This page has been archived and is no longer updated
Genetic enhancement of inflammatory pain by forebrain NR2B overexpression.
Author: Wei, F., et. al.
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"164 nature neuroscience ? volume 4 no 2 ? february 2001 articles NMDA receptors, which are expressed at excitatory synapses throughout the central nervous system (CNS), mediate a wide range of brain processes, including the synaptic plasticity asso- ciated with memory formation 1 , neuronal death in ischemia 2 and central sensitization during persistent pain 3?6 . Functional NMDA receptors contain heteromeric combinations of the NR1 subunit, plus one or more of NR2A?D 7?8 , of which the NR2A and NR2B subunits predominate in forebrain structures 9 . NR2A and NR2B subunits confer distinct properties to NMDA recep- tors; heteromers containing NR1 plus NR2B mediate a current that decays three to four times more slowly than receptors com- posed of NR1 plus NR2A 9 . At birth, forebrain NMDA receptors are composed almost exclusively of NR1 and NR2B subunits, gradually incorporating more NR2A subunits during postnatal development 10 . This developmental decrease in the forebrain NR2B:NR2A ratio, complete by the third or fourth postnatal week in rodents, parallels a decrease in the duration of NMDA recep- tor-mediated excitatory postsynaptic currents (EPSCs) 11?13 . This developmental change also parallels a decrease in sensitivity to formalin-induced pain 14 . In transgenic mice with forebrain-targeted NR2B overex- pression, this developmental change in NMDA receptor kinetics is reversed 15 . NR2B subunit expression, driven by the ?-cal- cium/calmodulin-dependent protein kinase II (?-CaMKII), is observed extensively in adult transgenic mice throughout the cerebral cortex, striatum, amygdala and hippocampus, but not in the thalamus, brainstem or cerebellum. Presumably by increasing the NR2B:NR2A ratio in NMDA receptor het- eromeric complexes, marked alterations occur in the physiology of mature hippocampal synapses, including a prolongation of NMDA receptor-mediated EPSCs and an enhancement of long- term potentiation. Adult NR2B transgenic mice exhibit superior performance on a battery of learning- and memory-related behavioral tasks, compared to wild type adults. Why, then, is the NR2B:NR2A ratio decreased during development, if learning and memory are hindered as a result? We proposed that fore- brain-targeted NR2B overexpression would have more broad effects on an animal?s behavior than reported previously, including altered responses to tissue injury and inflammation. RESULTS First, we examined how forebrain-targeted NR2B overexpression affected NMDA receptor function in two pain-related forebrain areas, the anterior cingulate cortex (ACC) and insular cortex. We prepared brain slices of these areas from adult mice, and we recorded excitatory postsynaptic field potentials (fEPSPs) upon local electrical stimulation 16,17 . In each region, after blockade of AMPA and kainate receptors by CNQX (20 �M), we observed a slow fEPSP (Fig. 1a) that could be entirely and reversibly blocked by the NMDA receptor antagonist AP-5 (100 �M; Fig. 1b). Con- sistent with the high levels of NR2B transgene expression found throughout the cerebral cortex in transgenic mice 15 , these mice, compared to wild type mice, exhibited enhanced NMDA receptor- mediated fEPSPs in both the ACC and insular cortex (Fig. 1c). In contrast, the spinal cord dorsal horn exhibited no change in NMDA receptor-mediated synaptic transmission. Intracellular recordings were done from dorsal horn neurons in slices from adult mice, and EPSPs were evoked by dorsal root stimulation. Slow AP-5- sensitive EPSPs, isolated in the presence of CNQX (20 �M; Fig. 2a), had similar slopes in wild type and transgenic slices Genetic enhancement of inflammatory pain by forebrain NR2B overexpression Feng Wei, Guo-Du Wang, Geoffrey A. Kerchner, Susan J. Kim, Hai-Ming Xu, Zhou-Feng Chen and Min Zhuo Washington University Pain Center and Departments of Anesthesiology, Anatomy & Neurobiology, and Psychiatry, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA The first three authors contributed equally to this work Correspondence should be addressed to M.Z. (zhuom@morpheus.wustl.edu) N-methyl-D-aspartate (NMDA) receptors contribute to many brain functions. We studied the effect of forebrain-targeted overexpression of the NMDA receptor subunit NR2B on the response of mice to tissue injury and inflammation. Transgenic mice exhibited prominent NR2B expression and enhanced NMDA receptor-mediated synaptic responses in two pain-related forebrain areas, the anterior cingulate cortex and insular cortex, but not in the spinal cord. Although transgenic and wild type mice were indistinguishable in tests of acute pain, transgenic mice exhibited enhanced respon- siveness to peripheral injection of two inflammatory stimuli, formalin and complete Freund?s adjuvant. Genetic modification of forebrain NMDA receptors can therefore influence pain perception, which suggests that forebrain-selective NMDA receptor antagonists, including NR2B- selective agents, may be useful analgesics for persistent pain. � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com nature neuroscience ? volume 4 no 2 ? february 2001 165 (Fig. 2b?d). Resting neuronal membrane potentials were not differ- ent in wild type (?68.7 � 2.7 mV, n = 27) and transgenic (?69.7 � 3.0 mV, n = 26) slices. To enhance sensitivity to possible changes in NMDA receptor-mediated EPSPs, we injected current through the recording electrode to depolarize neurons and relieve any voltage- dependent Mg 2+ blockade. EPSP slopes were similar in wild type and transgenic mice at 0 mV (wild type, 1.6 � 0.4 mV/ms, n = 5; trans- genic, 1.4 � 0.3 mV/ms, n = 18) and +20 mV (wild type, 1.6 � 0.4 mV/ms, n = 6; transgenic, 1.3 � 0.3 mV/ms, n = 16). Thus, we found no evidence that spinal NMDA receptor function was altered by fore- brain-targeted NR2B overexpression. Because electrophysiological experiments are sensitive to alter- ations in NMDA receptor function only in the sampled neurons, we did in situ hybridization with a probe for NR2B, to detect pos- sible changes in the expression of this NMDA receptor subunit in a subpopulation of spinal neurons. We found no detectable NR2B expression in the dorsal horn of wild type or transgenic mice (Fig. 3). In contrast, NR2B expression was significantly enhanced in two forebrain structures, the ACC and insular cor- tex (Fig. 3), consistent with a previous report 15 . Next, we asked whether NR2B overexpression affected acute noci- ception. No significant difference in tail-flick response latency was observed (wild type, 6.64 � 0.35 s, n = 7; transgenic, 6.16 � 0.31 s; n = 9), indicating that spinal nociceptive transmission was not sig- nificantly altered in transgenic mice. Likewise, wild type and trans- genic mice were indistinguishable in response latencies to noxious hot and cold stimuli. (Response latencies after placement on a hot or cold plate were, in wild type mice, 31.8 � 2.3 s at 50�C, 17.7 � 1.6 s at 52.5�C, 11.3 � 1.5 s at 55�C and 20.6 � 2.9 s at 0�C; in transgenic mice, 31.3 � 3.0 s at 50�C, 19.4 � 2.1 s at 52.5�C, 12.8 � 1.8 s at 55�C and 19.3 � 3.1 s at 0�C; n = 6?12 mice per condition.) We then examined the responses of transgenic and wild type mice to a more prolonged noxious stimulus, peripheral formalin injection, a common model for tissue injury and inflamma- tion 18,19 . First, we examined the pattern of formalin-induced neu- ronal activation in wild type mice by probing expression of the immediate-early gene product c-Fos. Although the physiological link between induction of c-Fos expression and nociceptive trans- mission is unclear, neuronal c-Fos expression is widely used as a correlative indicator for neuronal activity induced by noxious stimuli, including formalin injection in particular 20?22 . In adult wild type mice, subcutaneous injection of formalin into the dor- sum of a hind paw induced increased c-Fos expression, relative to saline-injected controls, in various CNS regions related to noci- ceptive transmission and modulation. Among forebrain areas, formalin injection induced prominent c-Fos staining in the ACC articles Fig. 1. Forebrain-targeted NR2B overexpression enhanced NMDA receptor-mediated synaptic responses in the ACC and insular cortex. (a) Traces of NMDA receptor-mediated fEPSPs recorded from the ACC and insular cortex in the presence of 20 �M CNQX. (b) Bath application of 100 �M AP-5 completely (top) and reversibly (bottom) blocked NMDA receptor-medi- ated fEPSPs in the ACC. Similar results were found in insular cortex (data not shown). (c) The input (stimulation intensity, 200-�s duration)?output (fEPSP slope) relationship in the ACC (wild type, n = 8; transgenic, n = 12) and insular cortex (wild- type, n = 6; transgenic, n = 9) reveals enhanced NMDA recep- tor-mediated responses in transgenic relative to wild-type mice (p < 0.001). Above, examples of fEPSP traces recorded from the ACC in wild-type and transgenic animals. We also integrated fEPSPs in the ACC and found an increased area under the curve for transgenic (33.2 � 4.3 mV?ms) relative to wild-type mice (19.1 � 3.3 mV?ms, p < 0.05). Similar results were found in the insular cortex (transgenic, 33.1 � 6.1 mV?ms versus wild type, 17.0 � 2.7 mV?ms; p < 0.05). Fig. 2. NMDA receptor-mediated synaptic responses in the spinal cord were not affected by forebrain-targeted NR2B overexpression. (a) Traces of EPSPs recorded in the presence of 20 �M CNQX, after bath applica- tion of 100 �M AP-5 (top) and after washout (bottom). (b) Examples of EPSP traces recorded from dorsal horn neurons in wild-type and trans- genic animals. (c) Summarized data of NMDA receptor-mediated responses to a single stimulation intensity (10 V) in spinal dorsal horn neurons (L50132, wild-type, n = 27; L50237, transgenic, n = 26). (d) In some experi- ments, synaptic responses were compared at three stimulation intensities (L50132, wild-type, n = 8?14; L50237, transgenic, n = 17?22), revealing no significant difference between wild-type and transgenic mice. a a b c b c d � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com 166 nature neuroscience ? volume 4 no 2 ? february 2001 (Figs. 4 and 5), lateral septal nucleus, secondary motor cor- tex, some nuclei in the amygdaloid complex (medial, baso- lateral and cortical nuclei), piriform cortex, retrosplenial cortex, several midline thalamic nuclei (lateral habenular, paraventricular, mediodorsal, centromedial, paracentral and anterodorsal nuclei) and various hypothalamic nuclei (paraventricular, periventricular, supraoptic and dorsome- dial nuclei). Less prominent but significant c-Fos expres- sion was observed in the somatosensory cortex (S1, S2 and hindlimb areas; Fig. 5), the hippocampal CA1 (Fig. 5) and CA3 subfields, insular cortex (Figs. 4 and 5), accumbens nucleus and lateral preoptic area. A small amount of for- malin-induced c-Fos expression was observed in other thal- amic nuclei, including the lateral posterior nucleus, posterior group, ventral lateral, ventral posterolateral, ven- tral posteromedial and ventral medial nuclei. No c-Fos expression was observed in the caudate-putamen nucleus. In the midbrain, formalin treatment augmented c-Fos expres- sion within all subareas of the periaqueductal gray (PAG; Fig. 5), dorsal raphe, interpeduncular nucleus, mesencephalic reticular formation, superficial gray layer of the superior colliculus and inferior colliculus. In the brainstem, c-Fos expression was also observed in the locus coeruleus, parabrachial nucleus and ros- troventral medulla. Abundant c-Fos expression was observed in the dorsal horn of lumbar spinal cord, particularly in superficial laminae and the deep neck of the dorsal horn ipsilateral to the injected hind paw, with much smaller (but nonetheless evident) expression contralaterally (Figs. 4 and 5). To determine whether forebrain-targeted NR2B overexpres- sion affected formalin-induced c-Fos expression, we examined the articles Fig. 3. Enhanced NR2B expression in the ACC and insular cor- tex but not the spinal dorsal horn in NR2B transgenic mice. Expression of NR2B was examined by in situ hybridization in wild-type and NR2B transgenic mice. Whereas the two fore- brain regions exhibited enhanced NR2B expression in transgenic compared to wild-type mice, no upregulation of NR2B expres- sion was seen in the spinal cord dorsal horn. Scale bars, 300 �m. Fig. 4. Enhanced forebrain c-Fos expression following formalin injection in NR2B transgenic mice. Immunohistochemical staining for c-Fos is illus- trated in the ACC, insular cortex and spinal cord in wild-type and NR2B transgenic mice 2 h after hind paw injection of saline or formalin (wild type, saline injected, n = 4 mice; wild type, formalin injected, n = 6; transgenic, saline, n = 4; transgenic, formalin, n = 4). Although the two forebrain regions exhibited enhanced c-Fos staining after formalin injection in transgenic compared to wild-type mice, the pattern of c-Fos expression in the spinal cord was not changed by forebrain-targeted NR2B overexpression. Scale bar, 300 �m. � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com nature neuroscience ? volume 4 no 2 ? february 2001 167 pattern of c-Fos immunoreactivity induced in NR2B transgenic mice after formalin injection in experiments done in parallel to those described above. In saline-injected control animals, baseline c-Fos expression was indistinguishable between wild type and transgenic mice (Figs. 4 and 5). After formalin treatment, the most prominent expression occurred in the ACC, insular cortex and hippocampal CA1 subfield, all of which exhibited significantly more c-Fos immunoreactivity in transgenic than wild type for- malin-treated mice (Figs. 4 and 5). Similar results were noted in the lateral septal nucleus, hippocampal CA3 subfield and accum- bens nucleus. No significant differences in c-Fos staining between transgenic and wild type mice after formalin injection were found in somatosensory cortex (Fig. 5), amygdala, thalamus or hypo- thalamus. Formalin-induced c-Fos expression was likewise unaf- fected by forebrain-targeted NR2B expression in the midbrain, brainstem and spinal cord (Figs. 4 and 5). The brain areas in which significantly greater c-Fos expression was observed in transgenic than in wild type mice were areas in which NR2B overexpression was present 15 (Figs. 1?3). Moreover, these areas of greater c-Fos expression encompassed some parts of the limbic system and other forebrain areas important for the central processing of pain infor- mation, including, in particular, the ACC and insular cortex 23,24 . We next asked whether forebrain-targeted NR2B overexpres- sion affected the behavioral responses of mice to tissue injury and inflammation. We used two models: peripheral injection of for- malin, and peripheral injection of complete Freund?s adjuvant (CFA). In the first set of experiments, formalin was injected sub- cutaneously into the dorsal side of a hind paw. Mice responded by licking the injected paw, and this behavior was typically concen- trated in two distinct phases: phase one (0?10 min) and phase two (10?55 min). We continued to observe mice until 120 min after injection, for two reasons. First, in previous studies (ref. 25 and unpublished data), we showed that mice continue to exhibit behav- ioral responses during the 55?120 min period (n = 50 mice, mean total response time 159 � 11 s), which we have called ?phase three.? Second, NMDA receptor-mediated, activity-dependent synaptic plasticity at central synapses can last for hours after induction. Thus, if forebrain-targeted NR2B overexpression alters the behav- ioral responses of mice to formalin injection, then it is reasonable that these alterations might be observed during phase three. Within the first 55 min after hind paw formalin injection, behavioral responses were similar between wild type and trans- genic mice (Fig. 6a and b). However, transgenic mice exhibited more pronounced phase-three responses compared to wild type articles Fig. 5. Enhanced forebrain c-Fos expression following formalin injection in NR2B transgenic mice. Numbers of c-Fos positive cells in the anterior cingulate cortex (ACC), the S1 and S2 regions of somatosensory cortex (S1,2), insular cortex, the CA1 subfield of the hippocampus, the peri- aqueductal gray (PAG) and spinal cord laminae I?VI (SCDH) are illus- trated in wild-type and NR2B transgenic mice with hind paw injection of saline (control; 4 wild-type, 6 transgenic) or formalin (4 wild type, 4 transgenic). Cells were counted contralateral to the injected hind paw except in the spinal cord, where cells were counted ipsilaterally. *Significant difference from wild type and transgenic controls; ?signifi- cant difference from wild type, formalin-treated mice. Fig. 6. Enhanced behavioral responses to formalin or CFA injection in NR2B transgenic mice. (a) The number of seconds during which wild-type (L50132, n = 16) and NR2B transgenic mice (L50237, n = 9) were engaged in noci- ceptive behavioral responses to hind paw formalin injection were plotted in 5-min intervals. Among nine NR2B transgenic mice, six of them were from transgenic line 1, and three were from line 2. Because no significant difference was found between them, the data were pooled. (b) Results from (a) were grouped into three phases (see Results). *Significant differ- ence from wild-type mice. (c) The responses of animals to a mechanical stimulus (a 0.4-mN von Frey fiber applied to the dorsum of a hind paw) that elicited no responses before dorsal hind paw CFA injection were recorded 1 and 3 days after injection. The data were plotted as percent positive responses to stimulation of the ipsilateral or contralateral hind paw (relative to side of injection) for wild-type (L50132, n = 4) and NR2B trans- genic (L50237, n = 4) mice. *Significant difference between wild-type and trans- genic mice in the indicated conditions. (d) Hind paw edema was measured with a fine caliper in wild-type and NR2B transgenic mice (white bars, wild-type, n = 4; black bars, NR2B transgenic, n = 4). No significant differ- ence was found between wild-type and transgenic mice. a b c d � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com 168 nature neuroscience ? volume 4 no 2 ? february 2001 mice (n = 6; Fig. 6a and b). Similar results were obtained from a second line of transgenic mice (n = 3). No obvious difference in the degree of hind paw edema was found between wild type and transgenic mice (data not shown). When a lower formalin con- centration was used (1%, 10 �l), no difference between the two groups of mice was observed at any time point (wild type, n =3, phase 1, 56 � 7 s; phase 2, 217 � 9 s; phase 3, 99 � 22 s; NR2B transgenic, n = 3, phase 1, 68 � 25 s; phase 2, 231 � 37 s; phase 3, 121 � 18 s). These results suggest that forebrain-targeted NR2B overexpression selectively enhanced delayed behavioral respons- es to hind paw formalin injection, and that this enhancement required a threshold dose. Finally, we tested the responses of wild type and transgenic mice to hind paw injection of CFA. Application of a 0.4 mN von Frey fiber to the dorsum of a hind paw elicited no response in untreated mice, but at one and three days after CFA injection, mice responded to stimulation of either the injected (ipsilateral) or, to a lesser extent, the contralateral hind paw by hind paw with- drawal. This mechanical allodynia, or display of a withdrawal response to a previously non-noxious mechanical stimulus, was significantly enhanced in NR2B transgenic mice (Fig. 6c). We also observed similar changes in hind paw edema in wild type and NR2B transgenic mice (Fig. 6d). DISCUSSION Our findings demonstrate that genetic manipulation of forebrain NMDA receptors could enhance persistent or chronic behavioral responses to tissue injury and inflammation, without affecting acute nociception. The finding that forebrain-targeted NR2B overexpression seemed to enhance phase-three responses selec- tively in the formalin test suggests that forebrain NMDA receptor- mediated phenomena may represent a mechanistic basis for these delayed behavioral responses. Taken together with the observation that transgenic mice also experienced enhanced mechanical allo- dynia one and three days after CFA injection, these data are con- sistent with the hypothesis that forebrain NMDA receptor activation is responsible, at least in part, for a prolonged noci- ceptive reaction to tissue injury and inflammation. Although our study does not identify the specific brain region(s) responsible for the altered behavior of transgenic mice, the observed phenotype is most likely explained by changes in NMDA receptor physiology within the forebrain, because ?-CaMKII promoter-driven overexpression of NR2B is expected to be restricted to the forebrains of mice 15 . Moreover, we present anatomical, electrophysiological and behavioral evidence in sup- port of this conclusion. Anatomical evidence was the enhanced expression of NR2B in the ACC and insular cortex, but not in the spinal cord, as detected by in situ hybridization. Also, we found enhanced formalin-induced c-Fos expression in the ACC, insu- lar cortex and other forebrain areas, but not in the midbrain, brainstem or spinal cord of transgenic mice. Our electrophysio- logical evidence was the enhanced NMDA receptor-mediated synaptic transmission in the ACC and insular cortex but not in the spinal cord dorsal horn of transgenic mice. Our behavioral evidence was that phase two formalin-induced behavioral respons- es, which are thought to depend on spinal NMDA receptors 19,26 , were not affected in transgenic mice. Taking together these four independent lines of evidence, the enhanced responsiveness of transgenic mice to inflammatory stimuli is most likely explained by changes in NMDA receptor composition in the forebrain alone. Our results suggest that a genetic manipulation conferring enhanced cognitive abilities may also provide unintended traits, such as increased susceptibility to persistent pain. We do not know why the forebrain NR2B:NR2A ratio undergoes a devel- opmental reduction. However, we note that although pain per- ception normally is a protective for an organism, it may, in some cases, be considered maladaptive, particularly when pain from a previous injury persists and cannot be avoided. Our data show that behavioral responses to inflammatory pain?not just learn- ing and memory?were affected by enhanced forebrain expres- sion of NR2B. Such a blunt manipulation likely affects an animal?s physiology in many ways. More selective regional manipulation of NR2B receptor expression (for example, targeting the CA1 subfield of the hippocampus exclusively) may help to avoid such unintended side effects. Our study implicates a molecular mechanism by which fore- brain activity could modulate behavioral responses to inflam- matory pain. In addition to the well characterized contributions of spinal or brainstem NMDA receptors, forebrain NMDA recep- tors may be important and unique in the response of animals to persistent pain. Because NR2B expression is restricted to fore- brain neurons in wild type adults 9 , NR2B-selective NMDA antag- onists, by preferentially targeting forebrain neurons, may prove beneficial in the treatment of chronic pain in humans. METHODS Transgenic mice. Both NR2B transgenic and littermate wild type mice were provided by Y. P. Tang and colleagues at Princeton University 15 . Adult male mice weighing 15?29 g were used for all experiments, and ?wild type,? as used throughout this manuscript, refers to littermate mice not segregating the NR2B transgene. Experimental protocols were approved by the Animal Studies Committee at Washington University. Electrophysiology. Mice were anesthetized with 1?2% halothane, and brain or spinal cord was isolated. Recordings from cortex were done as described 16 . Briefly, transverse cortical slices were maintained in an inter- face chamber at 28�C, where they were subfused with artificial cere- brospinal fluid (ACSF) consisting of 124 mM NaCl, 4.4 mM KCl, 2.0 mM CaCl 2 , 1.0 mM MgSO 4 , 25 mM NaHCO 3 , 1.0 mM Na 2 HPO 4 and 10 mM glucose, bubbled with 95% O 2 and 5% CO 2 . Stimulation with a bipolar tungsten electrode in layer V evoked extracellular field potentials in layer II/III. For recordings from spinal cord, transverse spinal slices with attached dorsal roots were maintained in ACSF at 34�C. Intracellular microelectrode recordings were made from dorsal horn laminae I/II neu- rons, and dorsal root stimulation evoked synaptic responses that were acquired and analyzed with the Axoclamp 2B amplifier and pCLAMP software (Axon Instruments, Foster City, California). In all experiments, bicuculline methiodide (10 �M) and strychnine hydrochloride (1 �M) were added to the perfusion solution. Anatomical analysis. In situ hybridization experiments were done as described 27 using an NR2B plasmid (gift from J.E. Huettner). For immuno- cytochemistry, mice were anesthetized with 3?4% halothane at 120 min following formalin or saline injection, perfused with fixative, and processed for immunostaining as described 16 using an anti-c-Fos rabbit antibody (1:20000; Oncogene Science, Uniondale, New York). Anatomical termi- nology is based on the atlas of Franklin and Paxinos 28 . The rostrocaudal levels corresponded to 0.98 to 0.5 mm (ACC), 0.7 to ?1.22 mm (the S1 and S2 regions of somatosensory cortex), 1.10 to 0.5 mm (insular cortex), ?1.70 to ?2.18 mm (the CA1 subfield of the hippocampus), and ?4.24 to ?4.72 mm (the periaqueductal gray), relative to bregma. Spinal sections analyzed were from L4?5. Behavioral experiments. To test acute pain responses, the latency of response to heating of the tail (tail-flick test) or to placement on a hot (50?55�C) or cold (0�C) plate was measured as described 29?30 . To test inflammatory pain, formalin (5%, 10 �l) or complete Freund?s adjuvant (CFA, 50%, 10 �l; Sigma, St. Louis, Missouri) was injected subcuta- neously into the dorsal side of a hind paw. For formalin, the total time spent licking or biting the injected hind paw was recorded during each articles � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com nature neuroscience ? volume 4 no 2 ? february 2001 169 subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368, 144?147 (1994). 11. Kato, N., Artola, A. & Singer, W. Developmental changes in the susceptibility to long-term potentiation of neurones in rat visual cortex slices. Brain Res. Dev. Brain Res. 60, 43?50 (1991). 12. Carmignoto, G. & Vicini, S. Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 258, 1007?1011 (1992). 13. Hestrin, S. Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse. Nature 357, 686?689 (1992). 14. Teng, C. J. & Abbott, F. V. The formalin test: a dose-response analysis at three developmental stages. Pain 76, 337?347 (1998). 15. Tang, Y. P. et al. Genetic enhancement of learning and memory in mice. Nature 401, 63?69 (1999). 16. Wei, F., Li, P. & Zhuo, M. Loss of synaptic depression in mammalian anterior cingulate cortex after amputation. J. Neurosci. 19, 9346?9354 (1999). 17. Sah, P. & Nicoll, R. A. Mechanisms underlying potentiation of synaptic transmission in rat anterior cingulate cortex in vitro. J. Physiol. (Lond.) 433, 615?630 (1991). 18. Dubuisson, D. & Dennis, S. G. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4, 161?174 (1977). 19. Tjolsen, A., Berge, O. G., Hunskaar, S., Rosland, J. H. & Hole, K. The formalin test: an evaluation of the method. Pain 51, 5?17 (1992). 20. Morgan, J. I. & Curran, T. Stimulus?transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci. 14, 421?451 (1991). 21. Herrera, D. G. & Robertson, H. A. Activation of c-fos in the brain. Prog. Neurobiol. 50, 83?107 (1996). 22. Munglani, R. & Hunt, S. P. Proto-oncogenes: basic concepts and stimulation induced changes in the spinal cord. Brain Res. Prog. Brain Res. 104, 283?298 (1995). 23. Casey, K. L. Forebrain mechanisms of nociception and pain: analysis through imaging. Proc. Natl. Acad. Sci. USA 96, 7668?7674 (1999). 24. Treede, R. D., Kenshalo, D. R., Gracely, R. H. & Jones, A. K. The cortical representation of pain. Pain 79, 105?111 (1999). 25. Kim, S. J., Calejesan, A. A., Li, P., Wei, F. & Zhuo, M. Sex differences in late behavioral response to subcutaneous formalin injection in mice. Brain Res. 829, 185?189 (1999). 26. Taylor, B. K., Peterson, M. A. & Basbaum, A. I. Persistent cardiovascular and behavioral nociceptive responses to subcutaneous formalin require peripheral nerve input. J. Neurosci. 15, 7575?7584 (1995). 27. Birren, S. J., Lo, L. C. & Anderson, D. J. Sympathetic neurons undergo a developmental switch in trophic dependence. Development 119, 597?610 (1993). 28. Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates (Academic, New York, 1997). 29. Calejesan, A. A., Kim, S. J. & Zhuo, M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur. J. Pain 4, 83?96 (2000). 30. Lee, D. E., Kim, S. J. & Zhuo, M. Comparison of behavioral responses to noxious cold and heat in mice. Brain Res. 845, 117?121 (1999). 31. Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. & Yaksh, T. L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55?63 (1994). articles five-minute interval, for two hours. For CFA, mechanical sensitivity was assessed with a set of von Frey filaments (Stoelting, Wood Dale, Illinois) using the up-down protocol 31 . Positive responses to application of a fil- ament to a hind paw included prolonged hind paw withdrawal followed by licking or scratching. In untreated wild type (n = 4) or transgenic mice (n = 4), the 0.4 mN (No. 2.44) filament, representing 50% of the thresh- old force, never produced responses, and was used to detect mechanical allodynia in CFA-injected mice. Hind paw edema was evaluated by mea- suring dorsal?ventral hind paw thickness with a fine caliper at three days after CFA injection. Data analysis. Results were expressed as mean � s.e.m. Statistical com- parisons were made with one- or two-way analysis of variance (ANOVA) with the post-hoc Scheffe F-test in immunocytochemical experiments, or the Student-Newmann-Keuls test in behavioral experiments, to identify significant differences. In all cases, p < 0.05 was considered significant. ACKNOWLEDGEMENTS We thank J.Z. Tsien (Princeton University) for his supply of mice, and G. Liu (M.I.T.) and other members of Zhuo lab for their comments and advice on the manuscript. RECEIVED 7 AUGUST; ACCEPTED 7 DECEMBER 2000 1. Collingridge, G. L. & Bliss, T. V. Memories of NMDA receptors and LTP. Trends Neurosci. 18, 54?56 (1995). 2. Choi, D. W. & Rothman, S. M. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13, 171?182 (1990). 3. Haley, J. E., Sullivan, A. F. & Dickenson, A. H. Evidence for spinal N-methyl- D-aspartate receptor involvement in prolonged chemical nociception in the rat. Brain Res. 518, 218?226 (1990). 4. Coderre, T. J., Katz, J., Vaccarino, A. L. & Melzack, R. Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52, 259?285 (1993). 5. Yaksh, T. L. The spinal pharmacology of facilitation of afferent processing evoked by high-threshold afferent input of the postinjury pain state. Curr. Opin. Neurol. Neurosurg. 6, 250?256 (1993). 6. Woolf, C. J. & Costigan, M. Transcriptional and posttranslational plasticity and the generation of inflammatory pain. Proc. Natl. Acad. Sci. USA 96, 7723?7730 (1999). 7. Nakanishi, S. Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597?603 (1992). 8. Hollmann, M. & Heinemann, S. Cloned glutamate receptors. Annu. Rev. Neurosci. 17, 31?108 (1994). 9. Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B. & Seeburg, P. H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529?540 (1994). 10. Sheng, M., Cummings, J., Roldan, L. A., Jan, Y. N. & Jan, L. Y. Changing � 2001 Nature Pub lishing Gr oup http://neur osci.nature . com � 2001 Nature Publishing Group http://neurosci.nature.com "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment