Calciumâcalmodulin-dependent protein kinase IV is required ... - Nature

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Calcium–calmodulin-dependent protein kinase IV is required for fear memory Feng Wei1, Chang-Shen Qiu1, Jason Liauw1, Daphné A. Robinson1, Nga Ho2,Talal Chatila2 and Min Zhuo1 1 Washington University Pain Center, Departments of Anesthesiology, Anatomy, and Neurobiology and Psychiatry, and 2 Departments of Pediatrics and Pathology and Immunology and the Center for Immunology, Washington University School of Medicine,

St. Louis, Missouri 63110, USA Correspondence should be addressed to M.Z. (zhuom@ morpheus.wustl.edu)

Published online: 13 May 2002, DOI: 10.1038/nn855 The ability to remember potential dangers in an environment is necessary to the survival of animals and humans. The cyclic AMP–responsive element binding protein (CREB) is a key transcription factor in synaptic plasticity and memory consolidation. We have found that in CaMKIV–/– mice—which are deficient in a component of the calcium–calmodulin-dependent protein kinase (CaMK) pathway, a major pathway of CREB activation—fear memory, but not persistent pain, was significantly reduced. CREB activation by fear conditioning and synaptic potentiation in the amygdala and cortical areas was reduced or blocked. We propose that cognitive memory related to a noxious shock can be disassociated from behavioral responses to tissue injury and inflammation.

Emotional learning and its expression in mammals, such as fear, require the involvement of higher brain structures, including the amygdala, hippocampus and related cortical areas1–5. Cumulative evidence consistently shows that within these areas, longterm changes in synaptic transmission and structure are important for the establishment and consolidation of such memory6–11. CREB is a major transcription factor12,13 that is centrally involved in the formation of long-term memory in both invertebrates and vertebrates14–22. CREB is activated by phosphorylation of the serine 133 residue, potentially within a short period of time12,13. CREB activation is mediated by two major pathways, the cAMP signaling pathway and calcium–calmodulin (Ca2+–CaM)-dependent protein kinase pathway23–29. Inhibition of CREB impairs behavioral performance in various memory tests across species15–19, whereas overexpression of CREB facilitates long-term fear memory20,21. Genetic manipulation of signaling pathways upstream of CREB has produced varying results. Inhibition of cAMP-protein kinase A signal pathways affects both spatial and fear memory in different species30–34. Different results were reported in regard to the function of CaMKIV, a component of the CaMK pathway, in behavioral memory. Genetic deletion of CaMKIV does not affect two common forms of spatial memory in mice35, whereas transgenic mice expressing a dominant-negative CaMKIV show deficits in various hippocampus-dependent tasks36. We hypothesized that different CREB activation pathways preferentially encode different forms of memory. We therefore examined the function of CaMKIV in behavioral acquisition of contextual and auditory fear memory using CaMKIV–/– mice, in which the gene encoding CaMKIV was abolished. We found that fear memory, but not behavioral responses to an acute noxious stimulus nature neuroscience • volume 5 no 6 • june 2002

or prolonged injury, was significantly reduced in CaMKIV–/– mice as compared with wild-type mice. Consistent with these findings, CREB activation by fear conditioning and synaptic potentiation in memory-related areas, including the amygdala and hippocampus, was blocked or attenuated in CaMKIV–/– mice.

RESULTS CaMKIV is crucial for fear memory but not nociception We assessed two forms of associative emotional memory in wild-type and CaMKIV–/– mice: contextual and auditory fear conditioning. Animals learn to fear a neutral conditioned stimulus (such as tone) that has been paired with an aversive unconditioned stimulus (such as a foot shock) and with the context in which the animals were conditioned by the pairing of conditioned and unconditioned stimuli. Early contextual and auditory fear conditionings are mediated by the hippocampus and/or amygdala, whereas late contextual memory may be mediated by areas of the cortex3,5. Contextual and auditory conditionings were measured at 1 hour, 1 day and 7 days after training (Fig. 1). We found no significant difference in contextual freezing immediately after training or 1 hour later (wild-type, n = 8 mice; CaMKIV–/–, n = 7). By contrast, at 1 and 7 days, we saw significantly less contextual freezing in CaMKIV–/– mice than in wild-type mice (Fig. 1a). Furthermore, when we tested auditory fear conditioning 1 day or 7 days after training, we saw significantly less freezing in response to the tone in CaMKIV–/– mice than in wild-type mice (Fig. 1b). No obvious difference in the behavioral response to the foot shock was found between wild-type and CaMKIV–/– mice. Next, we asked whether the lack of CaMKIV affected acute nociceptive transmission or persistent pain. We saw no signifi573

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Fig. 1. CaMKIV is required for fear memory but not behavioral responses to tissue injury. (a, b) Contextual and auditory fear conditioning at 1 h, 1 d and 7 d after training (, wild-type mice, n = 8; , CaMKIV–/– (KO), n = 7). (c) Nociceptive behavioral responses to hind-paw formalin injection during three different phases. No significant difference was found between wild-type (hatched) and CaMKIV–/– mice (black). (d) The responses of animals to a mechanical stimulus that elicited no responses before dorsal hind-paw CFA injection were recorded 1 and 3 d after CFA injection. The data are plotted as percentage positive responses to stimulation of the ipsilateral or contralateral hind paw for wild-type and CaMKIV–/– mice. *, P < 0.05 as compared with wild-type mice.

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cant difference in tail-flick latency (wild-type, n = 12 mice, mean 3.5 ± 0.3 s; CaMKIV–/–, n = 14, 3.9 ± 0.4 s), indicating that spinal nociceptive transmission was not significantly altered in CaMKIV–/– mice. Likewise, we found no differences in latencies to a hot-plate test (55°C) (wild-type, n = 17, 13.6 ± 1.1 s; CaMKIV–/–, n = 18, 15.7 ± 0.8 s, P > 0.05). Thus, the behavioral responses of CaMKIV–/– and wild-type mice to noxious thermal stimuli were indistinguishable. We then examined the response to a more prolonged nociceptive stimulus, hind-paw formalin injection. We had previously showed that formalin injection caused three phases of licking responses over 120 min after the injection37. We found no significant difference between wild-type and CaMKIV–/– mice in any of the three phases of response (wildtype, n = 11; CaMKIV–/–, n = 7, Fig. 1c). These results suggest that CaMKIV-dependent signal pathways are not required for behavioral responses to formalin injection.

To explore whether CaMKIV-dependent signal pathways contribute to behavioral responses in chronic inflammatory pain, we tested mechanical allodynia after hind-paw injection of complete Freund’s adjuvant (CFA). Application of a fine von Frey fiber to the dorsum of a hind paw elicited no response in untreated mice, but at 1 and 3 days after CFA injection (50%, 10 µl) into the dorsum of a single hind paw, mice responded to stimulation of either the same (ipsilateral) or, to a lesser extent, the contralateral hind paw by hind-paw withdrawal37. Again, we found that mechanical allodynia was similar in wild-type (n = 6) and CaMKIV–/– mice (n = 13, Fig. 1d) and that the degree of local inflammation in wild-type and CaMKIV–/– mice was similar. Together, these results indicate that CaMKIV is preferentially involved in fear memory induced by a noxious shock but not in the behavioral responses to acute noxious stimuli or tissue inflammation. Thus, behavioral responses to peripheral tissue inflammation commonly used in pain research do not reflect fear memory38. CREB activation by fear conditioning One major downstream target of CaMKIV is CREB, a key transcription factor involved in long-term memory, including fear memory16,18. Fear conditioning causes activation (phosphorylation) of CREB in the hippocampus and amygdala, as indicated by immunostaining for phosphorylated CREB (pCREB)39. However, the role of CaMKIV in fear conditioning–induced phosphorylation of CREB has not been examined. Because two forms of fear memory were significantly reduced in CaMKIV–/– mice, we predicted that the amount of fear conditioning–induced pCREB present should be reduced as well. As the distribution of CaMKIV immunoreactivity has been described only in the adult rat brain40, we first examined the distribution of CaMKIV in the hippocampus, amygdala and related cortical areas of wild-type mice (n = 3). Consistent with previous findings in the rat brain40, CaMKIV-labeled neurons were found in the hippocampus, amygdala, anterior cingulate cortex (ACC), somatosensory cortex and

Fig. 2. Distribution of CaMKIV in selected areas of adult mouse brain. Coronal section of brain showing the distribution of CaMKIV-labeled neurons in the basolateral amygdala, the CA1 region of hippocampus, ACC, primary and secondary somatosensory cortex (S1+2) and insular cortex of wild-type mice (n = 3 mice, from left to right, top and middle panels). High-magnification images from top row show staining in the basolateral amygdala (BL), pyramidal cell layer of the CA1, area 3 of ACC (Cg3), layer 4 of somatosensory cortex and agranular insular cortex (AI), respectively (middle panels). No immunostaining was seen in brain sections of the CaMKIV–/– mice (n = 3 mice, bottom panels). Cg1, area 1 of ACC; pir, pyriform cortex; GI, granular insular cortex. Scale bar, 200 µm (top and bottom panels), 50 µm (middle panels).

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Fig. 3. CaMKIV contributes to activation of CREB by fear conditioning. pCREB expression in the amygdala (BM, BL), hippocampus (CA1, DG), ACC (Cg1, Cg3), S1 and S2 and insular cortices (GI, AI) from wild-type and CaMKIV–/– mice in a control situation (Box), after shock–tone unpaired training or after tone–shock paired training. Notation as in Fig. 2; BM, basomedial amygdala. Scale bar, 300 µm.

insular cortex (Fig. 2). Higher-magnification confocal images showed strong CaMKIV immunoreactivity in neuronal nuclei and weak immunoreactivity in the cytoplasm throughout these areas. No CaMKIV immunostaining was seen in the brains of CaMKIV–/– mice (n = 3, Fig. 2). Next, we measured activation of CREB by fear conditioning in adult wild-type and CaMKIV–/– mice through immunocytochemistry. To demonstrate the associative specificity of pCREB expression in the various brain areas, we carried out a series of experiments in five different groups of wild-type and CaMKIV–/– mice: control (no stimulus) and those conditioned with tone alone, shock alone, shock–tone (unpaired training) and tone–shock (paired training). At 45 min after paired tone–shock presentations, greater pCREB immunoreactivity was found in the hippocampal CA1 and the basolateral part of the amygdala of wild-type mice (n = 5 mice) than in that of wild-type mice (n = 5) receiving unpaired training (Figs. 3 and 4). Auditory stimulation alone did not produce a significant change in pCREB levels in the hippocampus or amygdala (n = 4 mice), whereas shock alone caused modest increases in pCREB in the hippocampus and amygdala (n = 5 mice). In both wild-type (n = 5 mice) and CaMKIV–/– mice (n = 5 mice), similar basal levels of pCREB were found in different areas of the hippocampus, including CA1 (wild-type, 3.7 ± 0.4; CaMKIV–/–, 2.9 ± 0.1), CA3 (wild-type, 3.4 ± 0.2; CaMKIV–/–, 3.5 ± 0.2), dentate gyrus (DG) (wild-type, 3.7 ± 0.2, CaMKIV–/–, 3.0 ± 0.3) and amygdala (wild-type, 3.4 ± 0.1; CaMKIV–/–, 3.2 ± 0.5) (Figs. 3 and 4). This suggested that deletion of CaMKIV does not affect basal expression of pCREB in neurons. In the groups of mice receiving paired fear conditioning, pCREB levels in the CA1 region were significantly lower, but not absent, in CaMKIV–/– mice (n = 6 mice) as compared to wild-type mice (n = 5 mice, Figs. 3 and 4). This finding indicates that CaMKIV contributes only partially to the activation of CREB in the CA1 areas. How-

ever, paired conditioning activated pCREB in the basolateral nucleus of the amygdala (as compared to wild-type mice receiving paired training) was completely abolished in CaMKIV–/– mice (Fig. 4), suggesting that CaMKIV activity is essential to fear conditioning–induced CREB activation. We also examined three cortical areas involved in the processing of sensory information and possibly fear-related memory, the ACC, primary somatosensory cortex and agranular insular cortex. In wild-type mice, paired fear conditioning induced significantly larger increases in pCREB level in all three cortical regions than did unpaired training (Figs. 3 and 4). Tone or shock alone increased pCREB expression in the somatosensory and insular cortex of wild-type mice. However, CaMKIV seems to contribute differently to the activation of CREB by fear conditioning in the different cortical areas examined. In CaMKIV–/– mice, fear conditioning–induced pCREB was not seen in the somatosensory and insular cortex but was seen, although at a lesser level than in wildtype mice, in the ACC (Figs. 3 and 4). Basal expression of pCREB in these regions was similar in wild-type and CaMKIV–/– mice. This finding differs from our previous results obtained using primary cortical cultured cells35, indicating that in vitro–cultured cell preparations may not completely mimic physiological conditions in vivo. We also examined the expression of pCREB in auditory pathways, including the cochlear nucleus, lateral superior olivary complex, inferior colliculus and medial nucleus of the trapezoid body. Similar levels of pCREB after tone alone, unpaired shock–tone or paired tone–shock were found in wild-type and CaMKIV–/– mice (data not shown), indicating that the auditory sensory transmission is normal. This conclusion is supported by Fig. 4. CaMKIV contributes to activation of CREB by fear conditioning. Quantification of pCREB staining in basolateral amygdala, hippocampal CA1, ACC, S1 and AI are illustrated in wild-type and CaMKIV–/– mice receiving shock–tone unpaired or tone–shock paired training. *, significant difference from wild-type and CaMKIV–/– controls; #, significant difference from unpaired CaMKIV–/– mice.


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Fig. 5. Requirement of CaMKIV for amygdala and cortical synaptic potentiation. (a) TBS (arrow) induced synaptic potentiation in the amygdala in wild-type (, n = 11 slices/9 mice) but not CaMKIV–/– mice (, n = 6 slices/6 mice). (b) CaMKIV is also required for potentiation in the ACC by TBS (wild-type, n = 7 slices/5 mice; CaMKIV–/–, n = 6 slices/6 mice). Insets in (a) and (b), representative records of the EPSP before (pre) and 40 min after (post) TBS in a wild-type and CaMKIV–/– slice. (c–d) Synaptic potentiation in the insular (wild-type, n = 7 slices/6 mice; CaMKIV–/–, n = 8 slices/5 mice) and somatosensory cortices (wild-type, n = 5 slices/5 mice; CaMKIV–/–, n = 7 slices/6 mice).

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behavioral data indicating that immediate responses (such as freezing response measured after fear conditioning) are normal in CaMKIV–/– mice. These results indicate that CaMKIV is crucial for the activation of CREB in areas related to fear memory in vivo and that its contribution is region specific. Synaptic potentiation in the amygdala and cortex Synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), are a thought to be important for learning and memory 3,6–11. In the CA1 region of the hippocampus, LTP but not LTD is altered in CaMKIV–/– mice35. Here we wanted to examine synaptic potentiation in the amygdala, a structure that plays an important role in fear memory 1–4,7–11 . We examined synaptic potentiation at ‘thalamic’ input synapses

Fig. 6. CaMKIV is required for CaM translocation and activation of CREB in the hippocampus and amygdala. (a) Confocal images of CaM and pCREB staining in CA1 pyramidal neuron layer from either wild-type or CaMKIV–/– mice, with or without high-K+ stimulation. (b) Summary data of CaM translocation in CA1, CA3 and DG areas of hippocampus from wild-type (WT; control, n = 9 sections/3 mice; treated, n = 11 sections/3 mice) and CaMKIV–/– mice (KO; control, n = 7 sections/2 mice; treated, n = 11 sections/3 mice). (c) CaM and pCREB staining in neurons of the basolateral amygdala receiving similar treatment as described in (a). (d) Summary data of CaM translocation in basolateral amygdala neurons from CaMKIV–/– (control, n = 7 sections/2 mice; treated, n = 11 sections/3 mice) and wild-type mice (control, n = 9 sections/3 mice; treated, n = 11 sections/3 mice). Scale bar, 25 µm (a, c). *, P < 0.05 as compared with sections of wild-type mice.

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to the lateral amygdala by placing a stimulating electrode in the ventral striatum41. In contrast to the results for the CA1 region of hippocampus, we found in preliminary experiments that strong tetanic stimulation did not induce reliable potentiation (data not shown). We therefore used five trains of theta-burst stimulation (TBS) as a stimulus5. In slices from wild-type mice, TBS induced significant synaptic potentiation (n = 11 slices/9 mice; 169.4 ± 8.0%; Fig. 5a). In slices from CaMKIV –/– mice, however, this synaptic potentiation was significantly reduced or blocked (n = 6 slices/6 mice; 124.1 ± 14.1%, P < 0.01 compared to slices of wild-type mice). We also examined synaptic potentiation in the ACC, somatosensory cortex and insular cortex. In the ACC, strong tetanic stimulation induces only short-term potentiation42. Working with normal slices from wild-type mice, we found that TBS induced significant synaptic potentiation lasting at least 40 min (n = 7 slices/5 mice, 153.7 ± 17.6%). In CaMKIV–/– mice, we saw no potentiation after TBS (n = 6 slices/6 mice, 91.3 ± 8.6%), suggesting that CaMKIV is required for synaptic potentiation in the ACC (Fig. 5b). A similar defect in synaptic potentiation was found in the insular cortex (wild-type, n = 7 slices/6 mice, 139.3 ± 8.9%; CaMKIV–/–, n = 8 slices/5 mice, 118.1 ± 7.6%; P < 0.05

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Fig. 7. CaMKIV contributes to CaM translocation and activation of CREB in three cortical areas. (a–c) Confocal images of CaM and pCREB staining in (a) Cg1 of ACC, (b) S1 and (c) AI slices from either wild-type or CaMKIV–/– mice with or without high-K+ stimulation. Scale bar, 25 µm. (d) Summary data of CaM translocation in these regions after neuronal depolarization from wild-type (WT; for Cgl and AI, control, n = 15 sections/3 mice, treated, n = 12 sections/3 mice; for S1, control, n = 9 sections/3 mice, treated, n = 11 sections/3 mice) and CaMKIV–/– mice (KO; for Cgl and AI, control, n = 17 sections/3 mice, treated, n = 15 sections/3 mice; for S1, control, n = 7 sections/3 mice, treated, n = 11 sections/3 mice). *, P < 0.05 as compared with sections of wild-type mice.

comparing two groups) (Fig. 5c). In the somatosensory cortex of wild-type mice, TBS induced a long-lasting enhancement of synaptic responses persisting for at least 40 min after induction (n = 5 slices/5 mice, 150.2 ± 12.6%). In CaMKIV–/– mice, however, we saw no synaptic potentiation (n = 7 slices/6 mice, 111.9 ± 7.1%; P < 0.05 as compared with potentiation in wild-type mice) (Fig. 5d). These results provide the first evidence that CaMKIV contributes to synaptic potentiation in these cortical areas.

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CaM translocation from the cytoplasm to the nucleus Neural activity (for example, induced by the application of highKCl solution) causes translocation of CaM from the cytoplasm to the nucleus and subsequent activation of CREB in the nucleus of central neurons26. CaM-binding proteins in the nucleus may serve as a sink for Ca2+–CaM, leading to accumulation of CaM in the nucleus after elevation of intracellular calcium43. If this is true, CaM translocation might be affected in mice lacking CaMKIV, a CaM-binding protein in the nucleus. To test whether KCl depolarization causes CaM translocation from the cytoplasm to the nucleus, we measured the ratio of CaM immunoreactivity between neuronal nuclei and cytoplasm in brain slices from both wild-type and CaMKIV–/– mice treated with either control solution or 90 mM KCl (Figs. 6 and 7). We stained the same neurons to detect pCREB, because activation of CaMKIV leads to phosphorylation of CREB in the nucleus26. In wild-type mice, KCl application caused significant CaM translocation in neurons within the CA1 and DG but not the CA3 region of the hippocampus (Fig. 6a and b). In CaMKIV–/– mice, CaM translocation triggered by KCl was significantly lower than that in wild-type mice in the CA1, and completely absent in the DG. Consistently, pCREB was also reduced in the CA1 (Fig. 6a) and absent in the DG (data not shown). CaM translocation by neural activity was reported in cultured neurons and hippocampus26,44, and it is not known whether similar translocation occurs in neurons in other regions of the brain. Therefore, we carried out similar experiments in amygdala and cortical slices of wild-type and CaMKIV–/– mice. Application of 90 mM KCl solution to amygdala slices of wild-type mice caused significant CaM translocation and activation of CREB in neurons within the basolateral amygdala (Fig. 6c). Notably, just as in CA1 hippocampal neurons (Fig. 6a), both CaM translocation and pCREB were significantly lower in slices from CaMKIV–/– mice as compared with wild-type mice (Fig. 6c and d). CaM translocation and activation of CREB by KCl application were also found nature neuroscience • volume 5 no 6 • june 2002

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in three different areas of the cortex of wild-type mice, including the ACC, somatosensory cortex and insular cortex (Fig. 7a-c). Both CaM translocation and CREB activation were significantly reduced in the ACC and insular cortex and completely absent in the somatosensory cortex of CaMKIV –/– mice (Fig. 7d).

DISCUSSION Two key Ca2+–CaM-dependent protein kinases, CaMKII and CaMKIV, are important modulators of synaptic plasticity and behavioral memory27,45–48. CaMKII is highly expressed at postsynaptic sites, and its activation contributes to the phosphorylation of synaptic proteins, including glutamate receptors, as well as to the synaptic potentiation known as LTP 27,45. Genetically manipulated mice with or without abnormal CaMKII activity showed defects in the ability to learn and remember during spatial and fear memory tests46–48. Unlike CaMKII, CaMKIV is located mainly in the neuronal nuclei and is involved in the regulation of activity-triggered gene expression26,35. Our present results provide several new findings regarding the role of CaMKIV in neuronal activation of CREB, synaptic potentiation and behavioral memory. Previous in vitro experiments from cultures and slices have shown that CaMKIV contributes to the activation of CREB by neuronal activity35. Our in vivo results show that CaMKIV contributes to the activation of CREB in various memory-related areas, such as the amygdala and hippocampus. Furthermore, the contribution of CaMKIV to CREB activation differs across these areas. In the amygdala, activation of CREB by fear conditioning was completely absent in CaMKIV–/– mice. In the hippocampal CA1 areas, however, activation of CREB was less than in wild-type mice, but not absent. Consistent with these immunostaining results, behavioral changes in spatial (hippocampus-dependent learning) and fear memory from current and previous studies support the conclusion that CaMKIV may have a more important role in amygdala-related fear memory than in hippocampus-related spatial memory. Our electrophys577


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iological studies show that CaMKIV contributes to synaptic potentiation in the amygdala, a structure centrally involved in fear memory. Our data thus provide further evidence in support of a connection between synaptic potentiation of glutamatergic transmission in the lateral amygdala and fear memory. In addition to the amygdala, we found that CaMKIV also contributes to synaptic potentiation in several cortical areas, including the ACC, insular cortex and somatosensory cortex. These findings suggest that CaMKIV serves as a key intracellular protein kinase contributing to synaptic potentiation. Our findings provide further evidence supporting the role of CREB in fear memory. Spatial memory, as well as fear memory, is impaired in CREB–/– mice16. However, spatial memory is not affected in CaMKIV–/– mice35. A possible explanation is that CaMKIV-independent pathways such as the cAMP/PKA signaling pathway may have a more dominant role in CREB activation linked to spatial memory. Consistent with this proposition is the observation that the contribution of CaMKIV to CREB activation varies among different brain regions. In some areas, such as the hippocampal CA1 region, considerable residual pCREB can still be found in CaMKIV–/– mice. The results presented here, obtained using brain slices, indicate that CaMKIV is required for CaM translocation into the nuclei of central neurons. This translocation, which is triggered by neural activity, is not limited to hippocampal neurons but also occurs in neurons located in the amygdala, ACC, somatosensory cortex and insular cortex; thus, it is probably a common signaling mechanism for neurons in the central nervous system. Previous studies have shown that CaM translocation reflects the trapping of Ca2+–CaM complexes by nuclear CaM-binding proteins43. The absence of CaM translocation in CaMKIV –/–mice identifies CaMKIV as the crucial sink that traps Ca2+–CaM complexes in neuronal nuclei. This trapping leads to CaMKIV activation and subsequent CREB phosphorylation and activation. Consistently, we found in both in vitro and in vivo conditions that activation of CREB was significantly reduced or abolished in CaMKIV –/– mice. We also found that synaptic potentiation induced by TBS was reduced or abolished in the same areas. These findings support the existence of an integrated mechanism involving CaMKIV that links changes in cytosolic Ca2+ concentrations to nuclear transcriptional activation. Finally, our studies provide strong evidence that cognitive memory related to a noxious shock can be disassociated from behavioral responses to an acute insult, or prolonged injury, at the molecular level. Although many signaling pathways may contribute to normal physiological functions as well as to distorted pathological conditions37,38,49, we believe that it is possible to identify signaling molecules that preferentially contribute to one but not the other. Identification of such signaling molecules may have applications useful in the improvement of human health.

METHODS

Knockout mice. CaMKIV–/– mice were derived as described35 and bred for several generations (F8–F12) in a C57Bl/6 background. Control wildtype mice were littermates of mutant mice. The Animal Care and Use Committee of Washington University approved the mouse protocols. No visual difference between wild-type and CaMKIV–/– mice is noticeable, and experiments were performed blind. Immunocytochemistry and confocal imaging. For CaMKIV staining, brain sections were incubated with anti-CaMKIV mouse antibody (1:500; Transduction Laboratories, Lexington, Kentucky) and then with FITCconjugated AffiniPure goat anti-mouse IgG at 1:100 dilution (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania). Images were 578

obtained with an Olympus (Melville, New York) Fluoview laser-scanning confocal microscope. Anatomical terminology is based on the atlas of Franklin and Paxinos50. To detect nuclear translocation of CaM and CREB phosphorylation, we performed experiments using brain slices. Adult male mice were anesthetized with halothane and coronal slices of brain were prepared and maintained in interfaced chambers about 28°C, where they were subfused with ACSF (124 mM NaCl, 4.4 mM KCl, 2.0 mM CaCl2, 1.0 mM MgSO4, 25 mM NaHCO3, 1.0 mM Na2HPO4 and 10 mM glucose) bubbled with 95% O2 plus 5% CO2. High K+-containing ACSF (with 4.4 mM KCl replaced with 90 mM KCl) was applied through bath solution for 180 s. Tetrodotoxin (1 µM) was added to block neuronal activity. Brain slices were incubated with 1:250 anti-CaM mouse monoclonal antibody (Chemicon, Temecula, California) and 1:500 anti-pCREB rabbit antiserum (Calbiochem, San Diego, California), followed by 1:600 Cy-3 goat anti-mouse and 1:100 FITC-conjugated AffiniPure donkey anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania). Images were collected sequentially on the Olympus Fluoview laser-scanning confocal microscope. Staining for CaM was quantified by using NIH Image (Scion Image, Scion Corp., Frederick, Maryland) to obtain measurements of relative fluorescence intensity in the whole nuclear area as compared with that in the cytoplasm27,46. All data was shown as a ratio between the nuclear and cytosolic area of each neuron. Only neurons with sharp boundaries and a well-defined nucleus were considered. Thirty neurons were measured from three different sections, for each stimulation, and averaged. For illustration, images were assembled into montages with Adobe Photoshop software. For fear-induced pCREB expression, brain slices were incubated with a rabbit anti-pCREB antibody at 1:500 dilution and Vectastain ABC kit (Vector, Burlingame, California) was used, followed by development using nickel-enhanced diaminobenzidine (DAB; Sigma, St. Louis, Missouri). The integrated intensity for the selected regions was normalized to the corresponding integrated intensity in the adjacent white matter as described previously41. Measurements were made from three randomly selected non-contiguous sections of each region in each mouse, observed from coded slides and averaged so that each animal had a mean value for regional pCREB immunoreactivity. Slice electrophysiology. Transverse slices of amygdala and cortex were rapidly prepared and maintained in an interface chamber at 30°C, where they were subfused with ACSF35,37. In amygdala slices, a bipolar tungsten stimulating electrode was placed in the ventral striatum, and an extracellular recording electrode (3–12 MΩ, filled with ACSF) was placed in the lateral amygdala41. In the cortical slices, a bipolar tungsten stimulating electrode was placed in layer 5, and extracellular field potentials were recorded using a glass microelectrode placed in layer 2/3. Synaptic responses were elicited at 0.02 Hz. Fear conditioning. We used a fear-conditioning shock chamber. Mice were placed in the chamber for 2 min before fear conditioning. The conditioned stimulus (CS) used was an 85 dB sound at 2,800 Hz for 30 s, and the unconditioned stimulus (US) was a continuously scrambled foot shock at 0.75 mA for 2 s. During training, mice were presented with a 30 s tone (CS) and a shock (US) beginning at 28 s after the onset of US. After CS/US pairing, the mice were allowed to stay in the chamber for another 30 s for measurement of immediate freezing. Freezing was scored every 10 s. During the retention test, each mouse was placed back into the shock chamber and the freezing response was recorded for 3 min. Subsequently, the mice were put into a novel chamber and monitored for 3 min before the onset of the tone (pre-CS). Immediately after that, a tone identical to the CS was delivered for 3 min and freezing responses were recorded. To test the association specificity of pCREB, in some experiments, CS/US unpaired trainings were also performed. The US preceded the CS by 60 s. Behavioral experiments. To test acute pain responses, the latency of responses to heating of the tail (tail-flick reflex) or to placement on a hot-plate (55°C) was measured as described37. To test inflammatory pain, formalin (5%, 10 µl) or CFA (50%, 10 µl; Sigma) was injected into the nature neuroscience • volume 5 no 6 • june 2002

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dorsal side of a hind paw. For formalin, the total time spent licking or biting the injected hind paw was recorded during each 5 min interval for 2 h. For CFA, mechanical sensitivity was assessed with an innocuous 0.4 millinewton filament as described37. Hind-paw edema was evaluated with a fine caliper at 3 d after CFA injection. Data analysis. Results were expressed as mean ± s.e.m. Statistical comparisons were made with one- or two-way analysis of variance (ANOVA) with the post-hoc Scheffé 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 statistically significant. Acknowledgments Supported by grants from the National Institute of Neurological Disorders and Stroke 38680, the National Institute on Drug Abuse 10833, the McDonnell Center for Higher Brain Function and Alzheimer Disease Research Center at Washington University.

Competing interest statement The authors declare that they have no competing financial interests.

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Calciumâcalmodulin-dependent protein kinase IV is required ... - Nature

Calciumâcalmodulin-dependent protein kinase IV is required ... - Nature

Calciumâcalmodulin-dependent protein kinase IV is required ... - Nature

Calciumâcalmodulin-dependent protein kinase IV is required ... - Nature