The L-type calcium channel Cav1.3 is required for proper hippocampal neurogenesis and cognitive functions
Graphical abstract
Introduction
L-type calcium channels (LTCCs) represent a subfamily of voltage gated Ca2+ channels that are characterized by high sensitivity to dihydropyridine Ca2+ channel blockers. They regulate a number of physiological functions, including the control of muscle contraction, hormone secretion and cardiac function, and LTCC antagonists are well-established drugs to treat antiarrhythmics and antihypertensives in cardiovascular diseases [1], [2], [3]. In the CNS, the predominant LTCC isoforms are Cav1.2 and Cav1.3, which show widespread expression in several brain regions, such as the cerebral cortex, amygdala, cerebellum, and hippocampus [4], [5], [6]. L-type Ca2+ currents regulate neuronal excitability [7], synaptic plasticity [8], [9] and neuronal gene transcription [10]. Concerning the latter, elevated intracellular free Ca2+ concentrations trigger the transcription of genes associated with long-term changes in synaptic plasticity, cell proliferation, programmed cell death, and neuronal differentiation [11], [12], [13], [14].
Brain LTCCs are required for normal fear-, anxiety-, and depression-like behaviors [3], [15], [16], [17], [18], and play an important role in hippocampus-dependent cognitive performance. Several studies reported an impact of Cav1.2 and Cav1.3 on hippocampus-associated learning and memory function, however with controversial findings. Administration of LTCC antagonists has been demonstrated to improve spatial memory in rodents [19], [20], but has also been shown to impair learning and memory performance [21], [22]. Cav1.2 knockout animals display deficits in spatial learning in the Morris water maze [8]. Cav1.2 dependent changes in cognition are primarily related to changes in LTP [8], whereas the low-voltage activated Cav1.3 channels apparently function as modulators of neuronal spiking behavior [23], [24]. Yet, the role of LTCCs in learning and memory function, in particular the individual contribution of Cav1.2 and Cav1.3 and the underlying mechanisms of LTCC-induced changes in hippocampus-dependent cognitive skills, still remains to be deciphered.
A possible mechanism of LTCC-mediated effects on learning and memory might involve adult neurogenesis. Adult hippocampal neurogenesis, i.e. the generation of new neurons from proliferating neural stem (NSCs) and neural progenitor cells (NPCs) within the dentate gyrus of most mammals including humans [25], [26], [27], [28], has specific functions in learning and memory, especially in spatial cognition and pattern separation [3], [29], [30]. The fact that this process is tightly regulated by intrinsic molecules, e.g. transforming growth factor family proteins [31], [32] and by extrinsic factors, e.g. aging, stress and physical activity [26], [33], [34], added a new dimension in the possibilities of regulation and modulation of cognition.
Concerning LTCCs as potential modulators of adult neurogenesis and cognition, convincing evidence from in vitro studies [35], [36], [37], [38] and from work on rodent brain slices [39] has demonstrated that immature NSC and NPC carry functional LTCCs. Electrophysiological analyses have confirmed LTCC-induced Ca2+ currents in subventricular zone derived NSCs and NPCs from embryonic rats [37], in NPCs from postnatal mice [36], and in adult hippocampal NPCs [35]. Consistently, Ca2+ influx through LTCCs controls the neuronal differentiation of NSCs and NPCs into mature neurons [35], [36], [38]. Excitation of LTCCs induced premature differentiation of proliferating NPCs, while treatment with nifedipine, a dihydropyridine calcium channel blocker primarily blocking LTCCs [40] inhibited neuronal differentiation [36]. Based on these findings, Deisseroth and colleagues further examined possible effects of LTCCs on NSC and NPC behavior and on adult neurogenesis in vivo [35]. Indeed, a 1-week treatment of adult rats with nifedipine significantly reduced the fraction of newborn neurons in the hippocampal dentate gyrus, and administration of the LTCC activator BayK 8644 elevated the numbers of newborn dentate gyrus granular neurons [35]. Hence, these findings clearly demonstrated that hippocampal neurogenesis is regulated by Cav1.2/Cav1.3 channel activity in the adult brain. However, it is unclear which cell types in the dentate gyrus are targeted by LTCC modulation (e.g. NSCs, NPCs, mature neurons), and which steps in adult neurogenesis (proliferation, cell survival, cell differentiation and fate determination) are affected. Further, the impact of aging on LTCC expression and functionality is still controversially discussed, with some authors reporting increased [41] and others reduced [42], [43] LTCC expression levels in the hippocampus of aged animals. To what extent the presumed changes in LTCC properties in the aged hippocampus might contribute to the reduced levels of neurogenesis in aged individuals, has not been addressed so far. Additionally, possible consequences of LTCC-induced changes in hippocampal neurogenesis on learning and memory remain to be addressed.
Here we analyze the expression patterns of the LTCC isoforms Cav1.2 and Cav1.3 in the main neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone, of adult (2–3-month) and middle-aged (15-month) mice. Using homozygous Cav1.3 knockout mice, we also examine the relevance of Cav1.3 for hippocampal neurogenesis in adult and middle-aged mice, and address the importance of Cav1.3 in adult mice for hippocampal function by evaluating depressive-like behavior and spatial memory in Cav1.3−/− mice.
Section snippets
Animals
Experiments were carried out in accordance with the European Community Council Directive (86/609/EEC) and were approved by the local animal health commission. For the in vivo experiments, adult (3 months) and middle-aged (15 months) female homozygous Cav1.3 knockout mice were used [44]. Cav1.3−/− mice were backcrossed for at least five generations into a C57BL/6N background. Wildtype (WT) C57BL/6N mice were used as control animals. qRT-PCR analysis of Cav1.2 and Cav1.3 mRNA, and
The LTCC isoforms Cav1.2 and Cav1.3 are expressed within the adult neurogenic regions
The presence of Cav1.2 and Cav1.3 has been demonstrated in several brain regions on the mRNA and protein levels (e.g. [4], [6]). Here we confirmed and extended the present knowledge on LTCC brain expression by performing qRT-PCR of Cav1.2 and Cav1.3 mRNA in non-neurogenic (cortex) and neurogenic (hippocampus, subventricular zone (SVZ)) regions of adult (3-month) and middle-aged (15-month) mice. Further, we specified Cav1.2 and Cav1.3 distribution within the neurogenic regions by
Discussion
We examined the expression of LTCCs within the adult neurogenic regions, i.e. the hippocampal dentate gyrus and the SVZ, and found Cav1.2 and Cav1.3 immunoreactive cell populations at different and specific stages of neuronal differentiation. We addressed the significance of Cav1.3 for dentate gyrus neurogenesis using adult (3-month) and middle-aged (15 month) Cav1.3−/− mice, and we observed a crucial role of this LTCC isoform in proper neurogenesis functions. Further, we detected impaired
Conclusion
To date, LTCC antagonists are suggested as therapeutics to ameliorate cognitive deficits in Alzheimer's disease (AD), since in AD increased activity and expression of LTCCs has been reported [75], and Cav1.3 blockers are currently developed to ameliorate motor symptoms in Parkinson's disease [76]. In the light of our results, showing detrimental effects of Cav1.3 deletion on neurogenesis and cognition, the long-term effects of LTCC blockers in the neurogenic niche should be considered in future
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by the FWF Special Research Program (SFB) F44 (F4413-B23) “Cell Signaling in Chronic CNS Disorders”. Moreover, this work has been made possible through the support from the State Government of Salzburg (Austria) (Stifungsprofessur, and 20204-WISS/80/199-2014), the FWF Hertha-Firnberg Postdoctoral programme no. T736-B24, the foundation Propter Homines (Liechtenstein), through funding from the European Union's Seventh Framework Program (FP7/2007-2013) under grant agreement
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