Inhibition of Dendritic l-type Calcium Current by Memantine in Frog tectum

The aim of the study was to explore the effects of memantine on responses elicited in the frog tectum by the bursts of spikes of moderate strength of a single retina ganglion cell and to gain an insight about the effect of memantine on the L-type Ca2+ current. Material and Methods. The experiments were performed in vivo on adult frogs (Rana temporaria). An individual retina ganglion cell (or its retinotectal fiber) was stimulated by current pulses delivered through a multichannel stimulating electrode positioned on the retina. Responses to the discharge of a single retinal ganglion cell were recorded in the tectum by an extracellular carbonfiber microelectrode positioned in the terminal arborization of the retinotectal fiber in the tectum layer F. The solution of memantine (1-amino-3,5-dimethyladamantane) hydrochloride (30 or 45 μM) was applied onto the surface of the tectum by perfusion at a rate of 0.4 mL/min. Results. Memantine (30–45 μM) largely inhibited the L-type Ca2+ channel-mediated slow negative wave and late discharges seen in the tectum responses without any effect on fast synaptic retinotectal transmission. Conclusions. Our results suggest that the neuroprotective effect of memantine could arise not only through the inhibition of the NMDA receptor current but also through the suppression of the L-type Ca2+ current.

We have previously demonstrated that a discharge of action potentials of a single frog retina ganglion cell can evoke a long-lasting suprathreshold excitation of recurrent neurons of the tectum column (14) due to the activation of a slow dendritic L-type Ca 2+ current seen in the recordings of tectal responses as a slow negative wave (sNW) (15).The excitation of the recurrent neurons is evidenced in the recordings as recurrent synaptic potentials (rSPs) superimposed on the sNW.In the present study, we aimed at exploring the action of memantine on the sNW of frog tectal responses and thus at gaining an insight about the effect of memantine on the L-type Ca 2+ current.

Material and Methods
The experimental procedure is described in detail in our earlier papers (16,17).Here we shortly emphasize the main points.
The experiments were performed in vivo on 7 adult frogs (Rana temporaria).All the experiments in this study were carried out in accordance with the Principles of Laboratory Animal Care (NIH publication No. 86-23 revised in 1985) and the European Communities Council Directive (November 24, 1986; 86/609/EEC) and were approved by the Animal Care and Use Committee of the State Food and Veterinary Service of Lithuania (No. 0167).During surgical manipulations, frogs were anesthetized with a high concentration of CO 2 .The dorsal tectum was exposed in the manner described by Maturana et al. (18).The retina, contralateral to the opened tectum, was prepared in the way de-scribed by George and Marks (19).The eyeball cavity was filled, and the exposed dorsal tectum was perfused with Ringer's solution (in mM: 116, NaCl; 2.5, KCl; 1.8, CaCl 2 ; 1.0, MgCl 2 ; 1.2, NaHCO 3 ; and 0.17, NaH 2 PO 4 ×2H 2 O; pH, 7.3-7.4).Frogs were immobilized using an intramuscular injection of dtubocurarine (0.15-0.3 mg), intubated and ventilated by a mechanical ventilator with a tidal volume of 2-3 mL at a frequency of 6-10 breaths per minute.The subsequent injections of d-tubocurarine (0.05-0.1 mg) were applied every hour to keep the frog immobilized during the experiment.Frogs were slightly anesthetized by placing them into the solution of MS-222 at a concentration of 50 mg/L (20).All the recordings were carried out in the dark at ambient temperatures of 17°C-22°C.At the end of the experiments, the animals were sacrificed with an overdose of an anesthetic.
An 8-channel electrode was used for the stimulation of the retina.The electrode consisted of 8 tungsten wires (channels) 40 μm in diameter bunched at 50-150 μm distances between the centers of different wires.The stimulating electrode was placed on the nasoventral quadrant of the naked retina.The single current pulses of magnitude of 11.8-22.5 μA and duration of 50 μs, or a train of 2-4 of such pulses were applied to the retina through a pair of stimulating electrode channels using an isolator (World Precision Instruments).The excitation of a single ganglion cell or its axon was achieved by slightly moving the stimulating electrode, switching between 8 channels of the electrode, and changing the strength of the current pulse.
The solution of memantine (1-amino-3,5-dimethyladamantane) hydrochloride (30 or 45 μM) was applied to the surface of the tectum by perfusion at a rate of 0.4 mL/min.It took approximately 3 minutes to replace the solution superfusing the tectum.The solution of the drug was prepared just before the use.Memantine hydrochloride was purchased from Sigma-Aldrich Co.
Responses evoked in the tectum layer F were recorded using a carbon-fiber microelectrode with a 50-70-μm tip.Responses consisted of individual retinotectal presynaptic action potentials (APs) followed by individual fast synaptic potentials (fSPs), the sNW with a duration of hundreds of ms, and rSPs superimposed on this wave (Fig. 1A).The amplitudes of the AP, fSP, and sNW, and the duration of the sNW were measured.The amplitude of the sNW (A sNW ) was evaluated as an average voltage in the time interval of the response from 60 to 120 ms.The duration of the sNW (T sNW ) was measured as the time interval from the peak of the last fSP in the burst to the end of the wave that was determined as the time moment when the level of the wave approached the baseline within a noise level of ~10 μV.
Data are expressed as mean±SE.The paired t test with the confidence level of 0.95 was used for the evaluation of the statistical significance of the results.

Results
Recordings from 7 individual retinotectal axon terminals located in the dorsal tectum zones (areas 1, 3, 4, or 6) of 7 frogs were obtained.The stimulation of a single retina ganglion cell or its axon was considered reliable because an increase in the stimulation strength by up to 6 μA had no effect on the response, and a decrease by 0.2-0.8μA completely abolished it.It was also confirmed that recordings were taken precisely at the tectum layer F: the measured depth of the tip of the recording microelectrode from the tectum surface was 260-280 μm, and the latency of the individual action potentials in respect of the stimulus artifact was 7.47±0.19ms (range, 6.7-8.1 ms).
An individual retina ganglion cell was stimulated by a train of 2-4 current pulses with the interpulse intervals of 10 ms to evoke tectal responses with the sNW present (Fig. 1A).Then, 30 μM of memantine was added to the perfusion solution, and a change in the amplitude and duration of the sNW and fSPs was monitored during the perfusion when repeating the same stimulus (Figs. 1B and C).In 3 experiments, the concentration of memantine was increased afterward to 45 μM (Figs.1D and  E).Figs. 1 and 2A show that the amplitude of the fSP, A SP , did not change significantly during the perfusion with the memantine solution at a concentration of 30 μM (134±16 μV in control versus 140±17 μV with memantine; n=7; P=0.1).The amplitude and duration of the sNW, A sNW and T sNW , decreased from 93±21 μV and 157±20 ms in control to 36±9 μV (n=7, P=0.006) and 84±17 ms (n=7, P=0.0002) when the memantine solution (30 μM) was applied (Figs.1B, 2B, and 2C).An increase in the concentration of memantine to 45 μM virtually abolished the sNW (A sNW =0.8±5 μV, n=3, P=0.04; T sNW =31±12 ms, n=3, P<0.05) (Figs. 1E, 2B, and  2C).In all the experiments, the effect of memantine became obvious within the first 15 minutes of perfusion with the memantine solution.It took 12 to 50 minutes (31±6 minutes on the average) for the effect to develop fully.The dependence of amplitude of the fSP, and amplitude and duration of the sNW on the time of perfusion with the memantine solution is shown in Fig. 3, demonstrating that the amplitude of the retinotectal synaptic transmission remained unchanged and the amplitude and duration of the sNW decreased during the perfusion.
The surface of the frog brain was washed with Ringer's solution for 1 hour after the perfusion with the memantine solution, but no change in the tectal Fig. 1.Effects of memantine on tectal responses a, tectal response to the stimulation of an individual retina ganglion cell by a pair of current pulses with an interpulse interval of 10 ms under control conditions.The response consists of individual retinotectal action potentials (AP), fast retinotectal synaptic potentials (fSP), slow negative wave (sNW), and recurrent synaptic potentials (rSP) superimposed on this wave.A SP , amplitude of the first fSP; A sNW , amplitude of the sNW; T sNW , duration of the sNW; Art, stimulus artifact; t perf , duration of perfusion with the memantine solution.b, tectal response after 20-minute perfusion with 30-μM memantine (t perf =20 min).C, tectal response after 50-minute perfusion with 30-μM memantine (t perf =50 min).D, tectal response after 20-minute perfusion with the memantine solution at a greater concentration (45 μM) (t perf =50+20 min).Onset of the sNW can be delayed when the dendritic L-type Ca 2+ currents decreased sufficiently.e, tectal response after 50-minute perfusion with the memantine solution at a greater concentration (45 μM) (t perf =50+50 min).The application of memantine (30 μM) resulted in a decrease in the amplitude and duration of the sNW more than 3 and 2 times, respectively.An elevation in the concentration to 45 μM abolished the sNW completely.A number of the rSPs decreased following a decrease in the sNW.responses was seen.Therefore, the effect of memantine failed to be wash out.

Discussion
The obtained results clearly show that the sNW of frog tectal responses was decreased or even abolished by memantine.This indicates that memantine inhibits calcium influx through dendritic L-type Ca 2+ channels, since the sNW is generated by the activation of the L-type calcium current in the dendrites of recurrent pear-shaped neurons of the frog tectum column as it was demonstrated in our previous study (15).It is evident from the latter and other our studies (16,17) that the sNW remains stable within at least 2 hours of stimulation and recordings under control conditions.Thus, the time interval of 12 to 50 minutes (31±6 minutes on the average) of perfusion with the memantine solution, required for the effect to develop, was smaller than the time interval during which the stable control responses are usually recorded.Moreover, this matched the period needed for the development of the effect of nifedipine, a specific L-type Ca 2+ channel blocker (15,16).However, contrarily to the effect of nifedipine, we did not manage to wash out the effect of memantine.This agrees with the results of other studies.For example, Frankiewicz et al. (21) noted that the recovery of the response was seen 4 hours after the washout of memantine.If it took so long in the in vitro experiments on thin (~400 μm) hippocampal slices, it is hardly possible to expect washout of the effect of memantine in the in vivo experiments on the whole frog brain when only part of the upper brain surface was perfused with Ringer's solution.The failure to wash out the effect of memantine points out another important aspect of the action of memantine, namely, its potent noncompetitive nature of binding to receptors or other effectors.
It is important to note that the strength of the retinotectal synaptic transmission (amplitude of synaptic potential, A SP ) was not significantly affected by memantine (Fig. 2A).A small insignificant increase in this amplitude could be attributed to the enhanced tonic potentiation (22) of the retinotectal transmission due to an increased ambient concentration of acetylcholine induced by a relatively intense stimulation of the tectum column.
The concentrations of memantine (30 and 45 μM) used in our experiments were similar to the concentrations used by other researchers to investigate the effect of memantine on the NMDA receptor current in cell cultures (23) and brain slices (21).
The data of our experiments do not answer the question whether memantine directly blocks L-type Ca 2+ channels or whether its inhibitory effect on the L-type calcium current is indirect, through some signal transduction pathways targeting the gating and/or conductance of L-type Ca 2+ channels.A diverse action of memantine discussed in the introduction favors the latter point of view.For example, it has been established that the activity of L-type Ca 2+ channels is positively modified by neuromodulators such as acetylcholine, serotonin, noradrenaline, and glutamate metabotropic receptors, and negatively modified by GABA(B) receptors (24).However, we have not found in the literature any evidence on the activation of GABA(B) receptors by memantine.Considering approximately equal times required for the development of the effect of memantine and nifedipine, an L-type Ca 2+ channel blocker (15,16), the direct action of memantine on L-type Ca 2+ channels may be inferred.Certainly, in vitro experiments with the intracellular recordings of dendritic L-type Ca 2+ currents need to be done to address this question.
Experimental data show that L-type Ca 2+ channels are involved in the pathogenesis of Alzheimer's disease: amyloid-β peptides, a causative factor for Alzheimer's disease, potentiate the influx of calcium into neurons via L-type Ca 2+ channels (25).The blockers of those channels inhibit the accumulation and aggregation of amyloid-β peptides (26) and prevent neurons from amyloid-β peptideinduced apoptosis (27).Researchers suggest that the specific L-type Ca 2+ channel blockers could be used for the treatment of Alzheimer's disease (27,28).Our findings provide evidence to support this view.It has been established that L-type Ca 2+ channels are expressed in human neurons.Studies indicate that the activity of L-type Ca 2+ channels is altered by a number of neuromodulators in the brain of reptiles and mammals (24,25,29,30).Therefore, we expect that the effect of memantine we have described in frogs may be seen in human neurons, too.In such a case, our results may be useful for the development of new approaches to the treatment of Alzheimer's disease.

Conclusions
Memantine decreased the sNW elicited in frog tectal responses by a burst of action potentials of a single retina ganglion cell.As the sNW is generated by the dendritic L-type Ca 2+ current in pear-shaped neurons of the tectum column, this shows that memantine suppresses the L-type Ca 2+ current.Thus, the neuroprotective effect of memantine could be mediated not only through the inhibition of the NMDA receptor current but also through the suppression of the L-type Ca 2+ current.

acknowledgments
This study was supported by the Ministry of Education and Science of Lithuania.We are thankful to Julija Baginskaitė, the University of Edinburgh, for her help with the revision of the manuscript.

statement of Conflict of Interest
The authors state no conflict of interest.

Fig. 3 .
Fig. 3. Dependence of amplitude of the synaptic potential of retinotectal transmission, A SP (a), and the amplitude and duration of the slow negative wave, A sNW (b) and T sNW (C), respectively, on the time of perfusion with the memantine solution at a concentration of 30 μM Dashed lines show trends of the data points with R 2 indicated next to them.