Long-Term Potentiation of Glycinergic Synapses Triggered by Interleukin 1Β

Long-term potentiation of glycinergic synapses triggered by interleukin 1β

Anda M. Chirila1, Travis E. Brown1,2, Rachel A. Bishop, Nicholas W. Bellono, Francesco G. Pucci, and Julie A. Kauer3

Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912

Edited* by Gina G. Turrigiano, Brandeis University, Waltham, MA, and approved April 18, 2014 (received for review January 17, 2014)

Long-term potentiation (LTP) is a persistent increase in synaptic become apparent only when GABAAR and GlyRs are pharma- strength required for many behavioral adaptations, including learn- cologically blocked, indicating that under conditions of disinhibi- ing and memory, visual and somatosensory system functional devel- tion, nonnoxious mechanical stimuli can drive nociceptive-specific opment, and drug addiction. Recent work has suggested a role for projection pathways and elicit allodynia (21). The majority of LTP-like phenomena in the processing of nociceptive information in neurons tested in the dorsal horn receive glycinergic synapses, in- the dorsal horn and in the generation of central sensitization during cluding lamina I projection neurons, both excitatory and inhibitory chronic pain states. Whereas LTP of glutamatergic and GABAergic interneurons of lamina II (22, 23), and inhibitory glycinergic neurons synapses has been characterized throughout the central nervous (24). Given the diversity of afferent targets, it is likely that glycinergic system, to our knowledge there have been no reports of LTP at mam- synapses are differentially modulated in a cell type- and subregion- malian glycinergic synapses. Glycine receptors (GlyRs) are structurally specific manner. For example, during chronic inflammation, pros- related to GABAA receptors and have a similar inhibitory role. Here we taglandin E2 selectively depresses glycinergic synaptic inputs onto report that in the superficial dorsal horn of the spinal cord, glycinergic nonglycinergic neurons (24). Similarly, peripheral nerve injury sup- synapses on inhibitory GABAergic neurons exhibit LTP, occurring rap- presses glycinergic inhibition of a specific excitatory interneuron + idly after exposure to the inflammatory cytokine interleukin-1 beta. class [protein kinase C (PKC)γ neurons receiving Aβ fiber inputs], This form of LTP (GlyR LTP) results from an increase in the number allowing excitatory afferents carrying nonnociceptive tactile infor- and/or change in biophysical properties of postsynaptic glycine recep- mation to activate ascending projections of nociceptive pathways tors. Notably, formalin-induced peripheral inflammation in vivo poten- that are normally under strong inhibitory control (23). tiates glycinergic synapses on dorsal horn neurons, suggesting that Both hyperalgesia and allodynia occur within minutes of pe- GlyR LTP is triggered during inflammatory peripheral injury. Our ripheral inflammation, but the mechanisms underlying these results define a previously unidentified mechanism that could disin- rapid perceptual alterations are poorly understood. The proin- hibit neurons transmitting nociceptive information and may represent flammatory cytokine, IL-1β, is a potent hyperalgesic agent (25– a useful therapeutic target for the treatment of pain. 27), contributing both to peripheral and central sensitization after tissue damage (28–31). Following tissue trauma, nerve in- lycine mediates fast synaptic inhibition throughout the spi- jury, or inflammation, IL-1β levels are up-regulated in the spinal Gnal cord, brainstem, and midbrain, controlling normal mo- cord itself (29, 32, 33), and delivery of IL-1β intrathecally in- tor behavior and rhythm generation, somatosensory processing, creases the activity of superficial dorsal horn neurons that transmit auditory and retinal signaling, and coordination of reflex respon- pain signals to the brain (34, 35). Intrathecal delivery of an IL-1 ses (1). Strychnine-sensitive glycine receptors (GlyRs) are pen- receptor antagonist blocks allodynia in rodent models of inflam- tameric ligand-gated chloride channels of the Cys-loop receptor matory pain (36, 37). A recent study also found that IL-1β ap- family that together with GABAA receptors (GABAARs) dynam- plication rapidly potentiated primary afferent (glutamatergic) ically interact with the synaptic scaffold protein gephyrin to form synapses in dorsal horn slices, through unidentified signaling inhibitory synapses (1, 2). In the dorsal horn of the spinal cord, glycinergic synapses are essential for nociceptive and tactile sensory Significance NEUROSCIENCE processing both during adaptive and pathological pain states (3–7). However, compared with glutamatergic and GABAergic synapses, Whereas glycine is one of the three major neurotransmitters in little is known about the regulation of their synaptic strength. the central nervous system, glycinergic synapses are relatively Several studies have examined glycine receptor trafficking in understudied in intact tissue. Here we provide the first evi- cultured neurons and in heterologous expression systems (8, 9). + dence of long-term potentiation (LTP) at mammalian glycinergic Intracellular Ca2 appears important in the stabilization of GlyRs at 2+ synapses. In the spinal cord dorsal horn, glycinergic synapses on synapses in culture (10), and elevation of intracellular Ca can also inhibitory GABAergic neurons exhibit LTP, occurring rapidly after potently increase glycine receptor single channel openings in cultured exposure to the inflammatory cytokine interleukin-1 beta. This cells and in heterologous systems (11). However, the modulation form of LTP is mediated by postsynaptic alterations in glycine of glycinergic synaptic strength in native tissue remains relatively receptor function. We further show that peripheral inflam- unexplored. mation in vivo triggers glycine receptor LTP. Blocking glycine Following peripheral injury or inflammation, changes in tactile receptor LTP may represent a useful therapeutic strategy in the perception develop, including hyperalgesia (exaggerated pain upon treatment of inflammatory pain. noxious stimulation), allodynia (pain in response to innocuous stimuli), and secondary hyperalgesia (pain spreading beyond the Author contributions: A.M.C., T.E.B., and J.A.K. designed research; A.M.C., T.E.B., R.A.B., confines of the injured region). Inhibitory interneurons of the N.W.B., and F.G.P. performed research; A.M.C., T.E.B., R.A.B., and N.W.B. analyzed data; spinal dorsal horn have been proposed to gate the flow of innoc- and A.M.C., T.E.B., and J.A.K. wrote the paper. uous and nociceptive sensory information from the periphery to The authors declare no conflict of interest. higher brain centers (12), and supportive evidence for this idea is *This Direct Submission article had a prearranged editor. growing (13–17). Loss of GABAergic/glycinergic inhibition con- 1A.M.C. and T.E.B. contributed equally to this work. tributes to enhanced transmission of nociceptive signals through 2Present address: School of Pharmacy, University of Wyoming, Laramie, WY 82071. the dorsal horn circuit during pain states, resulting in hyperalgesia 3To whom correspondence should be addressed. E-mail: [email protected]. – and allodynia (3, 18 20). For example, polysynaptic A-fiber inputs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. onto neurokinin 1 receptor (NK1R)-expressing projection neurons 1073/pnas.1401013111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1401013111 PNAS | June 3, 2014 | vol. 111 | no. 22 | 8263–8268 Downloaded by guest on September 30, 2021 IL-1β Mediates a Postsynaptic Form of LTP. After brief IL-1β ap- ABplication, IPSC potentiation persisted for the duration of the 500 300 recording, suggesting that it represents a novel form of LTP. To rule out the possibility that residual IL-1β in the slice persistently 400 IL-1β IL-1β activated its receptors even upon wash out, we followed IL-1β 200 300 application with an antagonist of the IL-1 receptor (IL-1RN). Blocking the IL-1 receptor had no effect on established poten- 200 ± – β 100 tiation (IPSC amplitudes: 194.4 24.9% of pre IL-1 values n = P < A B 100 following IL-1RN application; 7; 0.005) (Fig. 2 and ), supporting the idea that IL-1β elicits persistent LTP at glycinergic

Normalized GlyR IPSCs Glycinergic IPSCs (pA) 0 0 synapses. The same concentration of IL-1RN entirely prevented 0 10 20 30 40 50 60 70 0 10 20 30 40 50 β Time (min) Time (min) potentiation when applied before IL-1 , as expected if the potentiation is mediated through the canonical IL-1 receptor (IPSC amplitudes 14–20 min after IL-1β application in the continued C D GAD65+ cells presence of IL-1RN: 100.3 ± 6.9% of control values; n = 5; not significant, n.s.) (Fig. 2 C and D). * * 500 300 Synaptic potentiation can be maintained by a long-lasting increase in neurotransmitter release, or instead by modifications 400 IL-1β IL-1β in postsynaptic receptor number or properties. To test whether 200 300 IL-1β increases postsynaptic glycine receptor sensitivity, we delivered exogenous glycine in the bathing medium instead of using 200 100 evoked glycine release from nerve terminals. In this experiment, 100 any potentiation must result from enhanced postsynaptic glycine GAD65+ cells receptor function or number. Pulses of exogenous glycine elicited

Glycinergic IPSCs (pA) 0 Normalized GlyR IPSCs 0 reproducible inward currents in the recorded neuron. Within 2 min 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 β Time (min) Time (min) of IL-1 application, however, glycine-induced inward currents increased and remained potentiated for the remainder of the re- Fig. 1. IL-1β induces rapid LTP of glycinergic synapses on GAD65-EGFP cording (125.3 ± 10.8% of control glycine induced current; n = 8, positive neurons. (A) Representative experiment illustrating that GlyR IPSCs BSA control; n = 6, IL-1β; P < 0.05) (Fig. 3 A and B). To test the in lamina II neurons are rapidly potentiated by addition of IL-1β (10 ng/mL) to effects of IL-1β on single quanta, we recorded miniature IPSCs β the ACSF bathing the spinal cord slice (IL-1 application denoted by blue bar). (mIPSCs) in GAD65-EGFP–labeled lamina II cells. Exposure to (Insets) Averages of 12 IPSCs before and at 20 min after IL-1β application. β ± Calibration: 60 pA, 5 ms. (B) Average responses of nine neurons. Error bars IL-1 increased quantal amplitudes significantly (132.6 12.6% of indicate mean ± SEM. (C) A single example showing that GlyR IPSCs recorded exclusively from GABAergic lamina II cells identified by expression of GAD65- EGFP robustly potentiate in response to IL-1β. Calibration for this inset and in all subsequent figures except where noted: 50 pA, 10 ms. (D) Average of 24 A B cells. (Inset) Differential interference contrast image and fluorescence image of a GAD65 positive neuron in an acutely prepared spinal cord slice. 800 300 IL-RN IL-RN IL-1β IL-1β 600 molecules released from glial cells (38). Here we report that 200 IL-1β rapidly elicits a postsynaptic form of long-term potentiation 400 (LTP) at glycinergic synapses on lamina II inhibitory neurons 100 (GlyR LTP), and that the same glycinergic synapses are potentiated 200 after peripheral inflammation.

Glycinergic IPSCs (pA) 0 Normalized GlyR IPSCs 0 Results 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 Time (min) Time (min) IL-1β Potentiates Glycinergic Synapses on Lamina II GABAergic Interneurons. We recorded evoked glycinergic synaptic currents from neurons in lamina II of the dorsal horn in mouse spinal C D cord slices. Bath application of IL-1β rapidly potentiated glyci- 1200 300 nergic inhibitory postsynaptic currents (IPSCs) (IPSC ampli- IL-RN IL-RN tudes: 160.8 ± 11.6% of pre–IL-1β values, n = 7, P < 0.005) (Fig. 1000 IL-1β IL-1β 1 A and B). In contrast, little or no potentiation of either glu- 800 200 tamatergic or GABAergic synapses was observed in similar 600 experiments after application of IL-1β, indicating that IL-1β se- 400 100 lectively modulates glycinergic synapses on lamina II neurons (Fig. S1). Although a proportion of the neurons in lamina II 200

Glycinergic IPSCs (pA) are GABAergic, glutamatergic interneurons predominate (39). To 0 Normalized GlyR IPSCs 0 0102030 40 50 60 0 10 20 30 40 50 focus on GABAergic lamina II interneurons, we recorded specif- Time (min) Time (min) ically from enhanced green fluorescent protein (EGFP)-labeled GABAergic neurons expressing glutamatic acid decarboxylase 65 Fig. 2. The IL-1 receptor mediates GlyR LTP. (A) Representative example β (GAD65) (40) (Fig. S2). We confirmed that IL-1β produced rapid showing that after potentiation by IL-1 , GlyR IPSCs remain potentiated even in the presence of the IL-1 receptor antagonist, IL-1RN (2 mg/mL). (B) Aver- and robust potentiation of glycinergic currents in this group of age of seven experiments. (C ) Single experiment illustrating that bath ap- identified GABAergic neurons (IPSC amplitudes: 165.0 ± 11.1% plication of IL-1RN before IL-1β addition entirely prevented potentiation. (D) of pre–IL-1β values, n = 24, P < 0.0001) (Fig. 1 C and D). Average of five experiments. Error bars indicate mean ± SEM.

8264 | www.pnas.org/cgi/doi/10.1073/pnas.1401013111 Chirila et al. Downloaded by guest on September 30, 2021 postsynaptic IL-1 receptors, followed by downstream activation 150 * AB) GLY l

o of the p38 MAPK pathway (41). Consistent with this model, bath GLY GLY GLY GLY rtn

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IL-1β c% 100 blocked IL-1β–induced GlyR LTP in GABAergic lamina II neurons

( tne (IPSC amplitude: 103.0 ± 10.6% of pre–IL-1β values, n = 8; n.s.),

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ucen whereas its inactive analog, SB202474, had no effect (205.0 40.6% 50 of pre–IL-1β values; n = 4; P < 0.05) (Fig. 4C).

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G 0 Activation of P2X7 Receptors Releases IL-1β to Potentiate Glycinergic Synapses on GABAergic Lamina II Cells. IL-1β is secreted from IL-1β CDControl microglia and astrocytes in response to peripheral inflammation Control IL-1β (42–45), and IL-1β release in the spinal cord can be elicited by activation of purinergic P2X7 receptors (45). We hypothesized * )lort 150 that when P2X7 receptors are activated, IL-1β released from glia

no within the spinal cord slice should induce GlyR LTP. As pre- c% dicted, glycinergic synapses did potentiate after brief exposure to (ed 100 the P2X7 receptor agonist, Bz-ATP (IPSC amplitudes: 135.0 ±

ut – – n = ilpm 13.0% of pre Bz-ATP values at 20 26 min after Bz-ATP, 11, 50 P < 0.05) (Fig. 4D). Preexposure to an IL-1β scavenging protein

C (IL-1Trap) entirely prevented the potentiation by Bz-ATP (IPSC SP ± – I amplitudes: 94.0 6.3% of pre-Bz-ATP values at 20 26 min m 0 after Bz-ATP in the continuous presence of IL-1Trap, n = 5, IL-1β Control n.s.), as expected if Bz-ATP potentiates glycinergic synapses via the release of IL-1β (Fig. 4E). Moreover, Bz-ATP application in P2X7 receptor null mice, animals known to have attenuated inflammatory pain (42), had no effect on glycinergic synapses Fig. 3. IL-1β potentiates glycinergic synapses by increasing postsynaptic A (IPSC amplitude 92.8 ± 8.0% of pre–Bz-ATP values, n = 4, n.s.) (Fig. responses to glycine. ( ) Bath application of glycine (1 mM, 30 s) induces an F inward current that is reproducible; a 2-min application of IL-1β triggers 4 ). These results suggest that even in the thin slice preparation, rapid potentiation of exogenous glycine responses. Calibration: 100 pA, activating endogenous P2X7 receptors releases sufficient amounts of 2min.(B) Averages of six experiments for IL-1β application; eight experiments IL-1β to potentiate glycinergic synapses. for BSA control experiments. Experiments for A and B did not use gad2-EGFP mice and therefore may include some non-GABAergic cells. (C) Miniature In Vivo Peripheral Inflammation Is Correlated with GlyR LTP. We next + glycinergic IPSCs in gad2 neurons were pharmacologically isolated and asked whether peripheral inflammation triggers potentiation of recorded before and after a 10-min exposure to IL-1β. Calibration: 50 pA, 100 glycinergic synapses on GABAergic dorsal horn neurons. Inflam- D β ms. ( ) Miniature IPSC amplitudes were significantly increased after IL-1 .Error mation was produced in the hind paws of mice by injecting for- bars indicate mean ± SEM. malin, whereas control mice received similar injections of saline, and we measured thermal and mechanical sensitivity. Thirty minutes control amplitudes, measured at 4–24 min after the start of IL-1β after injection, thermal sensitivity of saline-injected animals was application; n = 8; P < 0.05) (Fig. 3 C and D). IL-1β also had no at basal levels, whereas formalin-injected mice exhibited thermal A hyperalgesia consistent with inflammation (secondary hyperalgesia) effect on the paired-pulse ratio (Fig. S3 )oronmIPSCkinetics ± [mean rise time 1.21 ± 0.08 ms vs. 1.20 ± 0.05 ms following IL-1β; [paw withdrawal latency 35 min postsaline injection: 107 12.9% of τ ± ± baseline; n = 19; postformalin injection: 63.3 ± 7.7% of baseline, n.s.; mean decay time constant ( decay)3.69 0.23 ms vs. 3.91 ’ F = P < β one-way ANOVA with Bonferroni stest, (3, 32) 5.235, 0.005; 0.24 ms following IL-1 , n.s.]. Together, our data indicate that IL- NEUROSCIENCE n = 19] (Fig. 5A). Paw edema was notably increased in the formalin- 1β potentiates glycinergic synapses by increasing glycine re- treated group (paw weight/body weight ratio for the saline-treated ceptor number or function specifically in postsynaptic lamina group: 0.02 ± 0.001; n = 24; formalin-treated group: 0.03 ± 0.002; II interneurons. n = 27, P < 0.0005) (Fig. 5B). Mechanical hypersensitivity (allodynia) was also significantly greater 35 min after formalin [two-way Signaling Events Underlying IL-1β–Mediated GlyR LTP. Postsynaptic + ANOVA, F = 4.158, P < 0.05, Bonferroni post hoc analysis at Ca2 can increase glycine receptor-mediated currents, both by (1, 60) minute 35 (n = 16, P < 0.05)] (Fig. 5C). At 90 min postinjection, modulating open channel probability (11) and by increasing the animals from both groups were killed and spinal cord slices pre- dwell time of glycine receptors at synapses (10). We therefore 2+ pared. GABAergic neurons from saline-treated mice showed robust tested whether intracellular Ca is necessary for GlyR LTP by β 2+ GlyRLTPuponbathapplicationofIL-1 . In contrast, in cells from chelating intracellular Ca in the postsynaptic GABAergic formalin-treated mice, glycinergic synaptic currents did not poten- lamina II cell. Inclusion of BAPTA in the recording pipette tiate with IL-1β (IPSC amplitudes: saline-injected: 176.6 ± 16.7%, ± – β entirely blocked GlyR LTP (94.5 4.6% of pre IL-1 values; n = 9; formalin-injected: 104.1 ± 10.5%, n = 7; P < 0.005) (Fig. 5D). n = 5; n.s.), confirming our hypothesis that a rise in postsynaptic 2+ This result is consistent with the hypothesis that inflammation-in- intracellular Ca in the cell undergoing GlyR LTP is necessary duced release of IL-1β in vivo had already maximally potentiated β A for IL-1 to potentiate glycinergic synapses (Fig. 4 ). Further- (occluded LTP) at these glycinergic synapses. If so, we would expect 2+ more, exposure to thapsigargin, which depletes intracellular Ca an increase in mIPSC amplitude during inflammation like that seen stores, also prevented IL-1β–induced potentiation (IPSC amplitude: after exogenously applied IL-1β (Fig. 3 C and D). Miniature IPSC 106.9 ± 11.8% of pre–IL-1β values; n = 8; n.s.) (Fig. 4B). Taken + amplitudes in GAD65 positive neurons from formalin-treated mice together, these data suggest that intracellular Ca2 microdomains were markedly larger than those from saline-treated mice (mIPSC + provide a necessary rise in postsynaptic Ca2 concentration re- amplitudes, formalin-treated animals: 148.7 ± 12.4% of saline- quired to trigger GlyR LTP. treated control animals; n = 8, formalin; n = 5, saline; P < 0.05) (Fig. How does IL-1β regulate GlyR function at postsynaptic sites? 5 E–G). Together, these data support our hypothesis that inflam- The rapid induction of GlyR LTP indicates that IL-1β might mation in vivo potentiates glycinergic synapses on lamina II modulate glycinergic synaptic strength directly by binding to GABAergic neurons.

Chirila et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 8265 Downloaded by guest on September 30, 2021 ABCSB202474 SB 203580

200 Intracellular BAPTA 200 Thapsigargin 300 IL-1β IL-1β IL-1β

200

100 100 Fig. 4. GlyR LTP in GABAergic lamina II neurons + 100 requires intracellular Ca2 and p38 MAPK and can be induced by an activator of P2X7 receptors. (A)The + Ca2 chelator, BAPTA, was delivered into the post-

Normalized GlyR IPSCs Normalized GlyR IPSCs Normalized GlyR IPSCs 0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 synaptic cell via the pipette solution; 20 min after Time (min) Time (min) Time (min) initiating the recording, IL-1β application does not potentiate GlyR IPSCs. (B) Bath-applied thapsigargin D E F P2X7 -/- (10 μM) blocks GlyR LTP. (C) The p38 MAPK antagonist, SB203580 (filled symbols; 20 μM) but not its 200 200 IL1-Trap 200 inactive analog (SB202474, open symbols; 20 μM) Bz-ATP Bz-ATP Bz-ATP blocks GlyR LTP. (D) The P2X7 receptor agonist, Bz- ATP (100 μM), potentiates glycinergic IPSCs on GAD65 positive lamina II neurons. (E) Preapplication 100 100 100 of the IL-1β scavenger, IL-1Trap (1.32 nM) prevents Bz-ATP–induced potentiation. (F) Bz-ATP fails to potentiate glycinergic IPSCs in lamina II neurons from P2X7 null mice. These experiments did not use

Normalized GlyR IPSCs

Normalized GlyR IPSCs 0 0 Normalized GlyR IPSCs 0 0 10 20 30 40 50 0 10 20 30 40 0 10 20 30 40 GAD65-EGFP mice and therefore may include some Time (min) Time (min) Time (min) non-GABAergic cells. Error bars indicate mean ± SEM.

Discussion GlyR LTP, and ATP released in the dorsal horn as a result of LTP at glutamatergic C-fiber synapses onto lamina I projection peripheral inflammation may therefore mediate the local release neurons has been extensively studied as a cellular mechanism of IL-1β that modifies synaptic strength in vivo. accompanying hyperalgesia (18, 46–48), but relatively little is known about plasticity at dorsal horn inhibitory synapses (49). Glycine Receptor Trafficking and Channel Properties Modulated by 2+ 2+ To our knowledge, the LTP we describe here is the first example Ca . The regulation of glycine receptor trafficking by Ca - of LTP at glycinergic synapses in the mammalian CNS. Our dependent processes has been described using elegant single par- results from GABAergic lamina II neurons indicate a post- ticle tracking approaches (10, 50, 51). IL-1β can rapidly increase 2+ synaptic locus of potentiation, requiring either increased num- intracellular Ca in astrocytes and neurons (52, 53). Combining bers of postsynaptic glycine receptors, or long-lasting modulation these observations, we speculate that after IL-1β activates the + + of glycine channel function. Intracellular Ca2 in the post- IL-1R, there is a resulting rise in intracellular Ca2 that in- synaptic cell and p38 MAPK are both required for GlyR LTP. creases the scaffolding of glycine receptors to gephyrin at synaptic + Finally, we found that potentiation of the same glycinergic syn- sites. GlyR LTP may require Ca2 -mediated glycine receptor de- + apses accompanies peripheral inflammation, suggesting a possi- livery to synapses, as chelating postsynaptic Ca2 was sufficient to ble role for GlyR LTP in inflammatory pain. Local release of impair LTP induction. Alternatively, IL-1β may increase channel endogenous IL-1β elicited with an ATP analog also triggered open probability rather than number; glycinergic currents and

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0350 35 0350 35 N cinergic IPSCs on lamina II GABAergic neurons. (A)In- Saline Formalin Saline Formalin Saline Formalin 0 jection of formalin (red bars) but not saline (white bars) 0 10 20 30 40 into each hind paw induces secondary hyperalgesia Time (min) 35 min postinjection. (B) Formalin injection significantly E FG increases paw edema at 90 min. (C) Formalin injection Saline Formalin 200 decreases paw withdrawal threshold to von Frey fila- )%(eduti * 100 ments. (D) GABAergic lamina II neurons in slices prepared

ytilibabo from saline-treated mice (white) exhibit GlyR LTP in re-

lpmaCS sponse to IL-1β, whereas in slices from formalin-treated 100 mice, LTP is occluded (red). (E and F) Glycinergic miniature rp.muC 50 IPSCs recorded from gad2+ neurons from formalin-treated PIm mice (red) are larger than those from saline-treated mice. Calibration: 20 pA, 100 ms. (G) Cumulative probability 0 0 20 40 60 80 histogram comparing glycinergic IPSC amplitudes in sa- Saline mIPSC amplitude (pA) line-treated (black) and formalin-treated mice (red). Error Formalin bars indicate mean ± SEM.

8266 | www.pnas.org/cgi/doi/10.1073/pnas.1401013111 Chirila et al. Downloaded by guest on September 30, 2021 + single-channel openings are transiently increased by Ca2 (11), circuit is complex, and more work will be required to fully un- or by a number of neuromodulators downstream of intracellular derstand the role of GlyR LTP in nociception; in particular, we + Ca2 , including PKC, calcium/calmodulin-dependent protein need to determine which other dorsal horn neurons can also kinase II, and endocannabinoids (11, 54–62). Similar signaling undergo GlyR LTP. processes may mediate LTP described at goldfish Mauthner cell Levels of IL-1β are up-regulated in a variety of rodent pain glycinergic synapses, where underlying mechanisms have not been models over a time course of days or weeks, and it is possible that investigated (63). glycine receptor levels in lamina II interneurons temporally fol- low IL-1β expression. Glycine receptors have been suggested as IL-1β and GlyR LTP. The most extensively studied signaling cascades an important component of pain signaling that might be ame- downstream from the IL-1 receptor generally lead to transcription nable to drug therapy. However, blocking all glycine receptors factor activation and altered gene expression. The time course of with intrathecal administration of strychnine, for example, pro- GlyR LTP requires instead a rapid signaling pathway likely to in- motes pain hypersensitivity (70). Whereas this global block of volve p38 MAPK, a kinase strongly implicated in pain processing in glycine receptors in the entire network degrades somatosensory the dorsal horn, and which rapidly enhances voltage-gated sodium processing and elicits pain, modulation of synaptic strength β currents in nociceptors in response to IL-1 (31). Although our data in specific selected circuit elements provides a powerful tool β cannot rule out the possibility that IL-1 releases another signaling available to control signal flow without affecting all neurons at molecule that itself up-regulates glycine receptors in neurons, as once. Although global glycine receptor blockade is not a viable suggested for excitatory dorsal horn synaptic potentiation (38), the strategy for treating pain (3, 70), allosteric modulation of GlyRs β rapid time course of GlyR LTP makes this less likely. IL-1 can (1, 6, 71) or the signaling cascade that underlies GlyR LTP may modulate synaptic currents in unidentified superficial dorsal horn yield novel drug targets useful in treating intractable pain. neurons, transiently increasing (64) or decreasing IPSC amplitudes (35), and synaptic potentiation of excitatory C-fiber synapses on Methods β lamina I neurons requires the release of IL-1 (38) during peripheral Electrophysiology. Lumbar spinal cord slices (L4–L6) were prepared from C57Bl6 inflammation. Furthermore, intrathecal activation of glial cells mice, GAD65-EGFP mice, and P2X7−/− mice (P15–P32) and were perfused at produces mechanical allodynia and thermal hyperalgesia within ∼2 mL/min at room temperature with artificial cerebrospinal fluid (ACSF). 20 min, which are prevented by blocking IL-1 receptors in- Whole-cell voltage-clamp experiments were made from lamina II neurons using trathecally (65). GlyR LTP may contribute to the rapid nociceptive patch pipettes filled with a KCl-based internal solution. For glycinergic IPSCs, effects of intrathecal IL-1β (34, 36), a scenario consistent with the ACSF contained: 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μM) and bicucul- μ block of mechanical hyperalgesia by IL-1RN (66), the IL-1 receptor line (10 M), to block AMPA and GABAA receptors; the remaining synaptic μ B antagonist that blocked GlyR LTP in our experiments. currents were entirely blocked by 1 M strychnine (Fig. S3 ). For excitatory postsynaptic currents, ACSF contained picrotoxin (100 μM). Glycine-mediated Inhibitory Synapses in Nociception. There has been considerable IPSCs were stimulated at 0.1 Hz using a stainless steel stimulating electrode placed lateral to the recording site within lamina II. mIPSC were recorded at interest in the idea that the removal of GABAergic/glycinergic 2+ 2+ −70 mV in ACSF at 28–30 °C with 4 mM Sr replacing Ca ,andeightstimuliat inhibition contributes to nociception and central sensitization 40 Hz were used to elicit asynchronously released strontium mIPSCs. Asynchro- resulting from inflammation, peripheral nerve damage, or exper- nous mIPSCs were measured during the 400-ms period following the synchro- imental C-fiber excitation (2, 3, 6, 12, 21). Dorsal horn functional nous response and analyzed by an experimenter blind to animal treatment. circuitry is incompletely mapped out, but lamina II GABAergic interneurons are ideally located to gate the flow of nociceptive Behavior. Mice were tested for latency to paw flick or licking behavior on information from the periphery to supraspinal areas. Previous a hot plate (53 °C) or for paw withdrawal using von Frey filaments; mea- studies have shown that these interneurons receive both high- surements were made before a formalin (0.25 cc, 5%) or saline plantar in- threshold and low-threshold primary afferent input (67), and jection into both hind paws. Saline-treated animals were interleaved with innervate NK1R-expressing projection neurons (21, 68), there- formalin-treated littermates. N values represent the number of animals; one fore being positioned to provide inhibitory control over dorsal slice per animal was used for slice experiments.

β NEUROSCIENCE horn nociceptive circuitry. We hypothesize that IL-1 released ± from microglia or astrocytes rapidly potentiates glycinergic syn- Analysis and Statistics. Data are presented as means SEM of the percent change in IPSC amplitude. Potentiation was measured at 14–20 min fol- apses on GABAergic lamina II interneurons that normally inhibit lowing application of IL-1β or other manipulation except as noted. Signifi- the passage of nociceptive signals to the brain; after inflammation, cance was determined using a two-tailed Student t test or one-way ANOVA the nociception-transmitting neurons will in turn be rapidly dis- with a significance level of P < 0.05. mIPSC amplitudes were statistically inhibited via this mechanism, enhancing the perceived pain and compared using the Student t test and Kolmogorov–Smirnoff test. contributing to hyperalgesia and allodynia (44). GlyR LTP may operate in tandem with LTP at excitatory primary afferent syn- ACKNOWLEDGMENTS. We thank Drs. Edward Perl, Clifford Woolf, Jihong apses, causing disinhibition and opening the “gate” to enhanced Zheng, and Robert Gereau for assistance with developing spinal cord slice protocols, and Drs. Diane Lipscombe and Abigail Polter for advice on the project pain signaling, both by unmasking A-fiber inputs onto lamina I and manuscript. This research was supported by DA011289 (to J.A.K.) and and III projection neurons (21), and reducing the excitatory NS074612 (to T.E.B.) from Brown University, as well as by a pilot grant from the drive to GABAergic lamina II neurons (69). The dorsal horn Brown Institute for Brain Science and seed funds from Brown University.

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