Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts

Lena Rubi 0 Ulla Schandl 0 Michael Lagler 0 Petra Geier 0 Daniel Spies 0 Kuheli Das Gupta 0 Stefan Boehm 0 Helmut Kubista 0 0 L. Rubi U. Schandl M. Lagler P. Geier D. Spies K. D. Gupta S. Boehm H. Kubista (&) Department of Neurophysiology and Neuropharmacology, Center of Physiology and Pharmacology, Medical University of Vienna , Waehringerstrasse 13a, 1090 Vienna, Austria Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca2? influx to Ca2?-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of afterhyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological upregulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca2? homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on longlasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy. - L-type voltage-gated calcium channels (LTCCs) fulfill important neurological functions, for example as neuronal pacemakers, in synaptic plasticity and excitation-transcription coupling (Striessnig et al. 2006). However, elevated levels of LTCCs have been linked to pathology. LTCCs are up-regulated in aging neurons, and the incidence of several neurological diseases where LTCCs have been implicated, namely age-dependent memory deficits, Alzheimers disease (AD) and Parkinsons disease (PD), increases with age (Moyer et al. 1992; Thibault et al. 2001, 2007; Veng and Browning 2002; Davare and Hell 2003; Veng et al. 2003; Chan et al. 2007, 2010; Sulzer and Schmitz 2007; Anekonda et al. 2011; Dursun et al. 2011; Ilijic et al. 2011; Kim and Rhim 2011). Furthermore, a gain of function mutation in Cav1.2 has been linked to Timothy syndrome, which involves neurological dysfunction such as developmental delay and autism (Bidaud and Lory 2011). There is also evidence that hyperactive LTCCs play a role in epileptic disorders. For example, in a subpopulation of neurons of the spontaneously epileptic rat (SER), the group of Masashi Sasa found by comparison of current voltage relation curves that voltage-gated calcium currents are activated at considerably less depolarized voltages than in neurons of non-epileptic control rats (Yan et al. 2007). Indirect evidence from earlier studies of this group indicates that the channel responsible for this alteration in calcium current is an LTCC (e.g., Amano et al. 2001a and 2004). Furthermore, in neurons of the seizure prone gerbil, protein levels of Cav1.3 were found to be increased (Park et al. 2003; Kang et al. 2004). Similar to the above-named neurological dysfunctions, the incidence of epilepsies also increases with age (Werhahn 2009). LTCCs have long been suggested to act as important regulators of neuronal excitability, and their coupling to Ca2?-dependent conductances is known to play a crucial role in shaping neuronal discharge patterns (Moyer et al. 1992; Morisset and Nagy 1999). Enhanced LTCC-mediated afterhyperpolarizations were suggested to be causally linked to age-related cognitive impairment (see for example Gamelli et al. 2011). However, in a previous study (Geier et al. 2011), we showed by potentiation of LTCCs that these voltagegated calcium channels have both excitatory and inhibitory coupling modes in neurons of rat hippocampus, and both coupling modes can operate in a given neuron. Hence, it remained unknown whether, in which direction, and to what extent pathologically enhanced LTCC activities may affect the discharge properties of neurons. To address these questions, we performed patch-clamp recordings from various types of hippocampal neurons in primary culture and studied the effects of pharmacological up-regulation of LTCCs (denoted as LTCC: in the following) in current-clamp recordings of neuronal activity. Materials and Methods Primary Cell Culture of Hippocampal Neurons Hippocampi were dissected from neonatal SpragueDawley rats which had been killed by decapitation, and primary cultures of hippocampal neurons were prepared in the same manner as described previously (Geier et al. 2011). Hence, all experiments were performed ex vivo. (Molecular Devices, Sunnyvale, CA, USA) at a sampling rate of 20 kHz. Patch pipettes were made of borosilicate capillaries (GB150-8P, Science Products, Hofheim, Germany) with a Sutter P97 horizontal puller (Sutter Instrument Company, Novato, CA, USA). Tip resistances lay between 3.5 and 5 MX. Pipette solutions contained (in mM) 120 potassium gluconate, 1.5 sodium gluconate, 3.5 NaCl, 1.5 CaCl2, 0.25 MgCl2, 10 HEPES, 10 glucose and 5 EGTA. pH was adjusted to 7.3 by KOH. For perforated patch recordings, 500 lg/ml amphotericin B (from Streptomyces sp., compound purchased from Sigma-Aldrich, Vienna, Austria) was added to the pipette solution. Experiments were started only after the series resistance had dropped to the lowest achievable level (e.g., between 20 and 30 MX), which usually occurred within 1530 min. To assure that only viable cells were used, the following inclusion criteria had to be met: a membrane voltage of at least -50 mV and the capability of generating overshooting action potentials, which was always tested prior to the recordings. Typically, the neurons had a membrane resting potential of slightly less negative than -70 mV (67.3 6.3 mV, mean SD, as determined from 45 neurons used in this study). Experiments were performed at room temperature, and cells were superfused continuously with standard external solution containing (in mM) 140 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 20 glucose (pH was adjusted to 7.4 by NaOH). LTCC activity was modulated by application of the dihydropyridin (...truncated)