A novel Ca V 2.2 channel inhibition by piracetam in peripheral and central neurons

SCG sympathetic neuron culture and electrophysiology

SCG neurons were isolated from five-week-old male Wistar rats as described previously. ^14,15 Rats were obtained from the animal breeding facility of the School of Medicine at the Universidad Nacional Autonoma de Mexico (UNAM) and were handled according to the Mexican Official Norm for Use, Care and Reproduction of Laboratory Animals (NOM-062-ZOO-1999). Animals were anesthetized with CO₂ and decapitated with a guillotine. Following dissection, ganglia were desheated and sliced into small pieces. Tissue was dissociated with 20 U/mL papain, 1 mg/mL collagenase and 10 mg/mL dispase for 60 min and neurons were dissociated by gently pipetting. Cell suspension was centrifuged at 180 g for three minutes and washed twice in Leibovitz's medium (L15) and once in Dulbecco's modified Eagle's medium (DMEM). Neurons were plated on glass coverslips coated with poly-l-lysine and incubated at 37°C (5% CO₂) with DMEM supplemented with 10% heat-inactivated bovine serum and 1% penicillin–streptomycin. Cells were used up to 24 h after plating.

In our experiments, Ca_V2.2 current was defined as the component of calcium current that is sensitive to 100 μmol/L Cd²⁺ in the presence of 2 μmol/L nifedipine, since Ca_V2.2 channels carried above 90% of the calcium current in SCG neurons, with no detectable Ca_V2.1 (P/Q) component. ¹⁶ SCG neurons were constantly bathed during recording (1–2 mL/min) with a solution containing (in mmol/L) 165 tetraethylammonium chloride (TEA-CL), 2 BaCl₂, 1 MgCl₂, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 8 glucose, 0.002 nifedipine and 0.0001 tetradotoxin, pH adjusted to 7.4 with tetraethylammonium hydroxide, to isolate barium currents. The pipette solution (internal solution) contained (in mmol/L) 140 CsCl, 20 TEA-Cl, 5 MgCl₂, 10 HEPES, 0.1 Cs₄BAPTA (BAPTA = 1, 2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), 4 Na₂ATP, 0.3 GTP and 0.1 leupeptin, and 2 GDPβS (where indicated), pH adjusted to 7.2 with CsOH. Nifedipine-containing external solution was prepared daily from 25 mmol/L absolute ethanol stock solution and protected from light. Piracetam was added into the bath solution and was applied by using a focal perfusion system that exchanges the solution locally within 5–10 s. All experiments were performed at room temperature (23–25°C). Reagents were obtained as follows: BAPTA (Molecular Probes, Eugene, OR, USA), L15 and DMEM (Invitrogen Corp., Carlsbad, CA, USA); all other chemicals and reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Currents were recorded in whole-cell voltage-clamp configuration ¹⁷ with an HEKA EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). Pipettes made of borosilicate glass and with a resistance of 1.8–2.2 MΩ were coated with Sylgard 184 (Dow Corning, midland, MI, USA). Capacity transients were cancelled and series resistance (2–7 MΩ) was compensated to >60%. Voltage pulse command protocols were generated, and data were digitized, recorded and analyzed using Pulse + PulseFit (HEKA Instruments, Inc., Bellmore, NY, USA). Steady-state currents were sampled at 10 kHz and were low-pass filtered at 3 kHz (3-pole Bessel filter).

Rat brain slice preparation and electrophysiology

To study CA1 pyramidal neurons from the hippocampus, we used male Wistar rats (250–300 g body weight), individually housed and maintained under controlled conditions (12 h normal light/dark cycles, 22–25°C, and 38% relative humidity) with food and water ad libitum. Five-week-old male Wistar rats were killed by decapitation under ether anesthesia. Brains were extracted and sliced (400 μm) with a vibratome. Slices were maintained at room temperature (24–26°C) for one hour in artificial cerebrospinal fluid (ACSF) containing (in mmol/L) 126 NaCl, 3 KCl, 1.5 CaCl₂, 1.5 MgCl₂, 1.25 Na₂HPO₄ and 10 glucose. ACSF was continuously bubbled with 95% O₂/5% CO₂ mixture and equilibrated to pH 7.4 with NaOH. Rate perfusion was 1–2 mL/minute. Subsequently, brain slices were transferred to a recording chamber and continuously superfused with ACSF or ACFS with 10 μmol/L piracetam warmed at 34°C. Cells were visualized by differential interference contrast microscopy using a ×40/1.0 objective in an upright microscope (FN1; Nikon, Melville, NY, USA). Patch pipettes were pulled from borosilicate glass and filled with an internal solution containing (in mmol/L) 145 KCl, 5 HEPES, 5 ethylene glycol tetraacetic acid, adjusted to pH 7.3 with KOH.

Membrane potential and current recordings were made in voltage and current clamp whole-cell configuration using a List EPC7 amplifier (Heka Elektronik). Capacity transients were cancelled using EPC7 circuitry and series resistances (4–5 MΩ) was compensated to >70%. Voltage pulse command protocols were generated, and data were digitized, recorded and analyzed using Pclamp6 (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA). Membrane voltage and currents were digitized using a 12-bit A/D Tecmar Lab Master Board (Scientific Solutions, Inc., Mentor, OH, USA) with a maximum digitization rate of 125 kHz and sampled at 10 kHz. Recordings were filtered at 3 kHz (3-pole Bessel filter). Total currents were elicited with a voltage step from −60 to +30 mV during 20 ms. Current traces were not corrected for linear leak current. Voltage responses were generated with 800 pA square pulses during 20 ms. Also, spontaneous activity was recorded for 45 min and intervals of 20 s every 10 min were stored to count the number of action potentials (APs) or excitatory postsynaptic potentials (EPSPs) in the control conditions, under 10 μmol/L piracetam and under 1 μmol/L ω-conotoxin. ω-Conotoxin was used to determine indirectly if the piracetam effects could be explained by Ca_V2.2 channel inhibition.

Hyperexcitability model

To study the effect of piracetam on epileptiform discharges from hippocampal CA1, we used the hyperexcitability model based on the application of low magnesium and high potassium. ¹⁸ Brain slices were superfused with free Mg²⁺ ACSF containing (in mmol/L) 126 NaCl, 7 KCl, 1.5 CaCl₂, 0 MgCl₂, 1.25 Na₂HPO₄, 10 glucose and adjusted to pH 7.4 with NaOH. Sustained spontaneous neuronal discharges were evident within approximately 30 min of superfusion of free Mg²⁺ ACSF. Nine of 12 recorded neurons presented epileptic activity characterized by high-frequency discharges of APs. We recorded spontaneous activity for 45 min and quantified the number of APs during 20-s recordings in control, low-magnesium–high-potassium solution and low-magnesium–high-potassium plus piracetam (10 μmol/L) solution.

Data analysis

Current amplitudes were taken as the mean value of recorded points between 4 and 5 ms after onset of the test pulse prior to reaching the peak current. Due to the magnitude of the cell current being cell-size dependent, aggregate current data are presented as current densities normalized to cell capacitance.

Dose–response data for the piracetam effect on native Ca_V2.2 channels were fitted to the function:

The conductance G for barium, G _Ba, was calculated utilizing the following equation:

The Boltzmann equation was used as

Origin 8.0 software was utilized to perform fit of data, and for statistical analysis (OriginLab Corporation, Northampton, MA, USA). All data are expressed as mean ± standard error of the mean (SEM) and comparisons between groups were analyzed using Student unpaired t-tests. P < 0.05 was considered to be statistically significant.

Piracetam inhibits Ca_V2.2 calcium channels in sympathetic neurons

Cyclic derivatives of GABA affect calcium signaling in neurons. Levetiracetam inhibits Ca_V2.2 channels in hippocampal neurons ¹¹ and piracetam inhibits neuronal HVA calcium channels in invertebrates. ¹⁰ However, it is not well established whether piracetam can act on Ca_V2.2 channels in vertebrates. The calcium current in SCG neurons is mainly mediated by omega conotoxin-sensitive channels (Ca_V2.2) in recordings on which L-type calcium channels have been blocked with 2 μmol/L nifedipine. ^14,16 Figure 1 shows the percent of nifedipine- and ω-conotoxin-sensitive channels in SCG neurons (n = 8). In our hands, the 18 ± 3% of calcium current was blocked by 2 μmol/L nifedipine and the 78 ± 4% was blocked by 1 μmol/L ω-conotoxin GVIA. According to previous reports, these data show that SCG neurons express predominantly the Ca_V2.2 channels. Along this work, we recorded calcium currents in the presence of 2 μmol/L nifedipine, and in such conditions, the Ca_V2.2 current represents 90% of the calcium current. Here we characterized the effect of piracetam on Ca_V2.2 channels in rat SCG neurons. Voltage pulses were elicited every six seconds from −80 to −8 mV for 20 ms in voltage-clamp whole-cell configuration, using barium as the charge carrier. A rapidly activated inward barium current was inhibited by near 77% after bath application of 10 μmol/L piracetam (n = 7, Figure 2Aa). The effect of piracetam reached its maximum after 24 s, remained stable for one minute and reversed rapidly by washing-out (Figure 2Ab). Current amplitude was measured isochronally at 4–5 ms from depolarizing pulse onset. Figure 2Ac shows a summary of average currents amplitude density in control (7.79 ± 1.90 pA/pF) and under 10 μmol/L piracetam (1.90 ± 0.27 pA/pF). Therefore, piracetam induced strongly, rapidly and reversibly inhibition on Ca_V2.2 channels in SCG neurons. Also, inhibition of Ca_V2.2 current depended on piracetam concentration. We assayed piracetam concentrations of 2, 10, 20 and 2000 μmol/L (n = 4 for each concentration) and the dose–response curve was fitted to a sigmoid function by the least-squares method (Figure 2B). The half-maximal concentration of piracetam (IC₅₀) was 3.4 ± 0.6 μmol/L with a maximal inhibition of 94 ± 2% at 2000 μmol/L (Figure 2Bb). The calculated Hill coefficient for piracetam effect was 1.1 ± 0.2 (Equation 1), suggesting a bimolecular interaction without cooperativity. Inhibition of Ca_V2.2 channels might be explained by direct interaction with piracetam or by a mechanism mediated by G-proteins; we explored both possibilities in this paper. OPEN IN VIEWER

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Figure 2

Piracetam inhibited Ca_V2.2 channels in superior cervical ganglion (SCG) neurons. (Aa) Representative voltage-clamp whole-cell recordings of calcium current before (1), during application of 10 μmol/L piracetam (2) and at wash-out (3) in isolated SCG neurons. (Ab) Time course of calcium current amplitude showing piracetam-induced inhibition. Piracetam was applied to the cell when indicated by the gray area. (Ac) Summary of the effect of piracetam on barium current in seven cells. (Ba) Representative superimposed current traces before and during application of 2 μmol/L (a), 10 μmol/L (b), 20 μmol/L (c) and 2000 μmol/L (d) piracetam. (Bb) Dose–response curve of the inhibition induced by piracetam (n = 4 for each dose). Percent of inhibition was determined as the difference between current amplitude before and during piracetam application divided by current before piracetam. Smooth curve represents sigmoid fit to data (Equation 1). The Hill coefficient obtained from the curve was 1.1, and IC₅₀ = 3.4 μmol/L. Data are shown as mean ± SEM. *P < 0.05

Piracetam enhances facilitation ratio of Ca_V2.2 channels

To explore the effect of piracetam on the biophysical characteristics of Ca_V2.2 channels, we examined possible alterations in the voltage-dependence profile. We elicited currents with voltage pulses at different potentials from −50 to +38 mV in steps of 8 mV. Figure 3A shows family currents before (a) and during 10 μmol/L piracetam application (b). The I–V curves show that Ca_V2.2 channels are activated between −40 and +40 mV, with the maximal current amplitude at −10 mV (n = 5, Figure 3Ac). Piracetam inhibited Ca_V2.2 current ranging from 55% to 75% depending on the activation voltage. The maximal inhibition by piracetam occurs at 6 mV, suggesting that this inhibition depends on voltage activation of the channel. We analyzed the conductance–voltage relationship and found that piracetam inhibited the conductance by near 70% (Figure 3Ad), induced a significant moderate shift of the half conductance (V _h) by −3 mV (Figure 3Ae, Table 1) and did not significantly affect the steepness factor k value (see Equations 2 and 3). As the effect of piracetam depended on voltage and shifted voltage dependence of channels, it resembles the effect of voltage-dependent inhibition mediated by G-proteins. To address if such mechanisms are related, we used the conditioning prepulse protocol to evaluate the extent of prepulse facilitation. This paradigm represents a widely used tool for identifying voltage-dependent inhibition mediated by G-proteins. ^19,20 In these experiments, currents were evoked every 10 s with a pair of 20-ms depolarizing test pulses to −8 mV from a holding potential of −80 mV, one before (P1) and the other 50 ms after (P2) a 30-ms conditioning prepulse (PP) to +80 mV (Figure 3B upper panel). The current in control conditions, before piracetam application, was bigger in P2. This increase of calcium current after a conditioning prepulse is defined as tonic inhibition. Tonic inhibition is the basal inhibition of Ca_V2.2 channels without stimulation of any GPCR due to a free pool of Gβγ proteins bounded to the channels. ²¹ De-polarizing prepulses also relieved much of the piracetam-induced inhibition (Figure 3Ba). We analyzed the extent of voltage-dependent inhibition in piracetam-treated SCG neurons using a facilitation ratio by comparing currents in the second test pulse with those in the first pulse. In control conditions, this facilitation ratio was 1.48 ± 0.03 (n = 7) and during piracetam application it rose to 2.00 ± 0.12 (n = 7, Figure 3Bb). These results together suggest that piracetam induces a voltage-dependent inhibition of Ca_V2.2 channels and increases facilitation ratio like the membrane-delimited mechanism of G-protein inhibition. OPEN IN VIEWER

Piracetam-induced inhibition of Ca_V2.2 channels is independent of GPCR signaling pathway

GPCR activation induces a voltage-dependent inhibition of Ca_V2.2 channels which is mediated by the Gβγ subunits. ^22,23 Since our previous experiments showed that piracetam induces a voltage-dependent inhibition like the G-protein-mediated inhibition of Ca_V2.2 channels, we hypothesized that piracetam acts through the G-protein signaling pathway. To address whether or not G-proteins are involved, we applied 2 mmol/L GDPβS into the recording pipette. This non-hydrolyzable analog of GDP prevents dissociation of G-protein subunits ²⁴ and then Ca_V2.2 channels could not be inhibited by piracetam if Gβγ dimer is the underlying mechanism of this inhibition. In the absence of piracetam, dialysis of 2 mmol/L GDPβS decreased the facilitation ratio from 1.48 ± 0.03 to 1.21 ± 0.05, according to the block of tonic inhibition. ²¹ Surprisingly, in the presence of piracetam and 2 mmol/L GDPβS, the facilitation ratio was 1.87 ± 0.08 (Figure 4A), similar to that observed in neurons under piracetam and without GDPβS (2.00 ± 0.12). This result suggests that piracetam enhances the tonic inhibition of Ca_V2.2 channels by a mechanism independent of G-proteins or GPCR activation. OPEN IN VIEWER

We further investigated if the mechanism of piracetam differs from GPCR activation by testing additive effects of piracetam and noradrenaline on the inhibition of Ca_V2.2 channels. Figure 4Ba shows representative superimposed current traces from control conditions, under 100 μmol/L noradrenaline, and after addition of 10 μmol/L piracetam in the presence of noradrenaline. We used saturating concentration of noradrenaline to rule out possible Gβγ proteins that were not activated at lower concentrations. Piracetam application on neurons bathed with noradrenaline inhibits even more Ca_V2.2 current. While the inhibitory effect of noradrenaline was 78 ± 4%, inhibition by piracetam plus noradrenaline was 96 ± 1% and therefore the effect of piracetam was not occluded by noradrenaline (Figure 4B). This result supports that piracetam-induced Ca_V2.2 inhibition is independent of the G-protein signaling pathway and raises the possibility that piracetam alters basic biophysical properties of Ca_V2.2 channels by a novel mechanism.

Piracetam reduces the frequency of APs and EPSPs in CA1 pyramidal cells

Having established the effect of piracetam on Ca_V2.2 channels in isolated sympathetic neurons, we tested the effect of piracetam on naturally connected central neurons in hippocampal slices. One of the clinical uses of piracetam is for treatment of some epilepsies. Previously, we showed that in chronic epilepsy, changes in intracellular calcium equilibrium coexist with alterations in excitability. ¹³ Since piracetam inhibited Ca_V2.2 channels in SCG neurons and some antiepileptic drugs target this channel in central neurons, we did a first approach to relate this piracetam-induced inhibition of Ca_V2.2 channels with its effects on hippocampal neurons. We first tested the effects of piracetam on the excitability of pyramidal neurons in slices of the CA1 region of hippocampus, comparing the shape of APs during a current stimulus. In Figure 5A, APs were evoked by a current stimulus of 800 pA during 20 ms. We found that piracetam decreased the duration of APs (black dotted line) when compared with control cells (black line), a result that is evident during the third AP. Piracetam also induced changes in resting membrane potential and in after hyperpolarization potential. Changes in the shape of APs are consistent with alterations in underlying ionic currents. Thus, total currents were elicited with voltage stimulus for 20 ms from −60 to +30 mV and were recorded from control- and piracetam-treated cells, revealing a slow ionic current component, consistent with calcium channels (Figure 5B). Total currents are presented without leak and capacitive subtraction. If piracetam acts on the calcium current in hippocampal neurons, subtraction of current in control and in piracetam should eliminate ionic currents carried by sodium and potassium. Accordingly, the total current subtraction (control minus piracetam-treated cells) revealed a typical current phenotype for HVA calcium channels (Figure 5Bb). OPEN IN VIEWER

Ca_V2.2 currents are involved in neurotransmitter release, and therefore inhibition of Ca_V2.2 current by piracetam should alter the frequency of EPSPs recorded in CA1 pyramidal neurons. Accordingly, 10 μmol/L piracetam application revealed a remarkable blockade of synaptically mediated activity. Figure 5C shows representative one-second recordings of spontaneous activity, without eliciting a current stimulus, of CA1 pyramidal neurons in control conditions (n = 7), under piracetam (n = 7) and under 1 μmol/L ω-conotoxin (n = 4). Piracetam significantly decreased the frequency of APs (P < 0.05). While 133 ± 11 APs were counted during 20-s recordings under control conditions, 97 ± 10 and 89 ± 12 APs were counted under piracetam and ω-conotoxin, respectively (Figure 5Cb). Also, piracetam significantly decreased the frequency of EPSPs (P < 0.001). While 80 ± 7 EPSPs were counted during 20-s recordings under control conditions, 18 ± 3 and 18 ± 5 EPSPs were counted under piracetam and ω-conotoxin, respectively (Figure 5Cc). These results suggest that piracetam alters the firing pattern and synaptic transmission on hyppocampal pyramidal neurons. In addition, the fact that ω-conotoxin GVIA, a specific blocker of Ca_V2.2 channels, induced a similar effect of piracetam, reducing the frequency of APs and EPSPs in hippocampal neurons, suggests a relation between the piracetam-induced inhibition of Ca_V2.2 current, observed in SCG neurons, and the piracetam-induced reduction of excitability in hippocampal neurons.

Piracetam diminishes hyperexcitability

Since piracetam reduces the frequency of APs of CA1 pyramidal neurons under control conditions, likely through inhibition of calcium channels, and calcium homeostasis is relevant in hyperexcitability, ²⁵ we investigated if piracetam is able to attenuate discharges in hyperexcited hippocampal neurons. We used the well-documented model of hyperexcitability based on low magnesium and high potassium. Low-magnesium-induced hyperexcitability in rat hippocampal slices has been extensively used to achieve pharmacological characterization of antiepileptic drugs and experimental analgesics. ¹⁸ In our experimental conditions, sustained spontaneous neuronal discharges were evident within approximately 30 min of low-magnesium and high-potassium application (n = 9). The number of APs during 20-s recordings in low magnesium–high potassium significantly increased (77.5 ± 0.9 APs/20 s), and decreased after adding 10 μmol/L piracetam to low-magnesium–high-potassium solution (37.3 ± 1.8 APs/20 s, Figure 6). Then, under the same hyperexcitability condition, piracetam significantly reduced the frequency of APs to near levels of that observed before hyperexcitability onset (33.0 ± 1.0 APs/20 s). OPEN IN VIEWER

Taken together, piracetam inhibits Ca_V2.2 current in sympathetic neurons and reduces the frequency of APs in normal and hyperexcited hippocampal slices. These effects might be causally related and could be underlying the antiepileptic effect of piracetam.

Ca_V2.2 channel inhibition by piracetam in peripheral neurons

To properly study the actions of piracetam on Ca_V2.2 channels, we used a preparation of primary SCG neurons, in which the major component of the whole-cell calcium current is carried by Ca_V2.2 channels. ^16,26,27 In here, the contribution of L-type Ca²⁺ (Ca_V1) channels was excluded with nifedipine application, and the contribution of P/Q-type (Ca_V2.1) and R-type (Ca_V2.3) channels, which represents a negligible portion of the whole Ca²⁺ current in cultured rat SCG neurons, was not considered. This preparation, therefore, allowed us to examine the effects of piracetam on a homogeneous neuronal population of Ca_V2.2 channels in vitro. We found that piracetam suppresses dihidropyridine-insensitive Ca²⁺ current by about 90%. We previously described a similar suppression response with 10 μmol/L ω-conotoxin GVIA, ¹⁴ indicating that the whole population of Ca_V2.2 channels in SCGs is sensitive to piracetam. HVA calcium channel inhibition by piracetam has been previously reported in neurons from snail Helix pomatia. ¹⁰ However, there are some subtle differences. Near 19% of HVA current was inhibited by 2 mmol/L piracetam after more than one minute in snail neurons while 10 μmol/L piracetam inhibited Ca_V2.2 current by 77% after 30-s application in SCG neurons. We cannot rule out if these discrepancies depend on differences in taxonomical groups or in technical approach.

In addition, piracetam inhibited Ca_V2.2 channels in primary SCG neurons in a dose-dependent manner, with an IC₅₀ of 3.4 μmol/L, and a unitary value for the Hill coefficient, ruling out cooperative actions, which is consistent with a high selectivity and specificity for piracetam on Ca_V2.2 channels. In cultured hippocampal neurons, levetiracetam inhibits N-type calcium current with an IC₅₀ of 14.7 μmol/L, ¹¹ suggesting that both compounds act on the same concentration range and that piracetam could be even more potent than levetiracetam for inhibiting Ca_V2.2 channels. In fact, levetiracetam inhibits only half or less Ca_V2.2 channels at maximal doses, ¹¹ while piracetam inhibited the whole population of Ca_V2.2 channels in our experiments. Even though levetiracetam and piracetam are compounds related in structure, they have some chemical differences that can explain variations in their effects. Piracetam has the simplest structure of all of the nootropic compounds. Levetiracetam, on the other hand, has an additional ethyl group. Accordingly, levetiracetam has been grouped into antiepileptic drugs while piracetam has been grouped into cognitive enhancers. ¹ Subtle differences in chemical structures might explain their pharmacological differences and, in the same sense, might explain disparities between Ca_V2.2 channel inhibition induced by levetiracetam or by piracetam.

Possible mechanism of action of piracetam on Ca_V2.2 channels

We then sought to determine if the observed decrease in Ca_V2.2 current amplitude was caused by a direct action of piracetam on the channel, or if it was instead the result of indirect action involving a G-protein-mediated mechanism. Ca_V2.2 current traces in the presence of piracetam are kinetically different from traces in the absence of piracetam (see Figure 2Ba under letter b and Figure 3Ba under P1). Besides, piracetam induces a moderate shift of voltage-dependence and increases the facilitation ratio of Ca_V2.2 channels. Gβγ subunits coupled to GPCRs have similar effects. ^22,23 However, when we tested G-proteins on piracetam action, we found no relation. Piracetam action is not hampered by dialysis of GDPβS or occluded by application of saturating concentration of a GPCR agonist, suggesting that piracetam acts directly on Ca_V2.2 channels. Previous studies have demonstrated that piracetam interferes with the pore region of some ion channels, as in the case of GABA_A receptors. ²⁸ In addition, piracetam has great affinity for polar heads of phospholipids, ²⁹ inducing a new organization of lipids, and in such a way, piracetam increases neuronal membrane fluidity ^4,30 and facilitates mobile phospholipid-complex formation. ³¹ Thereby, we propose that these changes in membrane properties can cause interference with the intrinsic channel gating or the binding of regulatory subunits (i.e. Ca²⁺ channel β-subunit and G-protein βγ-subunit). In conclusion, piracetam revealed a novel Ca_V2.2 inhibition, mimicking some characteristics of voltage-dependent inhibition by G-proteins likely acting directly on the channel molecule.

Piracetam effect on hippocampal CA1 pyramidal neurons

Piracetam also has effects on central neurons. In hippocampal CA1 pyramidal neurons, we observed that piracetam altered the firing patter in response to a specific current stimulus. Taking into account inhibition of Ca_V2.2 channels in SCG neurons, we assessed, by indirect measurements, if the piracetam effects in CA1 neurons were a consequence of inhibition of Ca_V2.2 channels. We recorded total currents in the somatic region and found that most likely calcium currents are altered by piracetam, since subtraction of currents recorded in the absence or presence of piracetam gave a current with a HVA phenotype. However, Ca_V2.2 channels are mainly located in synaptic ends, being necessary for neurotransmitter release. ³² Accordingly, we found that the frequency of EPSPs diminishes in the presence of piracetam. We compared the effect of piracetam with the effect of a specific blocker of Ca_V2.2 channels, the ω-conotoxin GVIA. ω-Conotoxin GVIA also diminished the frequency of APs and EPSPs as piracetam did. Together, these results suggest that the effect of piracetam on the excitability of hippocampal CA1 neurons is related with the inhibition of Ca_V2.2 channels. Previous results have shown that nootropic drugs, such as levetiracetam, inhibit Ca_V2.2 channels, ^11,12 which can explain the reduction of vesicle release in synapses after a levetiracetam bath. ³³ Piracetam and levetiracetam might share this mode of action to diminish hyperexcitability, yet they are commonly used for different pathologies treatment. On the other hand, the fact that piracetam reduced excitability can be explained by additional alterations on other conductances such as sodium or potassium channels. For instance, it has been previously reported that piracetam inhibits calcium-activated potassium channels in invertebrate neurons. ¹⁰ We also observed that during a current stimulus, piracetam also induced changes in resting membrane potential, after hyperpolarization potential and AP duration, suggesting that piracetam could be targeting potassium channels such as delayed rectifiers, calcium-activated potassium channels or M-current potassium channels. Further studies should address the effect of piracetam on other ion channels which will lighten the whole mechanism of piracetam as a nootropic compound.

We found that piracetam also diminishes the frequency of APs in hyperexcited CA1 pyramidal neurons. Hyperexcitation is caused when excitatory neurotransmitter release is augmented or inhibitory neurotransmitter release is diminished. ^34,35 Since neurotransmitter release requires activation of voltage-gated calcium channels and Ca_V2.2 channels have been found in the hippocampus, ³⁶ inhibition of Ca_V2.2 channels could explain the effect of piracetam on hyperexcitability. Other antiepileptic drugs also target calcium channels ³⁷ or related subunits, such as alfa2delta auxiliary subunit. ^38–40 These results together support that piracetam diminishes the number of APs in control and hyperexcited states, and suggest a likely mechanism through the inhibition of Ca_V2.2 channels.

Our results provide evidence of a novel inhibition of Ca_V2.2 channels showing an increased facilitation ratio that is not mediated by G-proteins. This finding advances our understanding in calcium channel regulation, bridging the gap with clinical applications.

Author contributions: All authors contributed to the design of the studies, execution of the experiments and were directly involved in analysis of the data and preparation of the manuscript.