution containing the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.0 MgCl2, 2.0 CaCl2, and 25 glucose for P1.5 h at room temperature. 2.3. Patch-clamp recordings After recovery, slices were transferred to a submersion-type recording chamber (RC-22; Warner Instruments, Hamden, CT), mounted on the stage of an upright microscope (BX51WI, Olympus, Center Valley, PA) and perfused at room temperature with oxygenated aCSF at a rate of 1.5–3 ml/min. Patch electrodes were constructed from thin-walled single-filamented borosilicate glass (1.5 mm outer diameter; World Precision Instruments, Sarasota, FL) using a microelectrode puller (P-97; Sutter Instruments, Novato, CA). Pipette resistances ranged from 5 to 7 MX and seal resistances were >1 GX. Patch electrodes were filled

Fig. 1. Peripheral inflammation during the early postnatal period strengthens excitatory synapses in the neonatal superficial dorsal horn (SDH). (A) Subcutaneous injection of carrageenan (0.7 ll/g at 2%) into the left hindpaw at P2 (i.e. day 0 after injection) produced significant tissue edema lasting at least 4 days, as evidenced by an increase in the ipsilateral paw diameter compared to the contralateral side (p < 0.001 compared to naïve; two-way ANOVA). (B) Examples of traces illustrating mEPSCs isolated at a holding potential of 70 mV (top) and mIPSCs recorded in the same neuron from a holding potential of 0 mV (bottom). (C) Carrageenan (CARR) treatment at P2 evoked a significant increase in mEPSC frequency (left; *p = 0.011; Mann–Whitney test), but not mEPSC amplitude (right), in P3–4 SDH neurons compared to naïve littermate controls. (D) Neither the mIPSC frequency (left) nor the mIPSC amplitude (right) at P3–4 was affected by peripheral inflammation from P2.

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with a solution containing the following (in mM): 130 Cs-gluconate, 10 CsCl, 10 Hepes, 11 EGTA, 1.0 CaCl2, and 2.0 MgATP, pH 7.2 (305 mOsm). Dorsal horn neurons were visualized with infrared-differential interference contrast (IR-DIC) and patch-clamp recordings were obtained as described previously [5] using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA). Miniature postsynaptic currents (mPSCs) were isolated via the bath application of 500 nM TTX, and mEPSCs were isolated at a holding potential (hp) of 70 mV. Under these conditions, mEPSCs are abolished by bath application of the selective AMPAR antagonist NBQX [4]. mIPSCs were recorded at a hp of 0 mV, thus minimizing the contribution of NMDA and AMPA/kainate receptor-mediated events [58]. In some experiments, EPSCs were evoked via focal electrical stimulation (0–100 lA, 100 ls duration) delivered by a the second patch electrode placed near the cell of interest which was connected to a constant-current stimulator (Master-8, Jerusalem, Israel). Alternatively, to selectively stimulate primary afferent inputs to the dorsal horn, in some instances transverse slices (400–600 lm) of spinal cord were cut using a modified Vibroslice to preserve the attached dorsal root. Primary afferent fibers were then stimulated (0–1 mA, 100 ls–5 ms duration) with a patch electrode placed onto the dorsal root, which was coupled to a constantcurrent stimulator as described above. In all experiments, the threshold to evoke an EPSC was defined as the current intensity (at a duration of 100 ls) which evoked a measurable EPSC in 50% of the trials. A-fiber-mediated EPSCs were classified as monosynaptic based on their ability to follow repetitive stimulation (5 stimuli at 2 threshold delivered at 10 Hz) with a constant latency and absence of failures. High-threshold EPSCs were considered monosynaptic if no failures were observed during 1 Hz stimulation. To investigate whether inflammation altered the probability of glutamate release in the dorsal horn, pairs of identical stimuli (at 2 threshold at a frequency of 0.10 Hz) were delivered at various interstimulus intervals (50–250 ms; 10 trials each), and the paired-pulse ratio (PPR) was calculated as PPR = mean EPSC2/mean EPSC1 in order to correct for spurious facilitation which can be caused by random amplitude fluctuations [31]. To examine the current–voltage relationship of evoked AMPAR-mediated currents, spermine was added to the intracellular solution at 100 lM, and EPSCs were evoked (at 2 threshold) from a variety of holding potentials ( 70 mV to +40 mV) in the presence of 50 lM AP-5, 10 lM gabazine and 0.5 lM strychnine to block NMDARs, GABAARs and glycine receptors, respectively. To quantify the degree of rectification, a rectification index (RI) was calculated as RI = I hp+40/I hp 40 as described previously [13]. To calculate the ratio of AMPAR/NMDAR currents, EPSCs were evoked from a hp of +50 mV at a frequency of 0.10 Hz in the presence of 10 lM gabazine and 0.5 lM strychnine. Upon verification of a stable baseline current amplitude, AP-5 was bath applied at 50 lM to block the NMDAR component of the composite current, and the NMDAR-mediated response was subsequently obtained via electronic subtraction. To examine the relative contribution of NMDARs containing the NR2B subunit to the overall NMDAR response, NMDAR-mediated currents were isolated at a hp of +50 mV in the presence of 10 lM NBQX, 10 lM gabazine and 0.5 lM strychnine (to block AMPARs, GABAARs and GlyRs, respectively). The degree to which the NMDAR current was inhibited by the NR2B-selective NMDAR antagonist Ro 25-6981 maleate was measured and compared between experimental groups. All recordings and subsequent analysis of synaptic function in the SDH were performed by an experimenter who was blinded to the identity (i.e. naïve or CARR) of the experimental subject. Membrane voltages were adjusted for liquid junction potentials (approximately 14 mV) calculated using JPCalc software (P. Barry, University of New South Wales, Sydney, Australia; modified for

Molecular Devices). Currents were filtered at 4–6 kHz through a 3 dB, four-pole low-pass Bessell filter, digitally sampled at 20 kHz, and stored on a personal computer (ICT, Cincinnati, OH) using a commercially available data acquisition system (Digidata 1440A with pClamp 10.0 software; Molecular Devices). 2.4. Drugs Tetrodotoxin, NBQX, D( )AP-5, Ro 25-6981 maleate, and SR95531 hydrobromide (gabazine) were purchased from Tocris (Ellisville, MO). Strychnine hydrochloride was obtained from Sigma (St. Louis, MO). All drugs were bath applied at 1.5–3 ml/min. Spermine was purchased from Fisher Scientific (Florence, KY) and added to the patch solution on the day of the experiment. 2.5. Data analysis and statistics mPSCs were analyzed via visual inspection using Mini Analysis (version 6.0.3; Synaptosoft, Decatur, GA) software, while evoked EPSCs were analyzed using Clampfit (Molecular Devices). The threshold for mPSC detection was set at twice the mean amplitude of the background noise. Because we have previously observed that mPSC frequencies often fail to exhibit a normal distribution in newborn dorsal horn neurons, in these cases we used nonparametric statistical tests to determine whether significant differences in mPSC frequency existed between various groups (Mann–Whitney test; Prism 5.0; GraphPad Software, La Jolla, CA). Nonparametric tests were also used in cases in which the number of observations was insufficient (n < 24) to definitively conclude that data were distributed in a Gaussian manner. Where parametric tests were appropriate, t-tests and repeated measure two-way ANOVAs (with Bonferroni post-tests) were used unless otherwise stated. Data are expressed as means ± SEM.

3. Results 3.1. Peripheral inflammation during the early postnatal period selectively potentiates glutamatergic synapses in the developing rat dorsal horn Subcutaneous injections of carrageenan (0.7 ll/g at 2%) into the plantar surface of the rat hindpaw at postnatal day (P) 2 evoked significant edema which lasted for at least 4 days, as clearly evidenced by an increase in the diameter of the ipsilateral hindpaw compared to the contralateral side (p < 0.001 compared to Naïve; two-way ANOVA; n = 15 in each group; see Fig. 1A). Approximately 24–48 h after the injection, spinal cord slices were prepared from carrageenan(CARR)-treated pups or naïve littermates to allow for in vitro patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) in neonatal superficial dorsal horn (SDH) neurons at holding potentials of 70 mV and 0 mV, respectively (Fig. 1B). All sampled neurons were located in the SDH as judged by visual inspection and located between 50–150 lm from the edge of the dorsal white matter, suggesting that the majority of these neurons resided in lamina II [38]. As demonstrated in Fig. 1C, peripheral inflammation with carrageenan significantly increased mEPSC frequency at P3–4 compared to naïve controls (Naïve: 0.19 ± 0.03 Hz, n = 34; CARR: 0.28 ± 0.03 Hz, n = 34; p = 0.011; Mann–Whitney test) without significantly changing mEPSC amplitude (Naïve: 13.45 ± 1.24 pA; CARR: 15.29 ± 1.07 pA; p = 0.076; Mann–Whitney test). In contrast, CARR at P2 failed to alter mIPSC frequency (Naïve: 0.11 ± 0.01 Hz; CARR: 0.12 ± 0.02 Hz; p = 0.972; Mann–Whitney test) or amplitude (Naïve: 13.42 ± 0.71 pA; CARR: 15.31 ± 0.93 pA; p = 0.107; t-test) in

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the same neurons (Fig. 1D), suggesting a selective effect of tissue injury on glutamatergic synaptic function in the neonatal SDH. To determine if the facilitation in excitatory signaling following inflammation during the early postnatal period persisted beyond the duration of the original tissue damage, carrageenan was administered at P2 as described above and synaptic function in the SDH was subsequently characterized at P10–11, at which time the inflammatory response had largely subsided (Fig. 1A). We observed no difference in mEPSC frequency (Naïve: 0.28 ± 0.05 Hz, n = 26; CARR: 0.24 ± 0.03 Hz, n = 24; p = 1.0; Mann–Whitney test) or mEPSC amplitude (Naïve: 10.96 ± 0.63 pA; CARR: 11.43 ± 0.91 pA; p = 0.604; Mann–Whitney test) between the experimental groups (data not shown), suggesting that the injury-induced differences in glutamatergic synaptic strength had resolved by this time point. Inflammation at P2 also failed to modulate mIPSC properties at P10–11, as no significant differences were seen in the mean mIPSC frequency (p = 0.858) or mIPSC amplitude (p = 0.884) between groups (data not shown). To test the hypothesis that the consequences of tissue injury for synaptic efficacy in the SDH depend on postnatal age, we evoked a similar peripheral inflammation at later ages (P9 or P17) and subsequently characterized excitatory and inhibitory synaptic function in the SDH. Unilateral subcutaneous injections of carrageenan (0.7 ll/g at 2%) into the hindpaw during the second and third postnatal weeks produced an equivalent degree of paw edema as that seen following CARR injections at P2 (ratio of ipsilateral to contralateral paw diameter: P2: 1.55 ± 0.05, n = 15; P9: 1.52 ± 0.04, n = 5; P17: 1.53 ± 0.09, n = 4; data not shown). However, in contrast to the effects of carrageenan treatment during

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the first postnatal week, inflammation at P9 failed to potentiate glutamatergic signaling in the SDH at P10–11, as there were no significant differences in mEPSC frequency (Naïve: 0.50 ± 0.09 Hz, n = 34; CARR: 0.56 ± 0.09 Hz, n = 32; p = 0.492; Mann–Whitney test) or mEPSC amplitude (Naïve: 10.64 ± 0.69 pA; CARR: 11.68 ± 1.04 pA; p = 0.542) between the groups (Fig. 2A). In addition, the properties of mIPSCs were unchanged by tissue injury at this age (p = 0.555 for mIPSC frequency; p = 0.387 for mIPSC amplitude; Fig. 2B). Similarly, carrageenan at P17 did not significantly alter excitatory synaptic transmission in the SDH, as mean mEPSC frequency (Naïve: 1.33 ± 0.19 Hz, n = 33; CARR: 1.31 ± 0.20 Hz, n = 40; p = 0.614; Mann–Whitney test) and mEPSC amplitude (Naïve: 13.14 ± 0.61 pA; CARR: 12.63 ± 0.65 pA; p = 0.567; t-test) were similar between the groups at P18–20 (Fig. 2C). As seen at earlier ages (Figs. 1D and 2B), spontaneous synaptic inhibition in the SDH was not significantly affected by tissue damage during the third postnatal week (p = 0.306 for mIPSC frequency; p = 0.565 for mIPSC amplitude; Mann–Whitney tests; see Fig. 2D). Overall, the results suggest that the first postnatal week represents a sensitive period of postnatal development during which excitatory, but not inhibitory, synaptic strength in the SDH is modulated by tissue injury. 3.2. Tissue damage does not alter the paired-pulse ratio (PPR) of evoked EPSCs in the immature SDH An elevation in the frequency, but not amplitude, of mEPSCs after carrageenan treatment during the early postnatal period could be explained by an increase in the number of neurotransmitter

Fig. 2. Peripheral inflammation during the second or third postnatal week fails to modulate synaptic efficacy in the rat superficial dorsal horn. Hindpaw injections of carrageenan at P9 had no significant effects on spontaneous excitatory (A) or inhibitory (B) synaptic signaling in P10–11 SDH neurons. Synaptic function at P18–20 was similarly unaffected by carrageenan treatment at P17 (C and D), suggesting that the ability of tissue injury to facilitate glutamatergic synaptic efficacy is restricted to the early postnatal period.

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release sites and/or by an increased probability of glutamate release in the dorsal horn under pathological conditions. To investigate whether peripheral inflammation alters the probability of transmitter release (Pr) at primary afferent synapses within the neonatal SDH, CARR injections were administered at P2 and transverse spinal cord slices were subsequently prepared at P3–4 with the dorsal roots left attached. Pairs of electrical stimuli were delivered to the dorsal root at various interstimulus intervals (ISI; 50– 250 ms) and we measured the paired-pulse ratio (PPR = mean EPSC2/mean EPSC1) of primary afferent-evoked monosynaptic EPSCs (Fig. 3A). First, in order to verify that the PPR was indeed sensitive to manipulations in Pr under our experimental conditions, we measured the effect of lowering [Ca2+]external from 2.0 to 0.8 mM on the observed PPR at a variety of ISI. As would be expected from a reduction in Pr, the amplitude of the evoked EPSCs decreased in the presence of lower [Ca2+]external (from 150.1 ± 41.1 pA to 70.5 ± 29.5 pA for EPSC1; n = 7; p = 0.016; Wilcoxon signed-rank test), while the PPR was significantly increased in the same neurons (2 mM Ca2+: 0.55 ± 0.06; 0.8 mM Ca2+: 1.25 ± 0.25; at 50 ms ISI; n = 7; p = 0.011; Mann–Whitney test). A similar increase in PPR in the presence of reduced [Ca2+]external was observed across the range of ISIs examined (p = 0.0015; RM two-way ANOVA; data not shown). In contrast, there were no significant differences in the PPR of primary afferent-evoked EPSCs between the naïve and carrageenan groups at any ISI examined (Naïve: n = 18; CARR: n = 23; p = 0.908; RM two-way ANOVA; see Fig. 3B). Of course, a sizeable proportion of the mEPSCs recorded in P3–4 SDH neurons (Fig. 1B) will reflect glutamate release from intrinsic excitatory interneurons within the SDH, which would not be sampled in the above experiments evoking monosynaptic responses from the dorsal root. As a result, in order to address whether peripheral inflammation significantly modulated the probability of glutamate release from the terminals of these excitatory interneurons, we repeated the above paired-pulse analysis using focal stimulation within lamina II through a second patch electrode located near the cell of interest (see Section 2). Although the nature of focal extracellular stimulation makes it difficult to definitively identify the source of the presynaptic input, it should be noted that the average threshold for evoking monosynaptic EPSCs in immature SDH cells was approximately 50-fold lower when using focal stimulation within the SDH compared to the stimulation of primary afferents in the dorsal root (primary afferent: 2448 ± 546 lA, n = 41; focal: 47.6 ± 4.0 lA, n = 42; at 100 ls duration; p < 0.0001; Mann–Whitney test; see Fig. 3C). This is consistent with the stimulation of two different populations of synaptic inputs to neonatal SDH neurons, and is also consistent with the notion that focal stimulation within lamina II will preferentially activate cell bodies in the area, which have significantly larger diameters (and thus lower electrical thresholds) compared to axonal fibers. However, as was observed using primary afferent stimulation, carrageenan at P2 failed to alter the PPR of focally evoked EPSCs in P3–4 SDH neurons across a wide range of ISIs (Naïve: n = 23; CARR: n = 19; p = 0.691; RM two-way ANOVA; Fig. 3D). In summary, we can find no evidence that the enhanced mEPSC frequency produced by peripheral inflammation during the first postnatal week is accompanied by significant changes in the probability of glutamate release in the SDH under pathological conditions, suggesting that tissue injury may increase the number of glutamate release sites in the immature dorsal horn. 3.3. Carrageenan decreases the inward rectification of AMPARmediated currents in SDH neurons throughout the postnatal period

Fig. 3. Increased strength of excitatory SDH synapses after early tissue injury does not result from alterations in the probability of glutamate release within the neonatal dorsal horn. (A) Representative traces showing monosynaptic EPSCs evoked in a P3 dorsal horn neuron following paired electrical stimulation of the attached dorsal root. Ten pairs of stimuli were delivered (at 2 threshold) at each interstimulus interval (ISI; 50–250 ms), the traces averaged, and the paired-pulse ratio (PPR) calculated as mean EPSC2/mean EPSC1 at each ISI for a given neuron. (B) There were no significant differences in the mean PPR of primary afferent-evoked EPSCs between the naïve and carrageenantreated littermates across the range of ISIs examined. (C) Significantly lower stimulus intensities (at a duration of 100 ls) were required to evoke an EPSC in P3–4 SDH neurons using focal stimulation within the SDH compared to the use of primary afferent (i.e. dorsal root) stimulation (***p < 0.0001; Mann–Whitney test), suggesting the recruitment of different populations of synaptic inputs onto SDH neurons. (D) Inflammation at P2 also failed to alter the PPR of focally evoked EPSCs across the range of ISIs at P3–4.

Calcium-permeable AMPARs are expressed in the SDH and have been demonstrated to facilitate spinal nociceptive plasticity and

inflammatory hyperalgesia in the adult [23]. Increases in the expression of Ca2+-permeable AMPARs are accompanied by en-

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hanced inward rectification of AMPAR-mediated synaptic currents [13,53]. To determine if peripheral inflammation with carrageenan significantly increases the inward rectification of AMPAR-mediated responses in neonatal SDH neurons, as is known to occur in young adult lamina I neurons after hindpaw inflammation with cFA [53], we characterized the current–voltage relationship of AMPAR-mediated EPSCs in P3–4 SDH neurons following carrageenan injections at P2. Fig. 4A illustrates representative EPSCs evoked by focal stimulation at different holding potentials (from 70 to +40 mV). Sur-

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prisingly, we found that carrageenan treatment produced a slight but significant decrease in the inward rectification of AMPAR-mediated currents, as demonstrated by the significantly higher current amplitudes seen at a holding potential of +40 mV (n = 24; p < 0.001; RM two-way ANOVA) compared to naïve littermate controls (n = 24; see Fig. 4B). In addition, we quantified the degree of AMPAR rectification in each neuron by calculating a rectification index (RI), defined as RI = I hp+40/I hp 40 [13]. As shown in Fig. 4C, carrageenan injections at P2 evoked a significant increase in this rectification index at P3–4 (Naïve: 0.74 ± 0.05, n = 24; CARR: 0.89 ± 0.05, n = 24; p = 0.027; Mann–Whitney test). Finally, to determine if this reduction in the inward rectification of AMPARmediated currents is restricted to injuries occurring at early postnatal ages, we performed similar analyses in P18–20 SDH neurons following CARR injections at P17 as described above. We observed a similar decrease in AMPAR inward rectification following tissue injury during the third postnatal week (Naïve: n = 25; CARR: n = 23; p < 0.05; RM two-way ANOVA; Fig. 4D), suggesting that a subset of synaptic modifications following peripheral inflammation may occur throughout postnatal development. 3.4. Peripheral inflammation during the first postnatal week does not modulate AMPA/NMDA ratio or the contribution of NR2B-containing NMDARs in the neonatal SDH Alterations in the ratio of AMPAR-mediated to NMDAR-mediated currents may have important functional implications for the activity-dependent regulation of synaptic strength in CNS neurons [15]. To examine whether tissue injury acutely shifts the AMPA/NMDA ratio in developing SDH neurons, we measured the amplitude of AMPAR and NMDAR responses in P3–4 dorsal horn cells in the absence or presence of carrageenan injections at P2. EPSCs were evoked from a holding potential of +50 mV via focal stimulation in the presence of antagonists for GABAA and glycine receptors (see Section 2). The AMPARmediated component of the evoked response was isolated via the bath application of the selective NMDAR antagonist AP-5 (50 lM) and electronic subtraction used to visualize the NMDAR-mediated component of the composite current (Fig. 5A). We found that peripheral inflammation at P2 did not significantly change the AMPA/NMDA ratio at P3–4 (Naïve: 0.72 ± 0.10, n = 19; CARR: 0.65 ± 0.08, n = 21; p = 0.871; Mann– Whitney test; Fig. 5B). Previous work has demonstrated that peripheral inflammation reduces the relative contribution of the NR2B subunit to the overall NMDAR response in young adult lamina I neurons [53], which may reflect the increased phosphorylation of this subunit under inflammatory conditions [20,21]. To determine if similar changes occur in the neonatal SDH following tissue damage, we characterized the

3 Fig. 4. Carrageenan treatment decreases the inward rectification of AMPARmediated currents in SDH neurons throughout the first three postnatal weeks. (A) Example of protocol used to measure the rectification of AMPAR-mediated responses in SDH neurons. Monosynaptic EPSCs were evoked (at 2 threshold) from a variety of holding potentials ( 70 to +40 mV) via focal stimulation within the SDH in the presence of selective antagonists to NMDARs, GABAARs and glycine receptors (GlyRs). Each sweep represents the average of 10 evoked EPSCs. (B) Current–voltage plot of the normalized amplitude of AMPAR-mediated currents (normalized to the mean current amplitude observed at a holding potential of 70 mV) as a function of holding potential. Carrageenan injections at P2 significantly decreased the inward rectification of AMPAR currents in P3–4 SDH neurons compared to naïve littermate controls (***p < 0.001; RM two-way ANOVA). (C) The mean rectification index (defined as I hp+40/I hp 40) of AMPAR-mediated currents at P3–4 was significantly increased by carrageenan treatment at P2 compared to naïve controls (*p = 0.027; Mann–Whitney test). (D) Similar plot as described in (B), showing that carrageenan injections at P17 also significantly decreased the inward rectification of AMPAR responses in P18–20 SDH cells (*p < 0.05; RM two-way ANOVA).

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potential of +50 mV. However, Ro 25-6981 blocked a similar percentage of the NMDAR-mediated current in naïve and carrageenan-treated pups (Naïve: 63.1 ± 4.1%, n = 13; CARR: 71.7 ± 3.6%, n = 14; p = 0.139; Mann–Whitney test; see Fig. 5D), suggesting that the function of NR2B subunits at neonatal SDH synapses is not significantly modulated by tissue injury during early life. 4. Discussion These results demonstrate that peripheral inflammation during the first days of life leads to a selective increase in the strength of excitatory synapses in the immature superficial dorsal horn (SDH). This potentiation in glutamatergic efficacy is not observed following a similar tissue injury during the second or third postnatal week, which agrees with the previous work showing that inflammation fails to alter spontaneous excitatory neurotransmission in the mature dorsal horn [3]. In contrast, while a reduction in the efficacy of inhibitory SDH synapses has been implicated in inflammatory hyperalgesia at later stages of postnatal development [24,26,43], we find no evidence that tissue damage decreases inhibitory synaptic strength in the newborn dorsal horn. Collectively, the data indicate that the SDH network responds to tissue damage in an age-dependent manner, and thus suggest that the synaptic modifications which drive central sensitization may be developmentally regulated. 4.1. Potential mechanisms underlying the facilitation of glutamatergic SDH synapses after early tissue injury

Fig. 5. Tissue damage during the early postnatal period does not significantly modulate AMPA/NMDA ratios or NR2B function in neonatal dorsal horn neurons. (A) Example of AMPAR-mediated (black) and NMDAR-mediated (gray) EPSCs evoked by focal stimulation from a holding potential of +50 mV in the presence of GABAAR and GlyR antagonists. The NMDAR component was obtained by electronic subtraction of the AP-5-resistant current from the total evoked response (see Methods). Each sweep represents the average of 10 evoked EPSCs. (B) Carrageenan at P2 had no significant effect on the ratio of AMPAR-to-NMDAR current amplitudes in P3–4 SDH neurons. (C) Representative plot illustrating the peak amplitude of pharmacologically isolated NMDAR current (from a holding potential of +50 mV) as a function of time in a neonatal SDH neuron. Bath perfusion of the NR2B-selective NMDAR antagonist Ro 25-6981 maleate (gray bar) evoked a rapid decrease in current amplitude with a maximal effect observed within 4–5 min. (D) Ro 25-6981 inhibited the NMDAR-mediated current to a similar degree in the naïve and carrageenan groups, suggesting that early tissue injury does not influence the relative contribution of the NR2B subunit to overall NMDAR function in the immature SDH.

sensitivity of the NMDAR-mediated current to the selective NR2B antagonist Ro 25-6981 (5 lM) in P3–4 pups receiving CARR injections at P2 compared to naïve controls. As illustrated in Fig. 5C, bath application of Ro 25-6981 led to a rapid decrease in the amplitude of pharmacologically isolated NMDAR currents observed at a holding

Our experiments demonstrate that peripheral inflammation during early postnatal development significantly increases the frequency, but not amplitude, of mEPSCs in neonatal SDH neurons (Fig. 1C). A selective enhancement of mEPSC frequency could potentially be explained by one or more of the following alterations in synaptic function within the dorsal horn: (1) an increased probability of glutamate release from the terminals of primary afferents and/or intrinsic excitatory interneurons; (2) an increased number of excitatory synapses or neurotransmitter release sites; or (3) a conversion from ‘silent’ (i.e. pure NMDAR-only) synapses, which are found in the dorsal horn during the first two postnatal weeks [2,6,34], to functional synapses via the insertion of AMPARs into the postsynaptic membrane. However, the present data argue against any significant changes in the probability of glutamate release within the immature SDH under inflammatory conditions, as carrageenan treatment failed to influence the paired-pulse ratio (PPR) of evoked EPSCs regardless of whether the electrical stimulation was delivered to the dorsal root or within lamina II itself (Fig. 3). In addition, a postsynaptic mechanism involving the insertion of AMPARs to previously NMDAR-only synapses seems unlikely to explain the increased mEPSC frequency after tissue injury, as we observed no significant difference in the ratio of AMPARmediated to NMDAR-mediated currents between the naïve and the inflamed groups (Fig. 5B). Nonetheless, we cannot completely exclude a role for ‘silent’ synapses in the observed effects since we did not directly examine their prevalence in our experiments. As a result, we hypothesize that peripheral inflammation during the first postnatal week increases the number of glutamatergic synapses and/or release sites in the developing dorsal horn. This could result from an expansion of nociceptive primary afferent projections to the SDH, which occurs following neonatal tissue damage but not after injuries at later postnatal ages [51,54]. Under normal conditions, C-fiber inputs to SDH neurons are significantly strengthened during the first ten postnatal days [4,18], suggesting that the increased sensory input following tissue injury may accelerate the ongoing development of C-fiber synapses in the region. Given that the sprouting of sensory fibers within the dorsal horn

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following mild tissue damage was found to be reversible [54], it is interesting to note that the elevation in mEPSC frequency described here is also transient in nature. Additional changes in the number of synapses formed by excitatory interneurons within the SDH may also contribute to the higher mEPSC rate observed after peripheral inflammation during early life. It is possible that damage to deep tissue (i.e. muscles or joints) during the early postnatal period could result in distinct effects on synaptic function within the developing SDH. Indeed, it is known that the repetitive activation of muscle afferents produces greater facilitation of the flexion withdrawal reflex compared to the activation of cutaneous inputs [41], demonstrating that the ability of sensory afferents to evoke activity-dependent changes in the excitability of the spinal cord depends on the identity of their peripheral targets. This could be addressed in future experiments using the well-established models of muscle and joint inflammation [29,47,52]. 4.2. Carrageenan alters AMPAR rectification in neonatal SDH neurons The inward rectification of AMPAR currents reflects a voltagedependent block of Ca2+-permeable, GluR2-lacking receptors by intracellular polyamines [10,16,25,55]. We observed a decrease in the inward rectification of AMPAR-mediated currents in SDH neurons following hindpaw inflammation with carrageenan throughout the early postnatal period (Fig. 4). This suggests an increased contribution of GluR2-containing receptors at developing SDH synapses, which has also been documented in cerebellar stellate neurons after repetitive stimulation [37]. The mechanisms underlying this shift in rectification properties are presently unclear. One possible contributing factor is the activation of metabotropic glutamate receptors (mGluRs), which decrease AMPAR inward rectification in other areas of the CNS [7] and are known to be expressed in the developing SDH [9,11]. The decreased inward rectification and expected reduction in the Ca2+ permeability of AMPARs expressed by SDH neurons after injury might protect the cells from excitotoxic damage [33] during the barrage of sensory input occurring at the peak of the inflammatory response. Our results may seem surprising given the previous observations that hindpaw inflammation with cFA increased the inward rectification of AMPAR responses at primary afferent synapses onto young adult (P16–31) lamina I neurons, which reflected an increased role of Ca2+-permeable AMPARs [53]. Considering that the present experiments characterized synaptic function in the SDH 1–2 days after the tissue injury compared to the 3 day post-injury period used in the cFA study, it is interesting that the mRNA for the GluR2-flip splice variant was found to be upregulated in the rat spinal cord within 1 day after inflammation but subsequently returned to control levels by day 3 [59]. This suggests the possibility that a shift in the subunit composition of AMPARs may be occurring in SDH neurons during the first days after tissue damage. However, it should also be noted that recent in vivo patchclamp experiments suggest that the increased inward rectification of AMPAR currents occurs in SDH cells within 24 h after inflammation with cFA [28]. Thus, an alternative possibility is that the observed effects on AMPAR rectification depend on the chosen model of tissue damage. Indeed, lipopolysaccharide (LPS) injection into the ankle joint decreases spinal GluR1 expression within 24 h [44], which would be predicted to decrease AMPAR inward rectification as reported here after carrageenan treatment. 4.3. Contribution of NR2B subunit to NMDAR function in the immature SDH under pathological conditions Significant evidence suggests that the subunit composition of NMDA receptors in the developing CNS can be rapidly modulated

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by sensory experience. For example, visual stimulation decreases the relative proportion of NMDARs containing the NR2B subunit in neurons of the visual cortex, with a concomitant upregulation of NR2A-containing receptors, within hours [45,46]. To our knowledge, the present study is the first to examine whether the subunit composition of NMDARs in the immature dorsal horn is similarly modulated by the level of sensory input occurring during the early postnatal period. The relative expression of NR2A vs. NR2B subunits in the dorsal horn has critical implications for spinal nociceptive processing, since the identity of the NR2 subunit profoundly influences the kinetics and Mg2+ sensitivity of NMDARs [42] and could also determine the direction of activity-dependent plasticity (i.e. LTP vs. LTD) [36]. In addition, the phosphorylation of spinal NR2B subunits has been implicated in both inflammatory and neuropathic pain in the adult [1,21]. Our data indicate that receptors containing the NR2B subunit represent a sizeable proportion of the NMDARs expressed at neonatal SDH synapses (Fig. 5D). This is in agreement with the previous observations that mRNA for the NR2B and NR2D subunits, but not for NR2A or NR2C, was highly expressed in the spinal cord during early postnatal development [42,56]. Recent evidence suggests that these subunits may be combined in a novel stoichiometry within neonatal SDH neurons [19]. The present results demonstrate that the relative contribution of NR2B-containing receptors to overall NMDAR signaling in the immature SDH is independent of fluctuations in sensory input, as peripheral inflammation failed to alter the percentage of NMDAR-mediated current blocked by a selective NR2B antagonist. However, we cannot exclude the possibility that a more rigorous characterization of NMDAR properties (such as Mg2+ sensitivity) in neonatal SDH neurons will reveal other changes in NMDAR subunit composition and/or function following peripheral inflammation during early life, which could have significant effects on spinal nociceptive processing. 5. Conclusions It is clear that the superficial dorsal horn (SDH) is organized in a fundamentally different manner during early life, as evidenced by the enlarged receptive fields, lower thresholds, and prolonged spike after-discharges that characterize neonatal dorsal horn neurons [18]. The present study extends these findings by demonstrating that the immature SDH network also exhibits distinct responses to tissue damage at the synaptic level. A complete understanding of how neonatal pain circuits in the CNS are shaped by noxious insults during early postnatal development is not only critical to improving the clinical treatment of pain in infants and children, but may also yield important insights into the ongoing question of how early trauma causes changes in pain sensitivity throughout life. Acknowledgements This work was supported in part by the University of Cincinnati Millennium Fund. The authors declare that they do not have a conflict of interest with any of the work presented in this manuscript. References [1] Abe T, Matsumura S, Katano T, Mabuchi T, Takagi K, Xu L, Yamamoto A, Hattori K, Yagi T, Watanabe M, Nakazawa T, Yamamoto T, Mishina M, Nakai Y, Ito S. Fyn kinasemediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. Eur J Neurosci 2005;22:1445–54. [2] Baba H, Doubell TP, Moore KA, Woolf CJ. Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord. J Neurophysiol 2000;83:955–62.

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