Neuroscience. 2016 Jan 28;313:110-121.

Analysis of the protective effects of a neuronal Cav2.1 calcium channel in brain injury.

Kim TY, Yoshimoto T, Aoyama Y, Niimi K, Takahashi E.

Research Resources Center, RIKEN Brain Science Institute, Saitama 351-0198, Japan.



We previously reported that rolling Nagoya mice carrying a mutation in the α1 subunit of the Cav2.1 channel protective from ischemia- and kainate-induced neuronal damage. However, the protective effect of this mutation and its relationship to brain injury recovery have not been examined. To examine the relationship between Cav2.1 channel function and brain injury, we induced cryogenic brain damage in homozygous rolling Nagoya (rol/rol), control wild-type (+/+), ω-agatoxin IVA-pretreated +/+ (ω-aga +/+), and ω-agatoxin IVA-post-treated +/+ (ω-aga-post-treated +/+) mice. We measured the lesion area, blood brain-barrier permeability and performed immunohistochemistry and western blot analysis. The lesions of rol/rol and ω-aga +/+ mice were significantly smaller than those observed in +/+ mice at both day 1 and day 7 after injury. Similar results were shown in blood-brain barrier permeability. We observed more reactive astrogliosis in +/+ mice than in rol/rol or ω-aga +/+ mice. rol/rol and ω-aga +/+ mice had fewer degenerating cells due to cryogenic injury than did +/+ mice at both day 1 and day 7. ω-Aga-post-treated +/+ mice 24h after injury were sacrificed on day 7. The lesions were smaller in ω-aga-post-treated +/+ mice than those in vehicle-treated +/+ mice. We also examined phosphorylated p38 (pp38) at the injured site. ω-Aga-post-treated +/+ mouse brain slices showed weak pp38 signal; vehicle-treated +/+ mouse brain slices were pp38-positive. These findings demonstrate that the mutant Cav2.1 channel exerts a protective effect against cryogenic brain injury in rolling Nagoya mice. Our results indicate that inhibitors of the Cav2.1-dependent p38 signaling cascade would be useful as therapeutic agents in the treatment of brain injury.

PMID: 26616403



Traumatic brain injury (TBI) is a significant public health concern. It is one of the leading cause of death of disability in people under the age of 45. The pathophysiological mechanisms of TBI can be divided in 2 steps: (1) primary mechanical damage to cells and tissue as result of direct mechanical forces, and (2) secondary injury that involves additional molecular mechanisms of injury, degenerating neurons that continue to evolve (Namjoshi et al., 2013).


We used a cryogenic method to make a model for TBI (Fig. 1). Primary damage by cryogenic injury destroys the blood–brain barrier, increases tight junction permeability. Secondary injury leads to excitotoxicity, involving astrocytes and neurons (Murakami et al., 1999 and Lozano et al., 2015).



ET fig1Fig. 1. Stereotaxic equipment and the metal rod used for producing cryogenic TBI model. Cone-shaped copper cylinder is chilled with liquid nitrogen (-196°C) and placed stereotactically on the left parietal skull.


Protective effect of a Cav2.1 channel in brain injury

Neuronal voltage-gated Cav2.1 channel is located on presynaptic terminals and mediates a number of neuronal functions, including neurotransmitter release (Catterall and Few, 2008). We have previously suggested that mutant Cav2.1 channel disturbs N-methyl-D- aspartate (NMDA) signaling in ischemic incident (Tian et al., 2013) by using Cav2.1 channel mutant homozygous rolling Nagoya (rol/rol) mice (Fig. 2).



ET fig2Fig. 2. Cav2.1 mutant homozygous rolling Nagoya (rol/rol) mice. Spontaneous ataxic rol/rol mice, carrying R1262G mutation at S4 segment on the domain III of Cav2.1 channel α1 subunit, have reduced voltage sensitivity and activity of the Cav2.1 channel.


In our another study, we demonstrated that mutant Cav2.1 channel has protective effect against kainate-induced brain injury by suppressing kainate and/or α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) signal gate (Kim et al., 2014). Cav2.1 channels are blocked by ω-agatoxin IVA, the venom from the funnel web spide Agenelopsis aperta (Catterall and Few, 2008). Phosphorylated p38 expression were lower in ω-agatoxin IVA-treated mice comparted to vehicle-treated mice after brain injury. Taken these results together, abnormal Cav2.1 channel have protective effect against brain injury by disturbing glutamate related p38 mitogen-activated protein kinase (MAPK) cascade (Fig. 3).



 ET fig3

Fig. 3. Putative mechanism for mutant Cav2.1 channel related p38 MAPK signaling and size of lesion on day 1 after brain injury. Ca2+ concentration plays an important role in neuronal function. Abnormal Cav2.1 channel leads to disturbance of Ca2+ influx, resulting in disturbing p38 MAPK signaling.  The lesion size was bigger in wild-type mice than rol/rol mice on day 1 after brain injury.


Therapeutic agent in the treatment of brain injury

Although Cav2.1 has not been considered as a best therapeutic target, we found a potential to be used for therapy by performing experiment in Cav2.1 mutant mice and Cav2.1 inhibitor injection in a model of brain injury. Cav2.1 channel blocker may be used as protective and therapeutic agents against brain injury, and rolling Nagoya mice could serve as a useful model for brain injury research.



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Kim TY, Yoshimoto T, Aoyama Y, Niimi K, Takahashi E, Itakura C. Age-dependent kainate sensitivity in heterozygous rolling Nagoya Cav2.1 channel mutant mice. Pharmacol Biochem Behav. 2014;124:250-259.

Lozano D, Gonzales-Portillo GS, Acosta S, de la Pena I, Tajiri N, Kaneko Y, Borlongan CV. Neuroinflammatory responses to traumatic brain injury: etiology, clinical consequences, and therapeutic opportunities. Neuropsychiatr Dis Treat. 2015;11:97-106.

Murakami K, Kondo T, Yang G, Chen SF, Morita-Fujimura Y, Chan PH. Cold injury in mice: a model to study mechanisms of brain edema and neuronal apoptosis. Prog Neurobiol. 1999;57:289-299.

Namjoshi DR, Good C, Cheng WH, Panenka W, Richards D, Cripton PA, Wellington CL. Towards clinical management of traumatic brain injury: a review of models and mechanisms from a biomechanical perspective. Dis Model Mech. 2013;6:1325-1338.

Tian X, Zhou Y, Gao L, He G, Jiang W, Li W, Takahashi E. Analysis of ischemic neuronal injury in Cav2.1 channel α1 subunit mutant mice. Biochem Biophys Res Commun. 2013;434:60-64.



Tae Yeon Kim, DVM

Research Resources Center, RIKEN Brain Science Institute

2-1 Hirosawa, Wako, Saitama, 351-0198, Japan


TEL; +81-48-467-9754

FAX; +81-48-467-9692


Eiki Takahashi, DVM, PhD.

Research Resources Center, RIKEN Brain Science Institute

2-1 Hirosawa, Wako, Saitama, 351-0198, Japan


TEL; +81-48-467-5871

FAX; +81-48-467-9692



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