Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases
© Vadakkan et al; licensee BioMed Central Ltd. 2006
Received: 10 January 2006
Accepted: 17 February 2006
Published: 17 February 2006
The Ca2+/calmodulin-stimulated adenylyl cyclase (AC) isoforms AC1 and AC8, couple NMDA receptor activation to cAMP signaling pathways in neurons and are important for development, learning and memory, drug addiction and persistent pain. AC1 and AC8 in the anterior cingulate cortex (ACC) and the spinal cord were previously shown to be important in subcutaneous inflammatory pain. Muscle pain is different from cutaneous pain in its characteristics as well as conducting fibers. Therefore, we conducted the present work to test the role of AC1 and AC8 in both acute persistent and chronic muscle pain.
Using an acute persistent inflammatory muscle pain model, we found that the behavioral nociceptive responses of both the late phase of acute muscle pain and the chronic muscle inflammatory pain were significantly reduced in AC1 knockout (KO) and AC1&8 double knockout (DKO) mice. Activation of other adenylyl cyclases in these KO mice by microinjection of forskolin into the ACC or spinal cord, but not into the peripheral tissue, rescued the behavioral nociceptive responses. Additionally, intra-peritoneal injection of an AC1 inhibitor significantly reduced behavioral responses in both acute persistent and chronic muscle pain.
The results of the present study demonstrate that neuronal Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC and spinal cord are important for both late acute persistent and chronic inflammatory muscle pain.
In neurons, activity dependent cAMP synthesis is primarily mediated by membrane bound Ca2+/calmodulin-stimulated adenylyl cyclases (ACs). So far, ten members of the adenylyl cyclase family have been identified . Of these, AC 1–9 isoforms are present in the brain (reviews, [2, 3]). Two of the adenylyl cyclases, AC1 and AC8, are activated by calcium through the calcium-binding protein calmodulin . These enzymes link activity-dependent increases in intracellular calcium to the production of intracellular cAMP. In addition, these neuronal adenylyl cyclases are viewed as coincidence detectors due to their specific interaction with NMDA receptors and voltage-dependent calcium channels at the neuronal membrane.
The role of the cAMP pathway in neurons along the pain pathway, including the anterior cingulate cortex (ACC) and the spinal dorsal horn were demonstrated [4, 5] and discussed [6, 7]. Peripheral injuries activate a series of signaling molecules downstream of adenylyl cyclases in neuronal populations. These molecules include pMAPK [8, 9], transcription factor pCREB [10–12], and the immediate early genes Egr-1 [13, 14] and Arc . Neuronal adenylyl cyclases were shown to contribute to NMDA receptor-dependent synaptic potentiation in the hippocampus [16, 17]. Recently, we showed that Ca2+/calmodulin-stimulated adenylyl cyclases are required to trigger synaptic potentiation in the ACC neurons  and synaptic facilitation in the spinal cord dorsal horn .
Chronic myofascial pain represents a considerable health problem . Muscle pain is a symptom of various disorders including fibromyalgia, metabolic myopathies, myositis and also a side effect of medications, especially the statin group of cholesterol lowering drugs [21, 22]. Injury to the muscle results in diffuse aching pain that is difficult to localize , spreading to regions outside the area of innervation . Inflammation induced by intramuscular injection of capsaicin or carrageenan results in long-lasting (over weeks) bilateral secondary mechanical hyperalgesia. On the other hand, injection of these agents into the skin causes only a short-lasting, unilateral secondary mechanical hyperalgesia [25, 26]. Although the role of adenylyl cyclases was shown to be important in behavioral sensitization associated with chronic subcutaneous inflammation and neuropathic pain [5, 7, 27], their role in inflammatory muscle pain has not been studied.
In the present work, we adapted a chronic inflammatory muscle pain model  and modified it to induce acute muscle pain by intramuscular injection of formalin. To overcome the non-selective action of adenylyl cyclase inhibitors (e.g. SQ 22536) used in previous studies [25, 27], the present investigation used AC1 knockout (KO), AC8 KO and AC1&8 double knockout (DKO) mice to study the behavioral nociceptive changes after inducing acute persistent pain (formalin injection) and chronic pain (carrageenan injection) in comparison to the wild-type mice.
Effects of deletion of AC1 and AC8 on motor function
Role of AC1 and AC8 on acute persistent inflammatory muscle pain
Chronic inflammatory muscle pain
Inhibition of nociceptive responses in acute and chronic inflammatory muscle pain by the novel AC1 inhibitor
Activation of non-Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC 'rescued' behavioral sensitization
Activation of adenylyl cyclases in the spinal cord, but not in peripheral tissue, rescued mechanical allodynia
The present study examined the role of Ca2+/calmodulin-stimulated adenylyl cyclases AC1 and AC8 in both acute persistent and chronic muscle pain. AC1 and AC8 act downstream from NMDA receptors and contribute to NMDA receptor-dependent synaptic potentiation lasting several hours . Our results provide genetic, pharmacological and behavioral evidence that adenylyl cyclase isoforms AC1 and AC8 are important for behavioral sensitization after muscle inflammation. Previous studies have examined the role of AC1 and AC8 in behavioral allodynia in subcutaneous inflammation . Since the characteristics of pain arising from the muscle inflammation is different from that of cutaneous pain [23, 24], we investigated the role of Ca2+/calmodulin-stimulated adenylyl cyclases in both acute persistent and chronic muscle inflammation. The behavioral responses during the early phase, after intramuscular formalin injection, were not significantly reduced either in AC1 or in AC8 single KO mice compared to the wild-type (Fig. 2B &2D). The behavioral responses during the late phase were significantly reduced in AC1 KO as well as AC1&8 DKO indicating that AC1 has direct contribution towards the late phase of acute persistent muscle pain.
Adenylyl cyclases and synaptic enhancement
Ca2+/calmodulin-stimulated ACs are activated by NMDA receptor-mediated calcium entry and the reduction in the late phase of the formalin lick test may be due to the loss of NMDA receptor-dependent synaptic potentiation that normally lasts for several hours . Our recent study has demonstrated that Ca2+/calmodulin-stimulated adenylyl cyclases are required for triggering NMDA receptor dependent synaptic potentiation in ACC neurons . Therefore, one possibility is that the absence of Ca2+/calmodulin-stimulated adenylyl cyclases in KO mice prevents NMDA-dependent synaptic potentiation and leads to reduction in behavioral responses in the late phase of the formalin lick test. The activation of intra and/or extra-neuronal adenylyl cyclases in the ACC rescued mechanical allodynia. While the reason for this is not yet known, we hypothesize that direct activation of non-Ca2+/calmodulin-stimulated adenylyl cyclases may initiate cellular changes in neuronal and/or non-neuronal cells ultimately triggering downstream pathways following the cAMP step in neurons. In the spinal cord, the role of Ca2+/calmodulin-stimulated adenylyl cyclases is different. cAMP generated by adenylyl cyclase stimulated by forskolin acts synergistically with 5 HT to recruit functional AMPA receptors and provides a mechanism for long-lasting synaptic enhancement . Therefore, activation of adenylyl cyclases by forskolin at the spinal cord is also a contributory mechanism for the phenotypic rescue in AC1&8 DKO mice after intrathecal injection.
Role of adenylyl cyclases in maintaining chronicity of mechanical allodynia
Chronic inflammation caused by carrageenan induced a sensitization process (allodynia) in normal animals. Repeated injections of carrageenan on days 1 and 5 were sufficient to maintain this allodynia up to day 28. In DKO mice, nociceptive responses were reduced by day 9. While adenylyl cyclases are required for maintaining the late phases, it cannot be ruled out that initiation of the activity by these Ca2+/calmodulin-stimulated adenylyl cyclases in the early phase is required for activation of downstream cascades that maintains the late phases. The cAMP synthesis induced by nociceptive responses during persistent pain may have a dose-dependent effect on a downstream cascade that regulates adenylyl cyclase synthesis and activity. In addition, coupling of calcium and cAMP systems may result in an ordered activation or a positive feedback regulation of calcium and cAMP dependent protein kinases and possibly provide positive feedback regulation of calcium channels, thus maintaining mechanical allodynia. Elevated cAMP signals arising from activation of AC1 and 8 by calcium may therefore play an important role in synaptic plasticity associated with chronic muscle pain.
The present study demonstrates that Ca2+/calmodulin-stimulated adenylyl cyclases AC1 and 8 induce a sensitized state in the nociceptive pathway and provides evidence that they are critical in the maintenance of muscle inflammatory pain. Specifically, the role of highly calcium sensitive AC1 is critical as evidenced by the significant reduction of behavioral nociceptive responses in the late phase of both acute and chronic muscle pain following intra-peritoneal injection of the AC1 inhibitor. However, in animals with genetic deletion of AC1 and AC8, forskolin may rescue behavioral phenotypes by activating non-Ca2+/calmodulin-stimulated adenylyl cyclases in both the ACC and spinal cord. These results indicate that cAMP signaling in both the ACC and spinal cord are important for the maintenance of chronic muscle inflammatory pain. Taken together, the results of our previous report  and the present study consistently suggest that Ca2+/calmodulin-stimulated neuronal adenylyl cyclases play a key role in mediating the late phase of acute persistent as well as chronic inflammatory pain.
Adult (8–12 weeks) male mice lacking adenylyl cyclase isoforms AC1, AC8 and both AC1 and AC8 [5, 16, 31] were maintained on a C57BL/6 background and were crossed back at least 12 generations. Adult (8–12 weeks) C57BL/6 mice were used as controls. KO mice were well groomed and did not show any signs of congenital abnormalities or motor coordination defects. Experimenters were blind to the genotype. Animals were maintained on a 12 hour light dark cycle with food and water available ad libitum. The experimental protocols were approved by the Animal Care Committee at the University of Toronto.
Motor function test
Motor function was tested using an accelerating RotaRod (Med Associates). One hour before testing, animals were trained on the RotaRod at a constant acceleration of 16 rpm until they could remain on for a 30-sec period. The RotaRod test was performed by measuring the time each animal was able to maintain its balance walking on the rotating drum. The RotaRod accelerated from 4 to 40 rpm over a 5-min period. Mice were given three trials with a maximum time of 300 sec and a 5 min inter-trial rest interval. The latency to fall was taken as a measure of motor function. Some mice hold on to the rotating drum instead of walking. For these mice, the latency to fall was recorded after 2 complete rotations.
Formalin lick test for acute inflammatory muscle pain
Formalin (10 μL; 5% in normal saline) was injected deep into the gastrocnemius muscle on the left side. The needle was directed from the lateral side to avoid any bone penetration and the tip was stopped at the middle of the muscle for injection. The total time spent licking or biting the injected left leg, including the thigh and paw, was recorded every 5 min for 2 hours immediately following the injection. Responses from 0 to 10 min were plotted as early nociceptive responses, those from 10 to 55 min as intermediate, and those from 55 to 120 min as late nociceptive responses.
Chronic inflammatory muscle pain model
Mice were briefly anaesthetized with isoflurane. 20 μL of carrageenan (3%, in normal saline pH 7.2, 20 μL) or normal saline control (pH 7.2) was injected into the left gastrocnemius muscle, as described in the formalin lick test. This protocol was adapted from similar experiments that induced chronic muscle pain in rats . Care was taken during the injections due to the high viscosity of carrageenan. The injection site was mildly massaged to ensure proper diffusion of the drug from the injection site. To induce chronic muscle pain, two intramuscular injections of carrageenan were administered on days 1 and 5.
Measurement of mechanical allodynia
Mice were allowed to acclimatize to the chamber for 30 min before testing. A threshold stimulus was determined by an animal's paw withdrawal upon application of a von Frey filament (Stoelting, Wood Dale, Illinois) over the dorsum of the left hind paw to the point of bending. Mechanical sensitivity of the animal to the innocuous pressure of a 0.4 mN von Frey filament (No.2.44) was scored and repeated every 5 min for up to 10 times. Positive responses included prolonged hind paw withdrawal followed by licking or scratching and these responses were plotted as percentage positive responses. Injections were carried out on days 1 and 5. Mechanical allodynia was tested on days 1, 2, 5 (before and after injection), 6, 8, 9, 12 and 28. For rescue experiments with forskolin, mechanical allodynia was tested thirty minutes after forskolin microinjection. A two-way analysis of variance (ANOVA) was used to compare differences.
Intraperitoneal injection of AC1 inhibitor
The novel AC1 inhibitor NB001 was dissolved in 1% DMSO in normal saline and injected into the peritoneal cavity in doses varying from 0.1 to 5 mg/kg body weight in a volume of 10 μL/gram body weight. The effect of the drug was tested 30 minutes after the injection.
After anesthetizing with 2% halothane (30% O2 balanced with nitrogen), mice were placed in a Kopf stereotaxic instrument. A microinjection apparatus, consisting of a Hamilton syringe (50 μL) connected to an injector needle (30 gauge) by a thin polyethylene tube and a motorized syringe pump (Razel Scientific Instruments Inc., Stamford, Connecticut), was used to perfuse the drugs. The skin over the scalp was incised at the midline and bilateral openings were made in the skull to allow the insertion of a microinjection needle into the ACC. The coordinates of the injection were as follows: 0.7 mm anterior to Bregma, 0.3 mm lateral to the midline, and 1.75 mm ventral to the surface of the skull . Either 0.5 μl forskolin (12 nmolar) or a vehicle solution (20% DMSO in filter-sterilized phosphate-buffered saline, pH 7.4) was infused at a rate of 0.05 μl/min. The needle was withdrawn 5 min after completion of the injection. Upon completion of experiments, all animals were deeply anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde. Serial cryostat coronal sections (30 μm) of the ACC were mounted on glass slides, counterstained with hematoxylin and examined under the microscope to confirm the site of injection. All chemicals were purchased from Sigma (St. Louis, MO).
Spinal intrathecal injections
After anesthetizing with 2% halothane with 30% O2 balanced with nitrogen, mice were placed in a Kopf stereotaxic instrument. The anesthesia set-up was the same as that used for the ACC microinjection. The lumbar vertebrae were slightly flexed, spines were palpated and the needle was advanced manually through the inter-spinal space lateral to the spinous processes as described . Either 0.5 μl forskolin (12 nmolar) or a vehicle solution (20% DMSO in filter-sterilized phosphate-buffered saline, pH 7.4) was infused at a rate of 0.05 μl/min. After the completion of the injection, the needle was kept in place for 5 min before the withdrawal.
Results are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were performed by a two-way ANOVA. p < 0.05 was considered statistically significant. For the RotaRod test, data were analyzed using a one-way ANOVA.
This work was funded by EJLB-Canadian Institutes of Health Research (CIHR) Michael Smith Chair in Neurosciences and Mental Health in Canada, and Neuroscience Canada grants to Min Zhuo. Kunjumon Vadakkan was a recipient of James F. Crothers fellowship. We thank Drs. Louis J. Muglia and Daniel R. Storm for providing the AC1 KO, AC8 KO and AC1&8 DKO mice.
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