In general, αCaMKII-F89G transgenic (Tg) mice were visually indistinguishable from wild-type mice. Our previous study used biochemical and pharmacological techniques to screen five different lines of transgenic mice, and the CaMKII Tg-1 line was found to have the highest transgenic mRNA expression. CaMKII overexpression was limited to the forebrain areas, including the ACC, hippocampus, and amygdala, using a αCaMKII promoter-driven construct (see Methods and [14
]). No CaMKII overexpression was detected in the hindbrain or spinal cord [16
]. Furthermore, biochemical experiments showed that calcium-dependent CaMKII activity was increased by 2.6 fold in transgenic mice compared to wild-type mice. No changes in other protein kinase such as β CaMKII or CaMKIV were found. Administration of 0.5 μM 1NM-PP1 (a compound that inhibits transgenic CaMKII, see methods) in vitro or systemic application of 1NM-PP1 in Tg-1 mice was found to inhibit overexpressed activity without effecting basal CaMKII in wild-type mice. Thus we chose to use the Tg-1 line of transgenic αCaMKII-F89G mice in the present study.
We first performed anatomical experiments in wild-type and αCaMKII-F89G transgenic mice to check whether the gross development of several sensory related brain areas were affected. Analysis of serial coronal sections, examined by light microscopy, showed no visual detectable morphological differences in the ACC, insular cortex, brainstem, and spinal dorsal horn between CaMKII Tg and WT mice (Fig. ). Next we asked whether the neuronal spiking properties of ACC neurons might be affected in the transgenic mice. We performed intracellular recordings from ACC neurons (Fig. ). Similar to results found in the hippocampus, we did not find any significant changes in baseline synaptic responses in ACC neurons (wild-type, n = 16 neurons/10 mice; transgenic, n = 10 neurons/6 mice). No significant changes were observed in neuronal spiking in response to direct current injection (wild-type, n = 12 neurons/8 mice; transgenic, n = 9 neurons/6 mice). Resting membrane potentials were identical between cells recorded from wild-type (n = 14 neurons/10 mice) and transgenic mice (n = 12 neurons/6 mice). Furthermore, the current threshold for eliciting the first spike was similar in ACC neurons from wild-type and transgenic mice (n = 3–6 neurons for each group) and the number of spiking induced by injection of 0.3 nA was also identical (n = 6–10 cells). These results indicate that αCaMKII overexpression did not cause changes in the intrinsic neuronal electrophysiological properties of ACC neurons.
Figure 1 Brain morphology of wild-type and αCaMKII-F89G transgenic mice. Coronal sections showed no detectable morphological differences in the ACC, insular cortex, rostroventral medulla (RVM) and spinal cord dorsal horn. Scale bar: 250 μm (ACC, (more ...)
Figure 2 Comparison of neuronal properties in wild-type and αCaMKII-F89G transgenic mice. A. Evoked synaptic responses recorded to focal stimulation in the ACC of wild-type and αCaMKII-F89G transgenic mice. B. Action potentials of ACC neurons from (more ...)
CaMKII activity is important for synaptic plasticity. In a previous study of αCaMKII-F89G transgenic mice, we found that αCaMKII overexpression selectively affected the frequency-response relationship of synaptic plasticity in the CA1 region of the hippocampus [16
]. In addition to enhanced LTP induced by strong tetanic stimulation, synaptic responses in response to repetitive stimulation at lower frequencies were also altered [16
]. To study if cingulate plasticity may also be affected in these mice, we decided to investigate both LTP and LTD in ACC slices of wild-type and transgenic mice. We did not detect changes in synaptic potentiation induced by theta burst stimulation in ACC slices of transgenic mice (mean 157.2 ± 20.4% of control; n = 6 slices/5 mice) as compared to wild-type mice (mean 167.2 ± 12.8% of control, n = 12 slices/10 mice; P = 0.346) (Fig. ). To detect possible changes in frequency-dependent responses, we examined the effect of prolonged repetitive stimulation in cingulate slices. We previously showed that low-frequency repetitive stimulation at 1 Hz for 15 min induced LTD in the ACC of adult rats [9
]. Furthermore, repetitive stimulation at 3 or 5 Hz stimulation also induced LTD of synaptic responses in ACC slices [9
]. As shown in Fig. , we found that 1 Hz stimulation (15 min) induced robust LTD in the ACC of wild-type mice (8–12 weeks old) (mean 35.7 ± 14.1%, n = 6 slices/6 mice, P < 0.05 compared with baseline). Similar to ACC in adult rats, 5 Hz stimulation for 3 min also produced long-lasting depression of synaptic responses in ACC slices (mean 39.1 ± 5.5%, n = 5 slices/5 mice; P < 0.001 as compared with baseline) (Fig. ). We then tested if forebrain overexpression of CaMKII affects synaptic LTD in the ACC. As shown in Figure , we found that LTD induced by 1 Hz stimulation (15 min) was significantly reduced or completely abolished in transgenic mice (mean 84.4 ± 10.7%, n = 6 slices/6 mice; P < 0.01 as compared with LTD in wild-type mice). Similarly, in transgenic mice, 5 Hz stimulation did not induce any synaptic depression (mean 102.3 ± 8.9%, n = 5 slices/5 mice, Fig. ; P < 0.001 as compared with wild-type mice) (Fig. ).
Figure 3 Long-term potentiation (LTP) and long-term depression (LTD) in ACC slices of wild-type and αCaMKII-F89G transgenic mice. A. Model for the design of αCaMKII-F89G transgenic mice and the selective chemical inhibitor. B. Prolonged low frequency (more ...)
Results described above indicate that overexpressing CaMKII in the forebrain causes selective changes in synaptic LTD without causing any obvious anatomical abnormality or change in neuronal excitability. These findings suggest that αCaMKII-F89G transgenic mice may serve as an excellent model for investigating the role of CaMKII in forebrain plasticity. Since forebrain structures play a key role in sensory perception and plasticity, we next wanted to determine if forebrain CaMKII plays a role in behavioral responses to sensory stimuli and injury. To study the behavioral responses to acute noxious stimuli, we performed the tail-flick and hot-plate tests. We found that behavioral responses to noxious thermal stimuli were similar between wild-type and transgenic mice (Fig. , tail-flick test: wild-type, n = 6 mice; transgenic mice, n = 7 mice; thermal withdrawal: wild-type, n = 10 mice, transgenic mice, n = 6 mice), indicating that enhanced CaMKII activity in the forebrain did not significantly affect acute behavioral responses to noxious stimuli. To detect possible temperature-dependent changes in hot-plate responses, we measured responses at additional temperatures (50 and 52°C). Again, we did not find any significant difference (n = 12 mice for wild-type mice, n = 11 for transgenic mice) (Fig. ). We also measured hindpaw withdrawal to mechanical pressure and found that mechanical withdrawal thresholds were not affected in transgenic mice (n = 4 mice for each group, Fig. ). These results consistently demonstrate that behavioral responses to acute noxious thermal and mechanical stimuli were not affected by enhanced forebrain CaMKII activity.
Figure 4 Behavioral nociceptive responses to noxious heat, mechanical pressure and formalin injection were not altered in αCaMKII-F89G transgenic mice. A-B. Behavioral nociceptive responses to noxious heating in the tail-flick test at two different heating (more ...)
NMDA receptors and their related signaling pathways in the forebrain (including the ACC) have been implicated in injury-related behavioral sensitization [17
]. Thus, it is possible that CaMKII contributes to behavioral responses to tissue injury and inflammation, a long-lasting form of behavioral sensitization. The formalin test measures spontaneous responses to tissue injury and inflammation [17
]. Formalin-induced behavioral responses consist of three phases and depend on NMDA receptors at different levels of the brain [10
]. We tested formalin-induced nociceptive responses in wild-type and CaMKII transgenic mice and found that all the three phases did not differ between transgenic (n = 7 mice) and wild-type (n = 13 mice) mice (Fig. ). In animals with persistent pain, behavioral sensitization to non-noxious stimuli or mechanical allodynia happens after tissue injury. We next tested the role of enhanced CaMKII activity in the development of allodynia induced by a hind paw injection of CFA (50%, 10 μl). Application of a non-noxious von Frey fiber to the dorsum of a hind paw elicited no response in untreated wild-type mice, but at one and three days after CFA injection into the dorsum of a single hind paw, mice withdrew their hindpaw in response to stimulation of the ipsilateral or, to a lesser extent, the contralateral hind paw (Fig. ). This mechanical allodynia was significantly reduced in transgenic mice compared to wild-type mice (n = 5 mice for each group; Fig. ; P < 0.01 as compared between wild-type and transgenic mice). Similar results were observed in the contralateral hind paw. In wild-type mice, we found a significant decrease in hindpaw withdrawal latencies from a noxious heat source at 1 to 3 days after CFA injection in the ipsilateral hindpaw (so called thermal hyperalgesia) (n = 5 mice), and to a lesser extent in the contralateral hind paw (Fig. ). However, thermal hyperalgesia was significantly reduced in CaMKII transgenic mice (n = 5 mice) compared to wild-type mice (Fig. ). To be sure that the differences in pain behaviors were not attributable to differences in peripheral inflammation, we measured hind paw edema in both wild-type and transgenic mice. A similar degree of inflammation was found in wild-type and transgenic mice (n = 5 mice for each group, Fig. ), indicating that the peripheral responses to inflammation are likely identical in these mice.
Figure 5 Reduction of behavioral sensitization (allodynia) and hyperalgesia after CFA injection in αCaMKII-F89G transgenic mice. A. The behavioral responses of animals to a non-noxious mechanical stimulus (No. 2.44 von Frey fiber, which elicited no response (more ...)
Our results indicate that the forebrain overexpression of CaMKII significantly affected synaptic depression in the ACC in vitro and behavioral sensitization to inflammation in vivo. However, we cannot rule out the possibility that these changes in behavioral responses may due to developmental, long-term changes in CaMKII-dependent or related signaling pathways. In the present study, the chemically engineered mice provide us a chance to turn off the overexpressed CaMKII activity and examine if the observed changes can be reversed or 'rescued'. To test this, we treated CaMKII transgenic mice with the inhibitor 1NM-PP1 for several days to turn off the overexpressed CaMKII activity. We found that 1NM-PP1 treatment completely blocked the effects of CaMKII overexpression on behavioral allodynia and hyperalgesia in transgenic mice (n = 4–6 mice, see Fig. and , respectively). 1NM-PP1 treatment alone did not significantly affected hindpaw mechanical withdrawal thresholds (n = 6 mice).
Figure 6 Reduction in allodynia and hyperalgesia can be reversed by inhibiting the overexpression of CaMKII activity in αCaMKII-F89G transgenic mice. A. One-week pretreatment with the inhibitor 1NM-PP1 reversed the reduction of behavioral allodynia in (more ...)
Finally, we tested the effects of 1NM-PP1 on synaptic plasticity to determine if reversing CaMKII over expression could 'rescue' the loss of synaptic depression induced by 1 Hz repetitive stimulation. As shown in Figure , we found that 1NM-PP1 rescued synaptic depression in the transgenic mice (mean 53.2 ± 11.0% of control, n = 7 slices/6 mice, P < 0.05 as compared with transgenic mice). By contrast, the inhibitor did not significantly affect baseline synaptic responses in the ACC slices (n = 6 slices/6 mice) or synaptic depression in slices of wild-type mice (mean 40.9 ± 12.1% of control, n = 5 slices/5 mice).
Figure 7 'Rescued' LTD in αCaMKII-F89G transgenic mice by inhibiting the overexpression of CaMKII activity in αCaMKII-F89G transgenic mice. A. One-week pretreatment with 1NM-PP1 reversed the reduction of cingulate LTD in αCaMKII-F89G transgenic (more ...)