Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKM

Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKMζ in the Anterior Cingulate Cortex Xiang-Yao Li, et al. Science 330, 1400 (2010); DOI: 10.1126/science.1191792

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REPORTS 22. J. P. W. Letschert, thesis, Beta Section Beta: Biogeographical Patterns of Variation and Taxonomy (Wageningen Agricultural University Papers, 93-1, Wageningen, Netherlands 1993). 23. C. Jung, K. Pillen, L. Frese, S. Fähr, A. E. Melchinger, Theor. Appl. Genet. 86, 449 (1993). 24. We thank F. Domène, J. Bensefelt, A˚ . Karlsson, R. Sant, P. van Roggen, and A.-M. Nilsson for technical assistance, and Y. Fischer for help in preparing the manuscript. We also thank C. Bellini for critical reading of the manuscript. This work was supported by the Swedish Research Council and the Swedish Governmental Agency for Innovation Systems to O.N. and T.K., as well as by Südzucker. Phylogenetic data have been deposited with Dryad Digital Repository (DOI: 10.5061/dryad.4930).

Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKMz in the Anterior Cingulate Cortex Xiang-Yao Li,1,2* Hyoung-Gon Ko,3* Tao Chen,1,2* Giannina Descalzi,1 Kohei Koga,1 Hansen Wang,1 Susan S. Kim,1 Yuze Shang,1 Chuljung Kwak,3 Soo-Won Park,3 Jaehoon Shim,2,3 Kyungmin Lee,3,4 Graham L. Collingridge,2,5 Bong-Kiun Kaang,2,3† Min Zhuo1,2† Synaptic plasticity is a key mechanism for chronic pain. It occurs at different levels of the central nervous system, including spinal cord and cortex. Studies have mainly focused on signaling proteins that trigger these plastic changes, whereas few have addressed the maintenance of plastic changes related to chronic pain. We found that protein kinase M zeta (PKMz) maintains pain-induced persistent changes in the mouse anterior cingulate cortex (ACC). Peripheral nerve injury caused activation of PKMz in the ACC, and inhibiting PKMz by a selective inhibitor, z-pseudosubstrate inhibitory peptide (ZIP), erased synaptic potentiation. Microinjection of ZIP into the ACC blocked behavioral sensitization. These results suggest that PKMz in the ACC acts to maintain neuropathic pain. PKMz could thus be a new therapeutic target for treating chronic pain.

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ne key function of the brain is to learn and memorize sensory experiences and then adapt subsequent behavioral responses.

Investigations into the molecular mechanisms of such processes provide information for our basic understanding of brain functions (1). Synaptic

Nucleotide sequences have been deposited with GenBank under accession numbers HM448909 to HM448918 and HQ148107 to HQ148132. The use of BvFT1 and BvFT2 in control of sugar beet flowering is the subject of the patent application WO2010/025888.

Supporting Online Material www.sciencemag.org/cgi/content/full/330/6009/1397/DC1 Materials and Methods SOM Text Figs. S1 to S10 Tables S1 to S3 References 26 August 2010; accepted 22 October 2010 10.1126/science.1197004

plasticity is the major cellular model used for understanding these mechanisms. In addition to numerous studies on synaptic mechanisms for learning and memory, these same synaptic mechanisms may be responsible for pathological pain conditions (2–4). For example, nerve injury triggers long-term plastic changes along sensory pathways, from the peripheral sensory terminals to the spinal cord dorsal horn, amygdala, and cortex (3–7). These plastic changes contribute to pain perception, fear, and memory. The persistent nature 1 Department of Physiology, Faculty of Medicine, Center for the Study of Pain, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. 2Department of Brain and Cognitive Sciences, Seoul National University, Seoul 151 747, Korea. 3National Creative Research Initiative Center for Memory, Department of Biological Sciences, College of Natural Sciences, Seoul National University, San 56-1 Silim-dong, Gwanak-gu, Seoul 151-747, Korea. 4Department of Anatomy, School of Medicine, Kyungpook National University, 2-101 Dongin-Dong, Daegu 700-422, Korea. 5Medical Research Council (MRC) Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, UK.

*These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected] (B.-K.K.); [email protected] (M.Z.)

Fig. 1. Expression and phosphorylation of PKMz in the ACC during neuropathic pain. (A) Mechanical allodynia was tested in the sham and nerve injury groups from wild-type (WT) and AC1−/− animals 3 days after nerve injury. * indicates P < 0.05; error bars indicate SEMs. (B) Western blots for PKMz and p-PKMz in the ACC obtained between 3 days and 2 weeks after nerve injury. (The ACC of mice from the sham and nerve injury groups were used for Western blot analysis 90 min after the allodynia test.) (C) Level of PKMz in the ACC of mice from the sham and nerve injury groups. Level of PKMz increased significantly 3 days after nerve injury compared with PKMz in the sham group. (D) Level of p-PKMz increased significantly 3 days after nerve injury. This effect lasted over 2 weeks after nerve injury compared with p-PKMz in the sham group. (E) Peripheral nerve injury did not increase the levels of PKMz and p-PKMz in the ACC of AC1−/− mice. (F) Forskolin incubation increased PKMz (gray) and p-PKMz (blue) levels in the ACC of WT mice.

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12. D. Bradley, O. Ratcliffe, C. Vincent, R. Carpenter, E. Coen, Science 275, 80 (1997). 13. G. D. H. Bell, A. B. Bauer, J. Agric. Sci. 32, 112 (1942). 14. F. A. Abegg, J. Agric. Res. 53, 493 (1936). 15. Materials and methods are available as supporting material on Science Online. 16. P. Boudry et al., Theor. Appl. Genet. 88, 852 (1994). 17. S. Ohno, Evolution by Gene Duplication (Springer-Verlag, New York, ed. 1, 1970) 18. B. K. Blackman, J. L. Strasburg, A. R. Raduski, S. D. Michaels, L. H. Rieseberg, Curr. Biol. 20, 629 (2010). 19. H. Van Dijk, J. Exp. Bot. 60, 3143 (2009). 20. Y. Hanzawa, T. Money, D. Bradley, Proc. Natl. Acad. Sci. U.S.A. 102, 7748 (2005). 21. J. H. Ahn et al., EMBO J. 25, 605 (2006).

of chronic pain results in resistance to commonly used analgesics (6). Therefore, revealing the molecular mechanisms that are involved in the main-

tenance of pain-related long-term synaptic plasticity changes is critical for developing effective chronic pain treatments.

Fig. 2. Bilateral microinjections of ZIP into the ACC decreased allodynia in mice with peripheral nerve injury. (A) The schematic view of the microinjection experiments. (B) Location of microinjection sites in the ACC. Solid circles represent ZIP injection sites, whereas open circles represent saline injection sites. The right image shows an example of the location of the cannula tip in one ACC slice with hematoxylin and eosin staining. The injection site is indicated by an asterisk. (C) Microinjection of ZIP (blue) into the ACC decreased the positive response rate in the nerve injury animal, whereas saline microinjection (black) into the ACC had no effect on the response level. Open gray circles represent the summary control data of positive response level tested from the saline and ZIP group. The red arrows indicated the time point of ZIP injection. *P < 0.05; error bars, SEMs. (D) Two hours after injection, ZIP still has an analgesic effect. However, the allodynia response recovered to preinjection level 24 hours after injection. (E) ZIP has no effect on the traveled distance in the open field test. (F) The location of microinjection sites in the ACC in the conditioned place preference experiments. Solid or open circles indicate the location of the injection that did or did not induce place preference, respectively. (G) The changed time in the ZIP paired chamber was significantly different from that of the saline paired chamber. *P < 0.05. (H) The effect of ZIP infusion into the ACC on contextual fear memory. Representative brain slice shows the location of the injection site. ZIP was infused into the ACC 2 hours before memory retrieval, which was tested 3, 7, and 18 days after conditioning. (I) ZIP did not change the response latency in the tailflick (TF) test. (J) Microinjection of ZIP into the ACC had no effect on the paw-withdrawal threshold in normal mice. www.sciencemag.org

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Multiple protein kinases are thought to contribute to the induction of long-term potentiation (LTP) and initial consolidation of information storage. Among them, only protein kinase M zeta (PKMz) maintains persistent synaptic changes (8–10). PKMz, an atypical isoform of protein kinase C (PKC), has been detected in many regions of the brain, including the frontal cortex (11). In the hippocampus, LTP induction triggers the synthesis of PKMz, and activation of PKMz is critical for late-phase LTP (L-LTP) and memory consolidation (8, 12–15). The anterior cingulate cortex (ACC) is a key cortical area involved in chronic pain (2, 16, 17). We first examined whether, in the ACC, peripheral nerve injury causes changes in PKMz (18). As reported previously (17, 19), behavioral allodynic response was increased 3 days after nerve injury when compared with the response in sham-treated mice (Fig. 1A). The levels of PKMz in the ACC were significantly increased after nerve injury (sham-operated versus nerve injury group; n = 9 mice for each group, unpaired t test, t16 = –2.51, P < 0.05; Fig. 1, B and C). Because PKMz is activated by phosphorylation, we also conducted experiments to detect possible changes in the level of phosphorylated PKMz (p-PKMz). Consistently, the level of p-PKMz was also significantly increased (n = 8 for each group, unpaired t test, t14 = –2.26, P < 0.05; Fig. 1, B and D). These data suggest that peripheral nerve injury increases PKMz activity in the ACC. To determine whether such changes are longlasting, we examined PKMz and p-PKMz levels 7 and 14 days after nerve injury. Although behavioral allodynia persisted at these time points, the protein levels of PKMz returned to baseline (Fig. 1C), indicating that the regulation of the amount of PKMz is short-lasting. However, the p-PKMz level remained increased [two-way analysis of variance (ANOVA), F1,28 = 6.10, P < 0.05; Fig. 1D], suggesting that PKMz activity could contribute to the maintenance of neuropathic pain. To investigate whether the changes in PKMz protein levels or activity were a generalized phenomenon in the central nervous system, we also examined the levels of PKMz and p-PKMz in the hippocampus and spinal dorsal horn 3 days after nerve injury. PKMz and p-PKMz levels in the hippocampus and spinal cord did not change after nerve injury (fig. S1), suggesting that changes in PKMz are affected in a regionally specific manner in response to peripheral nerve injury. What could be the possible second messengers that trigger the activation and synthesis of PKMz in the ACC? In the ACC, calmodulinstimulated adenylyl cyclase 1 (AC1) is critical for ACC LTP and behavioral sensitization caused by peripheral nerve injury (16, 20). Thus, we examined whether AC1 acts upstream of PKMz in the ACC after nerve injury by using AC1 gene knockout mice (AC1−/−). AC1−/− mice did not show any significant behavioral sensitization 3 days after nerve injury (Fig. 1A). Similar amounts

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and regionally selective analgesic effect on nerve injury–related tonic pain. What is likely to be the synaptic mechanism responsible for the analgesic effects produced by ZIP in neuropathic pain? PKMz can potentiate postsynaptically the amplitude of a-amino-3hydroxy-5-methyl-4-isoxazole-propionate (AMPA)

receptor-mediated excitatory postsynaptic currents (EPSCs) (23). Because glutamatergic synaptic transmission in the ACC is increased after nerve injury (17), we speculated that PKMz may contribute to the maintenance of enhanced synaptic transmission induced by nerve injury. First, we recorded AMPA receptor-mediated EPSCs in layers II and


of PKMz and p-PKMz were found in shamoperated and nerve-injured AC1−/− mice 3 days after nerve injury (n = 9 for each group, unpaired t test, PKMz, t16 = –0.99, p-PKMz, t16 = –0.67, P > 0.05, Fig. 1E). To further examine the link between the AC1 and PKMz, we performed brain slice experiments. We used bath application of forskolin (5 mM), a cell-permeable activator of AC. Forskolin treatment increased the amount of PKMz and p-PKMz in a time-dependent manner (Fig. 1F). The increase in PKMz was rapid; the amount of PKMz was increased within 5 min after forskolin (5 mM) treatment (one-way ANOVA, F3,15 = 4.52, P < 0.05, n = 7 per group; Fig. 1F). This suggests that adenosine 3´,5´-monophosphate (cAMP) signaling increases PKMz levels in a transcription-independent manner, because transcription of PKMz pre-mRNA is likely to take more than 30 min (12, 21). So what could be the function of increased PKMz activity in the ACC? We wondered whether this increase contributes to behavioral sensitization after nerve injury. We performed intra-ACC microinjections of z-pseudosubstrate inhibitory peptide (ZIP) (10 nmol/ml, 0.5 ml per side), a selective cell-permeable PKMz inhibitor, in mice 3 days after nerve injury. Bilateral microinjections of ZIP into the ACC significantly reduced the allodynia (n = 8, paired t test, t7 = 8.78, P < 0.001; Fig. 2, A to C) unlike saline-injected controls (n = 6, paired t test, t 5 = 1.84, P > 0.05; Fig. 2C). Similar analgesic effects of ZIP were detected 7 days after nerve injury (n = 7, paired t test, t6 = 2.89, P < 0.05; Fig. 2C). The analgesic effects lasted for at least 2 hours, but there was recovery at 24 hours after the injection (n = 5, Fig. 2D). To determine whether the effect of ZIP is regionally selective, we microinjected ZIP into the primary somatosensory cortex. The same amount of ZIP did not produce any significant analgesic effect (paired t test, t3 = –2.30, P > 0.05, n = 4, fig. S2, A and B). To determine whether the effects of ZIP in ACC are sensory-selective, we examined the effects of this inhibitor in the open-field test and found no differences in the distance traveled between mice microinjected with ZIP or saline (Fig. 2E). Nerve injury can induce tonic pain (22). By using an injury-induced, conditioned place preference paradigm (22), we evaluated the effects of ZIP on nerve injury–related tonic pain. Microinjection of ZIP into the ACC also produced significant analgesic effects (n = 6, paired t test, t5 = –3.19, P < 0.05; Fig. 2, F and G). In addition, we examined whether ZIP infusion caused any memory impairment. When we infused the ACC with ZIP 2 hours before memory retrieval on 3, 7, or 18 days after contextual fear conditioning, we did not find any obvious defects in memory retrieval [n = 7 to 11 per group, unpaired t test, P > 0.05 in day 3 (t20 = 1.92), 7 (t16 = 1.56) or 18 (t13 = –1.75); Fig. 2H]. Moreover, microinjecting ZIP into the ACC did not affect the tail-flick reflex and the paw-withdrawal threshold in normal animals (Fig. 2, I and J). These data show that microinjection of ZIP into the ACC produces a modality

Fig. 3. Inhibition of PKMz selectively decreased the amplitude of eEPSCs in ACC neurons of mice with neuropathic pain by reducing the number of active AMPARs. (A and B) Samples showed the effect of ZIP (5 mM) on the amplitude of eEPSCs in the ACC neurons of animals from the nerve injury [black circles in (A)] and sham group [black circles in (B)]. The gray open circles represent the change of membrane resistance during recording. Black traces in the upper part of (A) and (B) indicate the averaged response at baseline, and blue traces indicate the average of 2 min responses collected 10 min after ZIP application. (C) Pooled data of effects of ZIP on the eEPSCs recorded from the ACC of mice in the sham (open) and nerve injury (solid) groups 10 min after ZIP application. *P < 0.05; error bars, SEMs. (D) The variance-current relationships of the eEPSCs recorded with (red) or without (black) CNQX (0.5 mM) for a representative neuron. (E) Voltage dependence of the single channel currents. Open gray circles indicate the individual data collected from nine neurons. (F) Example of peak-scaled NSFA performed on the eEPSCs before and after ZIP application from a neuron in a mouse with nerve injury. (G) ZIP reduced the numbers of active channels selectively in mice with nerve injury. The white and red bars represent the baseline of the sham and nerve injury groups, respectively. The blank blue and solid blue bars indicate the percent change in the number of active channels in sham and nerve injury, respectively, groups 10 min after bath application of ZIP. (H) Digitized photomicrograph showing neurons in laminae II of the spinal cord, which were recorded by stimulating dorsal roots. (I) Example eEPSCs from neurons in lamina II of spinal cord after Ad or C fiber stimulation. (J) The amplitudes of eEPSCs recorded in nerve injury mice were significantly larger than those in control mice at 1.5 (medium) and 2 times (high) the threshold (low)-stimulating intensity (sham, n = 11; nerve injury, n = 14; one-way ANOVA, F1,97 = 10.0, P < 0.01, Tukey test was used for the multiple comparisons). (K) Pooled data showed that ZIP had similar effects on EPSCs recorded in the spinal cord of nerve injury and sham mice.

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III of the ACC 3 or 7 days after nerve injury (24). After obtaining a stable baseline, we bath-applied ZIP (5 mM) to block PKMz activity. The evoked EPSCs (eEPSCs) were significantly decreased in six of seven experiments, and total mean responses were reduced to 83 T 6% (mean T SEM) of baseline after 10 min of bath application of ZIP (n = 7; Fig. 3, A and C). In contrast, ZIP did not affect the amplitude of eEPSCs recorded in neurons from sham-operated mice (n = 6; Fig. 3, B and C). The ZIP-induced reduction of eEPSCs may be due to a decrease in either the AMPA receptor unitary conductance (g) or number of active channels. Peak-scaled nonstationary fluctuation analysis (NSFA) has been developed to distinguish between these parameters (23, 25, 26). As a control, we performed manipulations that should affect either the number of active channels or the single-channel current amplitude but not the single-channel conductance. Similar to previous reports (26), NSFA was able to detect a reduction in the number of active channels induced by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (0.5 mM) (n = 4, paired t test, t3 = 6.60, P
0.05). In both cases, there was a reduction in sensory transmission (Fig. 3K), which may be due to a constitutive activity of PKMz in spinal sensory neurons. Consistent with these findings, intrathecal injection of ZIP (2 nmol/ml, 5 ml per animal) did not produce any analgesic effect (n = 4, paired t test, t3 = 0.52, P > 0.05). The ACC is a heterogeneous cortical area, in which not all neurons would be expected to respond to neuropathic pain. We therefore used transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the promoter of the c-fos gene (27, 28), because c-fos is known to be activated in sensory neurons after injury (29). In these mice, we found that many

Fig. 4. PKMz activity maintains L-LTP in the ACC. (A) Fos-immunopositive cells were found in layers II, III, V, and VI in the ACC of mice 7 days after nerve injury. (B) NeuN immunostaining in the ACC, with dashed line showing the structure of the ACC. The framed area in (A) is magnified in (a). (b) is the magnified image of (B). (c) is the merged result of (a) and (b). Yellow color in (c) represents the Fos/NeuN double-labeled neurons. Scale bar indicates 100 mm in (A) and (B) and 30 mm in (a) to (c). (C) The total number of Fos-positive neurons were counted in the layers II and III on both sides of the ACC (n = 5 mice in each group). *P < 0.001; error bars indicate SEMs. (D) An example of the recorded FosGFP-negative neuron from the ACC of the sham-treated mice. (E) An example showing the double-patched FosGFP-negative (1) and -positive (2) neurons from the ACC of the nerve injury group. (a) images show the expression of FosGFP; (b) images indicate the recorded neurons that were labeled by Alexa fluor 594. Yellow color in (Eb) indicates the overlap of GFP and Alexa fluor 594. Scale bar, 20 mm. (F) Pooled data showing the input-output curves of FosGFP-positive neurons from nerve injury mice (dark red) are different from FosGFP-negative neurons of nerve injury (red) and sham-treated (black) mice. Representative eEPSCs recorded from FosGFP-negative neurons (a) of sham-treated or nerve injury (b) mice or FosGFP-positive (c) neurons of nerve injury mice. (G and H) Pooled data showing the effects of ZIP (5 mM) on FosGFP-negative [black and (a)] neurons of sham-treated mice and FosGFP-negative [light blue and (b)] and FosGFP-positive [dark blue and (c)] neurons of nerve injury mice. Representative waveforms indicated eEPSCs before (red) and after ZIP (blue) application. *P < 0.05. (I) L-LTP of the field EPSP induced by the TBS in the ACC can last for at least 5 hours. L-LTP from naïve (n = 6) and sham-operated mice (n = 5) were similar, and data were pooled together (black). Blocking of the activity of PKMz by bath application of ZIP (5 mM) for 1 hour can reverse the synaptic potentiation (blue). L-LTP was occluded in nerve injury mice (red). Sample traces showing the field EPSP recorded at 0.5 hours before TBS conditioning (a) and 2.5 hours (b) and 4.5 hours (c, 0.5 hours after ZIP incubation) after TBS conditioning in the control, ZIP treatment, and nerve injury groups. The blue bar indicates the time scale of ZIP application. (J) Summary data showing ACC L-LTP 4 hours after the induction with ZIP treatment or nerve injury. White, control with TBS conditioning; blue, TBS conditioning with ZIP incubation; red, nerve injury with TBS conditioning. *P < 0.05.

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ACC neurons were activated 3 and 7 days after nerve injury (Fig. 4, A to C). We performed wholecell patch-clamp recordings from FosGFP-positive pyramidal cells (“green” cells) that were activated by allodynic pain and compared these with nonactivated neighboring neurons and neurons in sham-operated FosGFP mice (Fig. 4, D and E). The amplitudes of eEPSCs stimulated at the same intensity in the FosGFP-positive neurons were significantly greater than those of FosGFPnegative neurons in the ACC of nerve injury and sham-operated mice (two-way ANOVA, F2,59 = 17.25, P < 0.05, Fig. 4F). ZIP (5 mM) significantly reduced the amplitude of eEPSCs (n = 7, paired t test, t6 = 2.85, P < 0.05; Fig. 4, G and H) in FosGFP-positive pyramidal cells but had no effect on eEPSCs in either the FosGFP-negative neurons from the same nerve injury group or in neurons from the sham group (n = 5, nerve injury, t4 = –0.63; n = 6, sham, t5 = –1.45; paired t test, P > 0.05; Fig. 4, G and H). Furthermore, ZIP had no effect on the eEPSCs recorded on the FosGFP-positive neurons from the primary somatosensory cortex of mice with nerve injury (n = 6, paired t test, t5 = –0.55, P > 0.05; fig. S2C). In contrast to PKMz, cAMP-dependent protein kinase (PKA) is not required for the maintenance of enhanced EPSCs in the FosGFP-positive neurons, because the PKA inhibitor KT5720 (1 mM) did not affect EPSCs (n = 5, paired t test, t4 = –0.32, P > 0.05; fig. S4). These data suggest that neurons in the ACC that are affected by neuropathic pain have enhanced excitatory synaptic transmission and that this is expressed by a mechanism that is independent of PKA but involves persistent activation of PKMz. Cortical LTP has been proposed as a cellular model for chronic pain (2). One key function of PKMz is to maintain cortical potentiation at individual synapses (23). To test whether PKMz

may be required for maintaining L-LTP in the ACC, we recorded LTP in ACC slices. We induced LLTP by using a theta burst stimulation (TBS) protocol. The slope of the excitatory postsynaptic potential (EPSP) was significantly greater 3 hours after induction (n = 11, Fig. 4I). Bath application of ZIP (5 mM) 3 hours after induction significantly reduced the potentiation (n = 6; Fig. 4, I and J), suggesting that PKMz is required for the maintenance of L-LTP in the ACC. To test whether nerve injury–triggered plasticity shares some common mechanism with L-LTP in the ACC, we performed occlusion experiments. Indeed, synaptic potentiation was significantly reduced in slices of nerve injury mice as compared with those of control mice (unpaired t test, t12 = 3.52, P < 0.05; Fig. 4, I and J). We found that PKMz is critical for the maintenance of long-term plasticity in the ACC and contributes to neuropathic pain by modulating excitatory synaptic transmission within this cortical structure. Its role in maintaining synaptic potentiation is a critical part in allodynia resulting from nerve injury. Therefore, PKMz in ACC synapses provides a potential new therapeutic target for the treatment of chronic pain. References and Notes 1. E. R. Kandel, Science 294, 1030 (2001). 2. M. Zhuo, Trends Neurosci. 31, 199 (2008). 3. A. V. Apkarian, M. N. Baliki, P. Y. Geha, Prog. Neurobiol. 87, 81 (2009). 4. M. Costigan, J. Scholz, C. J. Woolf, Annu. Rev. Neurosci. 32, 1 (2009). 5. H. Ikeda et al., Science 312, 1659 (2006). 6. A. Latremoliere, C. J. Woolf, J. Pain 10, 895 (2009). 7. F. Wei, P. Li, M. Zhuo, J. Neurosci. 19, 9346 (1999). 8. T. C. Sacktor, Prog. Brain Res. 169, 27 (2008). 9. E. A. Drier et al., Nat. Neurosci. 5, 316 (2002). 10. P. V. Migues et al., Nat. Neurosci. 13, 630 (2010). 11. M. U. Naik et al., J. Comp. Neurol. 426, 243 (2000). 12. A. I. Hernandez et al., J. Biol. Chem. 278, 40305 (2003). 13. T. V. Bliss, G. L. Collingridge, S. Laroche, Science 313, 1058 (2006).

Micro-Optical Sectioning Tomography to Obtain a High-Resolution Atlas of the Mouse Brain Anan Li,* Hui Gong,* Bin Zhang,* Qingdi Wang, Cheng Yan, Jingpeng Wu, Qian Liu, Shaoqun Zeng, Qingming Luo† The neuroanatomical architecture is considered to be the basis for understanding brain function and dysfunction. However, existing imaging tools have limitations for brainwide mapping of neural circuits at a mesoscale level. We developed a micro-optical sectioning tomography (MOST) system that can provide micrometer-scale tomography of a centimeter-sized whole mouse brain. Using MOST, we obtained a three-dimensional structural data set of a Golgi-stained whole mouse brain at the neurite level. The morphology and spatial locations of neurons and traces of neurites could be clearly distinguished. We found that neighboring Purkinje cells stick to each other. ne of the most important aims in neuroscience is to obtain an interconnection diagram of the whole brain. In mammals, individual neurons are considered the basic units

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of the brain, but the complex functions of the brain depend more on the fine anatomical architecture of a very large number of neurons and their connections (1). Although modern neuroscience

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Supporting Online Material www.sciencemag.org/cgi/content/full/330/6009/1400/DC1 Materials and Methods Figs. S1 to S4 References 3 May 2010; accepted 18 October 2010 10.1126/science.1191792

has made great progress in brain studies at both the system and cellular levels, our empirical knowledge of neuroanatomical connectivity remains inadequate, limiting the progress of brain studies (2). Thus, it is necessary to gain new insights into the morphology, localization, and interconnectivity of neural circuits throughout the whole brain at an appropriate resolution. Individual synapses are the finest functional element in circuits, but it is currently not technologically feasible to study the brainwide connectivity of complex vertebrate organisms (e.g., mice) at the synaptic level. In contrast, mesoscale techniques are currently more feasible and applicable for understanding specific neural functions (2). Light microscopy has remained a key tool for neuroscientists to observe cellular properties at Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics–Huazhong University of Science and Technology, Wuhan 430074, P. R. China. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]

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