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Thus, we investigated to which extent Cav2. A Normalized current responses of Cav2. One of at least four experiments with similar results is shown. For statistics see Table 2. Source data provided in Figure 5—source data 1. We also investigated if post-burst afterhyperpolarizations would allow Cav2.

We first determined the recovery from inactivation of Cav2. This may be due to the different pulse protocols inducing channel inactivation over different time periods and may stabilize inactivated states with different recovery times.

Using whole-cell patch-clamp recordings Figure 6 , we therefore measured the steady-state inactivation of R-type I Ca in identified SN DA neurons in mouse midbrain slices. First, R-type current i. This observation is in accordance with the positive activation voltage-range of Cav2. The R-type current V 0. Since R-type currents may include current components not mediated by Cav2. Although the slope of the voltage-dependence of activation was different for the SNXsensitive current as compared to R-type current, steady-state inactivation was again observed at significantly more positive voltages as compared to control current — Left panel: representative current traces of recordings at —20 mV test potentials holding potential — mV ; notice that similar amplitudes of control and R-type current were chosen to facilitate comparison of current kinetics.

Right panel: voltage-dependence of steady-state activation and inactivation, curve fits to a Boltzman equation of all individual data points are shown. For parameters and statistics see Table 3. Left panel: Representative recording of a SNX sensitive current component compared to the respective control holding potential — mV, —10 mV test potential.

Steady-state activation and inactivation of control and SNXsensitive current. Test protocols: holding potential — mV; voltage-dependence of activation: ms test potentials to indicated voltages; voltage-dependence of inactivation: 5 s conditioning pulses to indicated voltages preceded I control and followed by 20 ms test pulses to V max.

C Exemplary neuron as seen under the patch-clamp microscope with patch pipette next to it left; BF, brightfield and a neuron after histochemical staining for tyrosine hydroxylase TH, green and neurobiotin Nb, red. Detailed parameters and statistics are given in Table 3.

Source data provided in Figure 6—source data 1. We independently also tested the availability of SNXsensitive R-type currents at more depolarized voltages using perforated patch recordings Figure 6—figure supplement 1 , which allow very stable recordings in identified SN DA neurons Figure 6—figure supplement 1I, J. We held cells at a more positive holding potential of —60 mV, a voltage within the spontaneous depolarization phase of the ISI, where a substantial fraction of Cav2.

Square pulses 50 ms were applied to 0 mV from a holding potential of —70 mV top B Current amplitude values plotted as a function of time.

E Left: Depolarizations of 50 ms were repeated every 10 s to the indicated potentials from a holding potential of —70 mV inset. Data were fitted to a Boltzman function. At each test potential V I Ca was estimated at the peak of the current trace. Left: To determine the voltage-dependence of steady-state inactivation, test pulses of 50 ms to 0 mV were preceded by 1 min prepulses to voltages from —80 to —10 mV.

This stimulation protocol inset was better tolerated by cultured DA midbrain neurons than the classical protocol used previously with other cells Calorio et al. Inactivation curves could be best fit to Boltzman functions with the following parameters: R-type: V 0. This is due to a run-down of SNX-sensitive current during the repeated depolarizations used to determine the voltage-dependence of gating. This has only minimal effects on gating parameters. Source data provided in Figure 7—source data 1.

We also determined the voltage-dependence of steady-state activation Figure 7E and inactivation Figure 7F before non-L, control and during application of nM SNX and calculated parameters for the SNXsensitive R-type current by subtracting the current resistant to nM SNX from the control current legend to Figure 7 for details.

SNXsensitive currents blue activated with a half maximal activation voltage V 0. To determine the steady-state inactivation, test pulses of 50 ms to 0 mV were preceded by 1 min prepulses to voltages from —80 to —10 mV Figure 7F.

SNXsensitive current inactivation was best fit with a V 0. Finally, we tested if SNX also affects the spontaneous AP firing of these cultured midbrain neurons.

In current-clamp recordings nM SNX significantly reduced the spontaneous firing frequency from 4. This may therefore also be of pathophysiological relevance given the previously reported pathogenic potential of Cav2.

The large shift of V 0. Our data predict that this contribution is higher in SN DA neurons than in cultured cells. However, this does not exclude a modulatory role of other posttranslational modifications of Cav2.

Direct proof for the existence of such complexes would require biochemical studies requiring immunoprecipitation of Cav2. We also found no suitable combination of antibodies raised in different species for such studies. This would be methodologically extremely challenging in these neurons.

We have previously shown that Cav2. At least in this rodent model of PD, the observed protection provides strong evidence for a role of these channels in PD pathology. Pharmacological inhibition of Cav2. Our previous preclinical findings identified also Cav2. Therefore, the inhibition of Cav2. Our findings reported here provide the rationale for exploring novel neuroprotective strategies based on Cav2. These could take advantage of the predicted strong dependence of continuous Cav2.

At present, it is unclear to which extent other high-voltage-activated Cav2 channels Cav2. While SNX is highly suitable to isolate Cav2. Therefore, one can argue that in current-clamp recordings, 50— nM SNX could alter pacemaking or the AP shape by effectively blocking Kv4. However, the observed SNXinduced shortening of APs and the reduction of spontaneous firing are difficult to reconcile with a block of I A channels, which typically induces a broadening of APs in several neuronal preparations Kim et al.

Therefore, the observed SNX effects in cultured neurons provided no final answer to the question about a potential role of Cav2. In brain slices of Cav2. Similarly, inconsistent findings for the contribution of LTCC currents for pacemaking in slice recordings Dragicevic et al.

The freshly extracted mouse brains from 12 to 14 weeks old male mice were snap-frozen in isopentane Carl Roth, catalog After cooling the coverslips on dry ice, regions of interest were punched under a dissection microscope using a pre-cooled sample corer Fine Science Tools, Germany VTA: inner diameter 0. The tissue punches were transferred into an Eppendorf tube and again snap-frozen in liquid nitrogen.

The sections were coverslipped immediately with Eukitt Sigma-Aldrich, catalog , left to harden overnight and imaged on a bright-field microscope at 2 x. Importantly, all assays specifically recognized the corresponding fragment even in the presence of a fold higher concentration of other splice variants Figure 2—figure supplement 1. Samples without template served as negative controls. The expression of seven different endogenous control genes was routinely measured and used for data normalization as previously described Vandesompele et al.

Briefly, data were normalized to the most stably expressed endogenous control genes Gapdh and Tfrc determined by geNorm Figure 2—figure supplement 1 , Supplementary file 1. Normalized molecule numbers were calculated for each assay based on their respective standard curve.

Standard curve parameters are given in Supplementary file 3. Details about assay specificity are given in Figure 2—figure supplement 1 and Supplementary file 3.

For transient transfections hCav2. Cell stocks of passage 8 were frozen and cultures were re-expanded from stocks for not more than 20 passages.

TsA cells were cultured as described Ortner et al. For splitting, cells were dissociated using 0. TsA cells were replaced and freshly thawed when they exceeded passage no. For electrophysiology, cells were plated on 10 cm culture dishes and subjected to transient transfections on the following day.

As described in Tomagra et al. Neurons were plated at final densities of cells per mm 2 on Petri dishes. Cultured neurons were used at 8—9 days in vitro DIV for current-clamp and voltage-clamp experiments. Petri dishes were coated with poly-L-Lysine 0. For whole-cell patch-clamp recordings, patch pipettes with a resistance of 1. All voltages were corrected for a liquid junction potential JP of —9 mV Lieb et al.

The pipette internal solution for recordings of Cav2. The pipette internal solution for recordings of Cav1. The bath solution for recordings of Cav2.

The bath solution for recordings of Cav1. Current-voltage I-V relationships were obtained by applying a 20 ms long square pulse protocol to various test potentials 5 mV voltage steps starting from a holding potential of — mV or —89 mV recovery of inactivation.

The resulting I-V curves were fitted to the following equation:. The voltage dependence of inactivation was assessed by application of 20 ms test pulses to the voltage of maximal activation V max before and after holding the cell at various conditioning test potentials for 5 s 30 s inter-sweep interval; 10 mV voltage steps; holding potential — mV.

Inactivation was calculated as the ratio between the current amplitudes of the 20 ms test pulses. Steady-state inactivation parameters were obtained by fitting the data to a modified Boltzmann equation:. The amount of inactivation during a 5 s depolarizing pulse from a holding potential of — mV to the V max was quantified by calculating the remaining current fraction after 50, , , , , and ms.

Recovery from inactivation was determined by 10 ms test pulses to V max at different time-points in s: 0. Window current was determined by multiplying mean current densities by fractional currents form steady-state inactivation curves to obtain the fraction of available channels at a given potential as described previously Hofer et al. Traces were sampled at 10 kHz and filtered using a low-pass Bessel filter set at 2 kHz.

Off-line data analysis was performed with pClamp Fast capacitive transients due to the depolarizing pulse were minimized online by the patch-clamp analog compensation.

For current-clamp experiments the pipette internal solution contained in mM: gluconic acid potassium salt: K-gluconate , 10 HEPES, 0. Data analysis was performed using Clampfit software. To study the contribution of Cav2. SNX nM was used in current- and voltage-clamp experiments. Whole-cell patch-clamp recordings were performed essentially as previously described Benkert et al.

Chemicals were obtained from Sigma Aldrich unless stated otherwise. Patch pipettes 2. Only TH and neurobiotin-positive cells were used for the statistical analysis. Inactivation was calculated as the ratio between the current amplitudes of the test versus control pulse. Data were not corrected for liquid junction potential —5 mV, measured according to Neher, Steady-state activation and inactivation curves were fitted as described above.

The brain was rapidly removed and a block of tissue containing the mesencephalon was immediately dissected. Brain slices were transferred into carbogenated artificial cerebrospinal fluid ACSF. Biocytin-streptavidin labeling was combined with TH-immuno-histochemistry Hess et al.

During the perforation process, the electrophysiological identification of the neuron was performed in current clamp mode. Perforated patch recordings were performed using protocols modified from Horn and Marty, and Akaike and Harata, ATP and GTP were omitted from the intracellular solution to prevent uncontrolled permeabilization of the cell membrane Lindau and Fernandez, The patch pipette was tip filled with internal solution and back filled with 0.

Amphotericin was dissolved in dimethyl sulfoxide final concentration: 0. The used DMSO concentration had no obvious effect on the investigated neurons.

In the analyzed recordings R a was comparable, did not change significantly over recording time, and was not significantly different between the distinct experimental groups. A change to the whole-cell configuration was indicated by a sudden change in R a and diffusion of tetraethylrhodamine-dextran into the neuron. Such experiments were rejected. GABAergic and glutamatergic synaptic input was reduced by addition of 0.

Target genes, visualized with Atto fluorophore, were co-stained with Tyrosine hydroxylase TH , visualized with AlexaFluor, as a marker for dopaminergic neurons. The algorithm delineates cell shapes according to the TH marker gene signal and quantifies the number and the area of target-mRNA-derived fluorescence dots. Target probe hybridization results in one fluorescent dot for each mRNA molecule, allowing determination of target-mRNA molecule numbers, independent from fluorescence-intensities.

Cryosectioning, laser-microdissection, reverse transcription: Cryosectioning, UV-laser microdissection UV-LMD and reverse transcription were carried out similarly as previously described in detail Benkert et al. Reverse transcription was carried out directly without a separate RNA isolation step in a one-tube procedure. All cDNA samples were precipitated as described Liss, Only cDNA pools expressing the correct marker gene profile i.

For Cav2. Primer sequences are given in Supplementary file 2. Outer duplex primer pairs were chosen using Oligo7. TaqMan assays were carefully established and performance was evaluated by generating standard curves, using defined amounts of cDNA derived from midbrain tissue mRNA , over four magnitudes of fold dilutions as templates, in at least three independent experiments, as described Duda et al.

Details of all qPCR assays TaqMan , and standard curve parameters used for analysis are provided in Supplementary file 4. Assay parameters were as follows: threshold for analysis: 0.

Data were analyzed using Clampfit Data were analyzed by appropriate statistical testing as indicated in detail for all experiments in the text, figure and table legends.

All data generated or analyzed during this study are included in the manuscript and supporting files. Raw data have been provided for mean population data shown in figures and tables. Our editorial process produces two outputs: i public reviews designed to be posted alongside the preprint for the benefit of readers; ii feedback on the manuscript for the authors, including requests for revisions, shown below.

We also include an acceptance summary that explains what the editors found interesting or important about the work. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Ideally, this would be provided by knockdown experiments showing reduced pacemaking and CaV2. As an alternative, a proximity ligation assay could be done to at least show assembly of b2 and CaV2. The reviewer makes some good suggestions for reorganizing the manuscript that should be considered. If possible, the isolated CaV2. Note that Supp Figure 1 is referenced in the paper after citing Supp Figure 2 and 3. Please revised. The action potential traces in Figure 1C do not seem to be a good representation of the maximum time-derivative of voltage.

Please revise. The reviewer’s suggestion is to make current Figure 5 the new Figure 3. Figure 4 should remain as such. Current Figure 3 and Figure 6 should be merged into new Figure 5. Alternative, current Figure 3 should become a supplemental figure of new Figure 5 current Figure 6. Panel C of this figure is not mentioned in the main text.

Please correct. In fact, this panel could go into a supplemental figure. Please verify. While the authors appropriately cite Jones et al. As currently written, the authors imply that these two papers validate the statement in line that R-type currents fully inactivate at voltages positive to mV. Reviewer recommends that this point be further clarified in the text.

This should be clearly described. Does it demonstrate similar properties? We fully agree with the reviewers that it is a relevant point but we do not see how this would be technically feasible. We ask the reviewers to consider the following:. We have already shown that, while SNX reduces pacemaking in cultured embryonic dopamine neurons this paper , neither Cav2. However, what is reduced by genetic or pharmacological Cav2.

To allow unperturbed activity and intracellular signaling, these experiments need to be carried out in perforated-patch configuration. Even without a knockdown approach, these are already challenging experiments in these highly vulnerable SN DA neurons but feasible, see 1. In order to identify transfected cells and still be able to carry out electrophysiology, one would need to co-transfect a marker like GFP.

Again, it would be hardly possible to detect successful recombination, in successfully transfected neurons, and still be able to record from them. With the help of Larry Zweifel Hunker et al. Alternatively, one could carry out e. These effects are likely to interfere with the effects on Cav2. Given all these considerations, we unfortunately feel the suggested more direct electrophysiological proof the reviewer asked for is, at present, not feasible.

This is indeed an option, which we thought we could pursue. Since this requires two antibodies from different species, we were searching for and tested suitable anti-Cav2.

The only anti-Cav2. It is raised in rabbits and works very nicely in Western-blots Author response image 1. We were unable to locate a commercial or non-commercial source for an anti-Cav2. We also tested different experimental conditions e.

Tris-acetate vs. Tris-glycine gels, blotting with or without SDS in transfer buffer which, however, still revealed this strong non-specific staining Author response image 2B , Tris-glycine gel. As mentioned above we considered this important criticism, when re-writing major parts of the manuscript, which was necessary because of additional data from novel experiments, some of them suggested by essential revisions 3 see below.

Our data now show different inactivation properties of SNXsensitive Cav2. Therefore, the data in cultured neurons are now presented together with those of SN DA neurons Figures 6 and 7 recorded in brain slices. This is outlined in more detail below.

The reviewer raised concerns about the isolation of R-type current components. His criticism hits an important point: R-type current is defined as current remaining after pharmacological block of all other Cavs.

However, several studies found that R-type current components are still present in some cells even after complete knockout of Cav2.

Therefore, we not only studied steady-state activation and inactivation of SNXsensitive currents in cultured neurons as suggested by the reviewer but also performed such experiments in SN DA neurons in vital brain slices. These findings now reveal:. Are you looking to buy Shiba Inu coin? Fortunately, you have a wide range of account options to buy the meme coin from popular crypto exchanges and online brokerages with Shiba Inu coins. The native cryptocurrency that fuels the network is called ether, or ETH for short.

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The expression of seven different endogenous control genes was routinely measured and used for data normalization as previously described Vandesompele et al. Briefly, data were normalized to the most stably expressed endogenous control genes Gapdh and Tfrc determined by geNorm Figure 2—figure supplement 1 , Supplementary file 1. Normalized molecule numbers were calculated for each assay based on their respective standard curve. Standard curve parameters are given in Supplementary file 3.

Details about assay specificity are given in Figure 2—figure supplement 1 and Supplementary file 3. For transient transfections hCav2. Cell stocks of passage 8 were frozen and cultures were re-expanded from stocks for not more than 20 passages. TsA cells were cultured as described Ortner et al. For splitting, cells were dissociated using 0.

TsA cells were replaced and freshly thawed when they exceeded passage no. For electrophysiology, cells were plated on 10 cm culture dishes and subjected to transient transfections on the following day. As described in Tomagra et al. Neurons were plated at final densities of cells per mm 2 on Petri dishes.

Cultured neurons were used at 8—9 days in vitro DIV for current-clamp and voltage-clamp experiments. Petri dishes were coated with poly-L-Lysine 0.

For whole-cell patch-clamp recordings, patch pipettes with a resistance of 1. All voltages were corrected for a liquid junction potential JP of —9 mV Lieb et al. The pipette internal solution for recordings of Cav2. The pipette internal solution for recordings of Cav1. The bath solution for recordings of Cav2.

The bath solution for recordings of Cav1. Current-voltage I-V relationships were obtained by applying a 20 ms long square pulse protocol to various test potentials 5 mV voltage steps starting from a holding potential of — mV or —89 mV recovery of inactivation. The resulting I-V curves were fitted to the following equation:. The voltage dependence of inactivation was assessed by application of 20 ms test pulses to the voltage of maximal activation V max before and after holding the cell at various conditioning test potentials for 5 s 30 s inter-sweep interval; 10 mV voltage steps; holding potential — mV.

Inactivation was calculated as the ratio between the current amplitudes of the 20 ms test pulses. Steady-state inactivation parameters were obtained by fitting the data to a modified Boltzmann equation:. The amount of inactivation during a 5 s depolarizing pulse from a holding potential of — mV to the V max was quantified by calculating the remaining current fraction after 50, , , , , and ms.

Recovery from inactivation was determined by 10 ms test pulses to V max at different time-points in s: 0. Window current was determined by multiplying mean current densities by fractional currents form steady-state inactivation curves to obtain the fraction of available channels at a given potential as described previously Hofer et al. Traces were sampled at 10 kHz and filtered using a low-pass Bessel filter set at 2 kHz.

Off-line data analysis was performed with pClamp Fast capacitive transients due to the depolarizing pulse were minimized online by the patch-clamp analog compensation. For current-clamp experiments the pipette internal solution contained in mM: gluconic acid potassium salt: K-gluconate , 10 HEPES, 0.

Data analysis was performed using Clampfit software. To study the contribution of Cav2. SNX nM was used in current- and voltage-clamp experiments. Whole-cell patch-clamp recordings were performed essentially as previously described Benkert et al. Chemicals were obtained from Sigma Aldrich unless stated otherwise. Patch pipettes 2.

Only TH and neurobiotin-positive cells were used for the statistical analysis. Inactivation was calculated as the ratio between the current amplitudes of the test versus control pulse. Data were not corrected for liquid junction potential —5 mV, measured according to Neher, Steady-state activation and inactivation curves were fitted as described above.

The brain was rapidly removed and a block of tissue containing the mesencephalon was immediately dissected. Brain slices were transferred into carbogenated artificial cerebrospinal fluid ACSF. Biocytin-streptavidin labeling was combined with TH-immuno-histochemistry Hess et al.

During the perforation process, the electrophysiological identification of the neuron was performed in current clamp mode. Perforated patch recordings were performed using protocols modified from Horn and Marty, and Akaike and Harata, ATP and GTP were omitted from the intracellular solution to prevent uncontrolled permeabilization of the cell membrane Lindau and Fernandez, The patch pipette was tip filled with internal solution and back filled with 0.

Amphotericin was dissolved in dimethyl sulfoxide final concentration: 0. The used DMSO concentration had no obvious effect on the investigated neurons. In the analyzed recordings R a was comparable, did not change significantly over recording time, and was not significantly different between the distinct experimental groups.

A change to the whole-cell configuration was indicated by a sudden change in R a and diffusion of tetraethylrhodamine-dextran into the neuron. Such experiments were rejected. GABAergic and glutamatergic synaptic input was reduced by addition of 0. Target genes, visualized with Atto fluorophore, were co-stained with Tyrosine hydroxylase TH , visualized with AlexaFluor, as a marker for dopaminergic neurons. The algorithm delineates cell shapes according to the TH marker gene signal and quantifies the number and the area of target-mRNA-derived fluorescence dots.

Target probe hybridization results in one fluorescent dot for each mRNA molecule, allowing determination of target-mRNA molecule numbers, independent from fluorescence-intensities.

Cryosectioning, laser-microdissection, reverse transcription: Cryosectioning, UV-laser microdissection UV-LMD and reverse transcription were carried out similarly as previously described in detail Benkert et al. Reverse transcription was carried out directly without a separate RNA isolation step in a one-tube procedure. All cDNA samples were precipitated as described Liss, Only cDNA pools expressing the correct marker gene profile i.

For Cav2. Primer sequences are given in Supplementary file 2. Outer duplex primer pairs were chosen using Oligo7. TaqMan assays were carefully established and performance was evaluated by generating standard curves, using defined amounts of cDNA derived from midbrain tissue mRNA , over four magnitudes of fold dilutions as templates, in at least three independent experiments, as described Duda et al.

Details of all qPCR assays TaqMan , and standard curve parameters used for analysis are provided in Supplementary file 4.

Assay parameters were as follows: threshold for analysis: 0. Data were analyzed using Clampfit Data were analyzed by appropriate statistical testing as indicated in detail for all experiments in the text, figure and table legends.

All data generated or analyzed during this study are included in the manuscript and supporting files. Raw data have been provided for mean population data shown in figures and tables. Our editorial process produces two outputs: i public reviews designed to be posted alongside the preprint for the benefit of readers; ii feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Ideally, this would be provided by knockdown experiments showing reduced pacemaking and CaV2. As an alternative, a proximity ligation assay could be done to at least show assembly of b2 and CaV2. The reviewer makes some good suggestions for reorganizing the manuscript that should be considered.

If possible, the isolated CaV2. Note that Supp Figure 1 is referenced in the paper after citing Supp Figure 2 and 3.

Please revised. The action potential traces in Figure 1C do not seem to be a good representation of the maximum time-derivative of voltage. Please revise. The reviewer’s suggestion is to make current Figure 5 the new Figure 3. Figure 4 should remain as such. Current Figure 3 and Figure 6 should be merged into new Figure 5. Alternative, current Figure 3 should become a supplemental figure of new Figure 5 current Figure 6.

Panel C of this figure is not mentioned in the main text. Please correct. In fact, this panel could go into a supplemental figure. Please verify. While the authors appropriately cite Jones et al. As currently written, the authors imply that these two papers validate the statement in line that R-type currents fully inactivate at voltages positive to mV. Reviewer recommends that this point be further clarified in the text.

This should be clearly described. Does it demonstrate similar properties? We fully agree with the reviewers that it is a relevant point but we do not see how this would be technically feasible. We ask the reviewers to consider the following:. We have already shown that, while SNX reduces pacemaking in cultured embryonic dopamine neurons this paper , neither Cav2. However, what is reduced by genetic or pharmacological Cav2.

To allow unperturbed activity and intracellular signaling, these experiments need to be carried out in perforated-patch configuration.

Even without a knockdown approach, these are already challenging experiments in these highly vulnerable SN DA neurons but feasible, see 1. In order to identify transfected cells and still be able to carry out electrophysiology, one would need to co-transfect a marker like GFP.

Again, it would be hardly possible to detect successful recombination, in successfully transfected neurons, and still be able to record from them. With the help of Larry Zweifel Hunker et al. Alternatively, one could carry out e.

These effects are likely to interfere with the effects on Cav2. Given all these considerations, we unfortunately feel the suggested more direct electrophysiological proof the reviewer asked for is, at present, not feasible. This is indeed an option, which we thought we could pursue. Since this requires two antibodies from different species, we were searching for and tested suitable anti-Cav2.

The only anti-Cav2. It is raised in rabbits and works very nicely in Western-blots Author response image 1. We were unable to locate a commercial or non-commercial source for an anti-Cav2. We also tested different experimental conditions e. Tris-acetate vs. Tris-glycine gels, blotting with or without SDS in transfer buffer which, however, still revealed this strong non-specific staining Author response image 2B , Tris-glycine gel. As mentioned above we considered this important criticism, when re-writing major parts of the manuscript, which was necessary because of additional data from novel experiments, some of them suggested by essential revisions 3 see below.

Our data now show different inactivation properties of SNXsensitive Cav2. Therefore, the data in cultured neurons are now presented together with those of SN DA neurons Figures 6 and 7 recorded in brain slices. This is outlined in more detail below.

The reviewer raised concerns about the isolation of R-type current components. His criticism hits an important point: R-type current is defined as current remaining after pharmacological block of all other Cavs. However, several studies found that R-type current components are still present in some cells even after complete knockout of Cav2. Therefore, we not only studied steady-state activation and inactivation of SNXsensitive currents in cultured neurons as suggested by the reviewer but also performed such experiments in SN DA neurons in vital brain slices.

These findings now reveal:. SNXsensitive currents in SN DA neurons inactivate at positive potentials Figure 6, Table 3 very similar to the R-type currents reported in our original manuscript. These data are in agreement with a substantial fraction Cav2. Save my name, email, and website in this browser for the next time I comment.

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