Involvement of N-type Ca2+ channel in microglial activation and its implications to aging-induced exaggerated cytokine response.

Voltage-dependent calcium channel (VDCC) is generally believed to be active only in excitable cells. However, we have reported recently that N-type VDCC (Cav2.2) could become functional in non-excitable cells under pathological conditions. In the present study, we show that Cav2.2 channels are also functional in physiological microglial activation process. By using a mouse microglial cell line (MG6), we examined the effects of a Cav2.2 blocker on the activation of MG6 cells, when treated with lipopolysaccharide (LPS) / interferon γ (IFNγ) or with interleukin-4 (IL-4). As a result, blocking the activation of Cav2.2 enhanced so-called alternative activation process of microglia (transition to neuroprotective M2 microglia) without changing the efficacy of the transition to neuroinflammatory M1 microglia. This enhanced M2 transition involved the activation of a transcription factor hypoxia inducible factor 2 (HIF-2), since a specific blocker of HIF-2 completely abolished this enhancement. We then examined whether Cav2.2 activation was involved in aging-related neuroinflammation. Using primary culture of microglia, we found that the efficacy of microglial M1 transition was enhanced but that M2 transition was reduced by aging, in agreement with a general notion that aging induces enhanced neuroinflammation. Finally, we show here that the moderate blockade of Cav2.2 expression in microglia restores this age-dependent reduction of microglial M2 transition and reduces the aging-induced exaggerated cytokine response, as revealed by a fast recovery from depressive-like behaviors in microglia-specific Cav2.2 deficient mice. These results suggest a critical role for microglial Cav2.2 channel in the aging-related neuroinflammation.

Upregulation of casein kinase 1epsilon in dorsal root ganglia and spinal cord after mouse spinal nerve injury contributes to neuropathic pain.

BACKGROUND: Neuropathic pain is a complex chronic pain generated by damage to, or pathological changes in the somatosensory nervous system. Characteristic features of neuropathic pain are allodynia, hyperalgesia and spontaneous pain. Such abnormalities associated with neuropathic pain state remain to be a significant clinical problem. However, the neuronal mechanisms underlying the pathogenesis of neuropathic pain are complex and still poorly understood. Casein kinase 1 is a serine/threonine protein kinase and has been implicated in a wide range of signaling activities such as cell differentiation, proliferation, apoptosis, circadian rhythms and membrane transport. In mammals, the CK1 family consists of seven members (alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon) with a highly conserved kinase domain and divergent amino- and carboxy-termini. RESULTS: Preliminary cDNA microarray analysis revealed that the expression of the casein kinase 1 epsilon (CK1epsilon) mRNA in the spinal cord of the neuropathic pain-resistant N- type Ca2+ channel deficient (Cav2.2-/-) mice was decreased by the spinal nerve injury. The same injury exerted no effects on the expression of CK1epsilon mRNA in the wild-type mice. Western blot analysis of the spinal cord identified the downregulation of CK1epsilon protein in the injured Cav2.2-/- mice, which is consistent with the data of microarray analysis. However, the expression of CK1epsilon protein was found to be up-regulated in the spinal cord of injured wild-type mice. Immunocytochemical analysis revealed that the spinal nerve injury changed the expression profiles of CK1epsilon protein in the dorsal root ganglion (DRG) and the spinal cord neurons. Both the percentage of CK1epsilon-positive neurons and the expression level of CK1epsilon protein were increased in DRG and the spinal cord of the neuropathic mice. These changes were reversed in the spinal cord of the injured Cav2.2-/- mice. Furthermore, intrathecal administration of a CK1 inhibitor IC261 produced marked anti-allodynic and anti-hyperalgesic effects on the neuropathic mice. In addition, primary afferent fiber-evoked spinal excitatory responses in the neuropathic mice were reduced by IC261. CONCLUSIONS: These results suggest that CK1epsilon plays important physiological roles in neuropathic pain signaling. Therefore CK1epsilon is a useful target for analgesic drug development.

Peripheral-type benzodiazepine receptor antagonist is effective in relieving neuropathic pain in mice.

cDNA microarray analysis showed the expression of peripheral-type benzodiazepine receptor (PBR) mRNA is slightly enhanced in the spinal cord of mice with spinal nerve injury (SNL) as compared with sham-operated mice. PBR transports cholesterol to the mitochondria, where cholesterol is converted to pregnenolone. Pregnenolone is then metabolized to progesterone, an activator of progesterone receptor, and further metabolized to produce allopregnanolone and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THDOC), positive allosteric modulators and activators of the GABA(A) receptor. In the present study, we first tested whether the enhanced PBR expression is causally related to neuropathic pain, and we found that the PBR antagonist PK11195 is effective in reducing SNL-induced mechanical allodynia and thermal hyperalgesia. Next we tested whether the PK11195-induced antinociception is attributable to reduced neurosteroid synthesis, which may possibly lead to reduced activation of the progesterone receptor and/or GABA(A) receptor. We found that allopregnanolone and 3alpha,5alpha-THDOC are effective in reducing the anti-hyperalgesic effect of PK11195, suggesting a partial contribution of reduced GABA(A)-receptor activation to PK11195-induced antinociception.

Properties of human Cav2.1 channel with a spinocerebellar ataxia type 6 mutation expressed in Purkinje cells.

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

Progesterone receptor antagonist is effective in relieving neuropathic pain.

Injury to the spinal nerves of mice induces allodynia and hyperalgesia. Intrathecal injection of the progesterone/estrogen receptor antagonist ICI 182,780 produced antinociceptive effects. Co-administration of estrogen did not reduce but tended to enhance the antinociceptive effect of ICI 182,780. On the other hand, co-administration of progesterone dose-dependently reduced the antinociceptive effect of ICI 182,780, indicating that the antinociceptive effect is through antiprogesterone receptor activity of ICI 182,780. These results suggest that spinal progesterone receptors play an important role in neuropathic pain, and that controlling the activity of progesterone receptors may be of great importance in the treatment of neuropathic pain.

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel.

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.

Effects of glucocorticoid receptor antagonists on allodynia and hyperalgesia in mouse model of neuropathic pain.

Injury to the spinal nerves of mice induces mechanical allodynia and thermal hyperalgesia. In the injured spinal cord, the expression of glucocorticoid receptor mRNA was increased, whereas it was decreased in N-type Ca(2+)-channel-deficient mice, in which neuropathic pain is eliminated. Intrathecal and intraperitoneal injection of the glucocorticoid receptor antagonist RU486 produced antinociceptive effects, whereas intracerebroventricular injection was without effect. The more selective antagonist dexamethasone 21-mesylate suppressed both mechanical allodynia and thermal hyperalgesia. These results suggest that spinal glucocorticoid receptors play an important role in neuropathic pain, and that controlling the activity of glucocorticoid receptors may be of great importance in the treatment of neuropathic pain.

Blocking the R-type (Cav2.3) Ca2+ channel enhanced morphine analgesia and reduced morphine tolerance.

Morphine is the drug of choice to treat intractable pain, although prolonged administration often causes undesirable side-effects including analgesic tolerance. It is speculated that voltage-dependent Ca(2+) channels (VDCCs) play a key role in morphine analgesia and tolerance. To examine the subtype specificity of VDCCs in these processes, we analysed mice lacking N-type (Ca(v)2.2) or R-type (Ca(v)2.3) VDCCs. Systemic morphine administration or exposure to warm water swim-stress, known to induce endogenous opioid release, resulted in greater analgesia in Ca(v)2.3(-/-) mice than in controls. Moreover, Ca(v)2.3(-/-) mice showed resistance to morphine tolerance. In contrast, Ca(v)2.2(-/-) mice showed similar levels of analgesia and tolerance to control mice. Intracerebroventricular (i.c.v.) but not intrathecal (i.t.) administration of morphine reproduced the result of systemic morphine in Ca(v)2.3(-/-) mice. Furthermore, i.c.v. administration of an R-type channel blocker potentiated morphine analgesia in wild-type mice. Thus, the inhibition of R-type Ca(2+) current could lead to high-efficiency opioid therapy without tolerance.

Increased sensitivity to halothane but decreased sensitivity to propofol in mice lacking the N-type Ca2+ channel.

Volatile anesthetics are known to depress excitatory synaptic transmission. Inhibition of voltage-dependent Ca2+ channels is speculated to underlie this mechanism, which remains to be clarified in vivo. We examined the sensitivity to halothane in mice lacking the N-type Ca2+ channel, a major contributor of presynaptic neurotransmitter release. Sensitivity to halothane was significantly increased in the knockout mice compared with the wild-type littermates. Halothane also depressed field excitatory postsynaptic potentials recorded from the Schaffer collateral-CA1 hippocampal synapses more greatly in the knockout mice. We further examined sleep time induced by injection of propofol, an intravenous anesthetic that mainly affects inhibitory synaptic transmission. In contrast, sensitivity to propofol was significantly decreased in the knockout mice. We suggest that inhibition of the N-type Ca2+ channel underlies mechanisms of halothane anesthesia but counteracts propofol anesthesia.

Anesthetic sensitivities to propofol and halothane in mice lacking the R-type (Cav2.3) Ca2+ channel.

UNLABELLED: Because inhibition of voltage-dependent Ca(2+) channels can be a mechanism underlying general anesthesia, we examined sensitivities to propofol and halothane in mice lacking the R-type (Ca(v)2.3) channel widely expressed in neurons. Sleep time after propofol injection (26 mg/kg IV) and halothane MAC(RR) and MAC (50% effective concentrations for the loss of the righting reflex and for the tail pinch/withdrawal response, respectively) were determined. Significantly shorter propofol-induced sleep time (291.6 +/- 16.8 s versus 344.4 +/- 12.1 s) and larger halothane MAC(RR) (1.11% +/- 0.04% versus 0.98% +/- 0.03%) were observed in Ca(v)2.3 channel knockouts (Ca(v)2.3(-/-)) than in wild-type (Ca(v)2.3(+/+)) litter mates. To investigate the basis of the decreased anesthetic sensitivities in vivo, field excitatory postsynaptic potentials and population spikes (PSs) were recorded from Schaffer collateral CA1 synapses in hippocampal slices. Propofol (10-30 micro M) inhibited PSs by potentiating gamma-aminobutyric acid-ergic inhibition, and this potentiation was markedly smaller at 30 micro M in Ca(v)2.3(-/-) mice, possibly accounting for the decreased propofol sensitivity in vivo. Halothane (1.4%-2.2%) inhibited field excitatory postsynaptic potentials similarly in both genotypes, whereas 1%-2% halothane depressed PSs more in Ca(v)2.3(-/-) mice, suggesting the postsynaptic role of the R-type channel in the propagation of excitation and other mechanisms underlying the increased halothane MAC(RR) in Ca(v)2.3(-/-) mice. IMPLICATIONS: Because inhibition of neuronal Ca(2+) currents can be a mechanism underlying general anesthesia, we examined anesthetic sensitivities in mice lacking the R-type (Ca(v)2.3) Ca(2+) channels both in vivo and in hippocampal slices. Decreased sensitivities in mutant mice imply a possibility that agents blocking this channel may increase the requirements of anesthetics/hypnotics.

Altered cocaine effects in mice lacking Ca(v)2.3 (alpha(1E)) calcium channel.

Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Ca(v)2.3 (alpha(1E)) calcium channel to cocaine effects using Ca(v)2.3 knockout mice (Ca(v)2.3-/-). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Ca(v)2.3+/+), but failed to produce any response in Ca(v)2.3-/- mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Ca(v)2.3+/+ mice; however, it had no significant effect in Ca(v)2.3-/- mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Ca(v)2.3-/- mice. These data indicate that Ca(v)2.3 channel contributes to the locomotor-stimulating effect of cocaine, and the deletion of Ca(v)2.3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist.

Ca(v)2.3 (alpha1E) Ca2+ channel participates in the control of sperm function.

To know the function of the Ca2+ channel containing alpha(1)2.3 (alpha1E) subunit (Ca(v)2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Ca(v)2.3 channel. The averaged rising rates of Ca2+ transients induced by alpha-D-mannose-bovine serum albumin in the head region of Ca(v)2.3-/- sperm were significantly lower than those of Ca(v)2.3+/+ sperm. A computer-assisted sperm motility assay revealed that straight-line velocity and linearity were greater in Ca(v)2.3-/- sperm than those in Ca(v)2.3+/+ sperm. These results suggest that the Ca(v)2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.

Role of Ca(v) 2.3 (alpha1E ) Ca2+ channel in ischemic neuronal injury.

We investigated the role of the Ca(v)2.3 (alpha1E) channel in ischemic neuronal injury using Ca(v)2.3 mutant mice. In focal ischemia model with a complete occlusion of the middle cerebral artery in vivo, infarct at 24 h was significantly larger in Ca(v)2.3 mutant mice compared with that in wild-type controls. In vitro Ca2+ imaging studies using hippocampal slices revealed that oxygen-glucose deprivation induced a [Ca2+]i increase in the hippocampal CA1 region more vigorously in Ca(v)2.3 mutant mice than in wild-type controls, and that tetrodotoxin or bicuculline application abolished the difference between the genotypes. These results suggest that the Ca(v)2.3 channel plays a protective role in ischemic neuronal injury by a mechanism in which GABAergic neuronal actions are involved.