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Compensatory T-type Ca2+ channel activity alters D2-autoreceptor responses of Substantia nigra dopamine neurons from Cav1.3 L-type Ca2+ channel KO mice.

Poetschke C, Dragicevic E, Duda J, Benkert J, Dougalis A, DeZio R, Snutch TP, Striessnig J, Liss B - Sci Rep (2015)

Bottom Line: This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA.Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults.This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Physiology, University of Ulm, 89081 Ulm, Germany.

ABSTRACT
The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

No MeSH data available.


Related in: MedlinePlus

Larger amplitudes of fast-inactivating low-voltage activated, T-type calcium channel currents in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overlay of representative currents recorded from a juvenile WT SN DA neuron in response to a single step to −30 mV from a LVA/HVA composite protocol (holding current of −100 mV, black trace) and from a HVA protocol (holding current of −60 mV, gray trace), to discriminate between low and high voltage activated currents (LVA/HVA). The red trace represents the subtracted current. Currents were evaluated for their time to peak. The subtracted LVA currents and the LVA/HVA composite current were consistently fitted with a two exponential decay (tau fast [τfast] and tau slow [τslow]). Scale bar 100 pA/1s. (b/c) Representative traces of subtracted, fast-inactivating T-type calcium channel blocker Z944-sensitive barium currents in juvenile SN DA neurons from a WT and a Cav1.3 KO mouse (response to 10 mV incremental depolarising pulses to 0 mV from a holding potential of −100 mV). Dotted boxes indicates the expanded view of the left hand traces shown on the right. Currents exhibited a voltage-dependent fast-activation and voltage-dependent fast-inactivation. Scale bars: left traces: 200 pA/1 s; right traces: 200 pA/500ms. (d) Maximal current amplitude of T-type barium currents in juvenile WT and Cav1.3 KO SN DA neurons at a test voltage of −30 mV. Note that the peak amplitude is significantly (about 3-fold) larger in Cav1.3 KO, suggesting elevated T-type currents (WMWU = 0, p = 0.02). (e) Steady-state activation curves for putative T-type barium currents in WT and Cav1.3 KO mice. Plot represents the ratio of conductance (G) to the maximal conductance (Gmax) and has been fitted with a single Boltzmann equation to identify the voltage for half-maximal activation (V50) and the slope (s) of the steady-state activation curve. Note similar steady-state activation of T-type currents in WT and KO. (f) Kinetic properties (time to peak and inactivation time constants τfast and τslow) of T-type currents at a test voltage of −30 mV are similar in SN DA neurons from juvenile WT and Cav1.3 KO mice. All data are shown as mean ± SEM, WT data in black and KO data in green. Significant differences are marked by asterisks.
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f5: Larger amplitudes of fast-inactivating low-voltage activated, T-type calcium channel currents in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overlay of representative currents recorded from a juvenile WT SN DA neuron in response to a single step to −30 mV from a LVA/HVA composite protocol (holding current of −100 mV, black trace) and from a HVA protocol (holding current of −60 mV, gray trace), to discriminate between low and high voltage activated currents (LVA/HVA). The red trace represents the subtracted current. Currents were evaluated for their time to peak. The subtracted LVA currents and the LVA/HVA composite current were consistently fitted with a two exponential decay (tau fast [τfast] and tau slow [τslow]). Scale bar 100 pA/1s. (b/c) Representative traces of subtracted, fast-inactivating T-type calcium channel blocker Z944-sensitive barium currents in juvenile SN DA neurons from a WT and a Cav1.3 KO mouse (response to 10 mV incremental depolarising pulses to 0 mV from a holding potential of −100 mV). Dotted boxes indicates the expanded view of the left hand traces shown on the right. Currents exhibited a voltage-dependent fast-activation and voltage-dependent fast-inactivation. Scale bars: left traces: 200 pA/1 s; right traces: 200 pA/500ms. (d) Maximal current amplitude of T-type barium currents in juvenile WT and Cav1.3 KO SN DA neurons at a test voltage of −30 mV. Note that the peak amplitude is significantly (about 3-fold) larger in Cav1.3 KO, suggesting elevated T-type currents (WMWU = 0, p = 0.02). (e) Steady-state activation curves for putative T-type barium currents in WT and Cav1.3 KO mice. Plot represents the ratio of conductance (G) to the maximal conductance (Gmax) and has been fitted with a single Boltzmann equation to identify the voltage for half-maximal activation (V50) and the slope (s) of the steady-state activation curve. Note similar steady-state activation of T-type currents in WT and KO. (f) Kinetic properties (time to peak and inactivation time constants τfast and τslow) of T-type currents at a test voltage of −30 mV are similar in SN DA neurons from juvenile WT and Cav1.3 KO mice. All data are shown as mean ± SEM, WT data in black and KO data in green. Significant differences are marked by asterisks.

Mentions: To compare TTCC currents in SN DA neurons from juvenile WT and Cav1.3 KO mice, we performed whole-cell voltage-clamp recordings. To isolate fast-inactivating, T-type Ca2+ currents we exploited current subtraction between two voltage-clamp protocols used to discriminate between low- and high-voltage-activated currents (HVA and LVA), together with specific intracellular/extracellular solutions that block sodium, potassium and synaptic conductances, as described previously31. To reduce the amount of inactivation seen when isolating Ca2+ currents in SN DA neurons under voltage-clamp protocols (e.g.32 and for easier discrimination between fast-inactivating and persistent currents), we used barium ions instead of calcium ions as the charge carrier33. Neurons were held for 5 seconds at either −100 mV (LVA/HVA composite current protocol) or −60 mV (HVA protocol), and were depolarised in 10 mV increments (for 2 s, every 6 s) up to +20 mV to construct full current-voltage (I–V) curves. Current subtraction was used to compute the peak amplitude of the fast-inactivating LVA currents recruited with the LVA/HVA composite protocol by subtracting the slowly-activating currents recorded with the HVA protocol (Fig. 5). Subtraction of currents resulted in isolation of fast activating, fast-inactivating conductances in SN DA neurons from WT mice (Fig. 5a). The subtracted current exhibited voltage-dependence in its activation, becoming faster at more positive voltages (time to peak at −50 mV: 62.1 ms ± 16.2, n = 5; at 0 mV, 26.0 ms ± 6.4, n = 5, p = 0.049, paired t-test, data not shown). The inactivation phase was consistently better fitted with the sum of two exponentials (τfast and τslow) that contributed on average 50–70% and 30–50% of the total current amplitudes. The fast inactivation time constant exhibited some voltage-dependency in most cells becoming faster throughout the −50 to 0 mV voltage range, the slow inactivation constant did not exhibit changes in the range studied (τfast and τslow at −50 mV: 152.1 ms ± 46.4 and 646.6 ms ± 162.5 respectively; at 0 mV: 75.1 ms ± 6.4 and 676.0 ms ± 67.6 respectively; τfast p = 0.2, τslow p = 0.9 with paired t-tests for inter-voltage τfast and τslow comparisons, n = 5, data not shown). Since we pharmacologically occluded fast-activating sodium and potassium conductances, and since biophysical properties of the fast-inactivating subtracted current are very similar to the T-type calcium currents recorded previously in SN DA neurons in brain slices32, and are pharmacologically inhibited by 10 µM Z944 (n = 14, data not shown), we refer to them henceforth as T-type (barium) currents. Comparing maximal T-type (barium) current amplitudes obtained at −30 mV from juvenile SN DA neurons from WT (n = 5 neurons from 4 mice, representative traces in Fig. 5b) and Cav1.3 KO mice (n = 4 neurons from 3 mice, representative traces in Fig. 5c) revealed that Cav1.3 KO mice displayed significantly larger peak currents (compare Fig. 5d; current amplitude at −30 mV: juvenile WT: −294.4 p ± 77.1, n = 5; juvenile Cav1.3 KO: −686.1 pA ± 118.4, n = 4; WMWU = 0, p = 0.02). SN DA T-type currents from juvenile WT and Cav1.3 KO mice however exhibited similar parameters for the steady-state activation (WT and Cav1.3 KO: voltage for half-maximal activation (V50): −40.1 mV ± 0.8, n = 5 and −42.3 mV ± 1.3, n = 4, WMWU = 6, p = 0.4; slope (s): 3.6 ± 1.0, n = 5 and 3.2 ± 1.2, n = 4, WMWU = 5, p = 0.3, see Fig. 5e) and activation/inactivation kinetics (WT and Cav1.3 KO at −30 mV: time to peak: 39.1 ms ± 11.3 and 18.6 ms ± 2.3, WMWU = 3, p = 0.1; τfast: 86.3 ms ± 15.9 and 52.5 ms ± 6.2, WMWU = 2, p = 0.06; τslow: 827.2 ms ± 95.9 and 604.9 ms ± 123.8, WMWU = 4, p = 0.2; n = 5 and 4 respectively, Fig. 5f). These data suggest that the steady state biophysical and kinetic properties of SN DA T-type (barium) currents are not per se affected in the Cav1.3 KO mice, but the peak current amplitude is about 3-fold larger. This would be in line with an elevated expression of TTCCs in SN DA neurons from juvenile Cav1.3 KO mice.


Compensatory T-type Ca2+ channel activity alters D2-autoreceptor responses of Substantia nigra dopamine neurons from Cav1.3 L-type Ca2+ channel KO mice.

Poetschke C, Dragicevic E, Duda J, Benkert J, Dougalis A, DeZio R, Snutch TP, Striessnig J, Liss B - Sci Rep (2015)

Larger amplitudes of fast-inactivating low-voltage activated, T-type calcium channel currents in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overlay of representative currents recorded from a juvenile WT SN DA neuron in response to a single step to −30 mV from a LVA/HVA composite protocol (holding current of −100 mV, black trace) and from a HVA protocol (holding current of −60 mV, gray trace), to discriminate between low and high voltage activated currents (LVA/HVA). The red trace represents the subtracted current. Currents were evaluated for their time to peak. The subtracted LVA currents and the LVA/HVA composite current were consistently fitted with a two exponential decay (tau fast [τfast] and tau slow [τslow]). Scale bar 100 pA/1s. (b/c) Representative traces of subtracted, fast-inactivating T-type calcium channel blocker Z944-sensitive barium currents in juvenile SN DA neurons from a WT and a Cav1.3 KO mouse (response to 10 mV incremental depolarising pulses to 0 mV from a holding potential of −100 mV). Dotted boxes indicates the expanded view of the left hand traces shown on the right. Currents exhibited a voltage-dependent fast-activation and voltage-dependent fast-inactivation. Scale bars: left traces: 200 pA/1 s; right traces: 200 pA/500ms. (d) Maximal current amplitude of T-type barium currents in juvenile WT and Cav1.3 KO SN DA neurons at a test voltage of −30 mV. Note that the peak amplitude is significantly (about 3-fold) larger in Cav1.3 KO, suggesting elevated T-type currents (WMWU = 0, p = 0.02). (e) Steady-state activation curves for putative T-type barium currents in WT and Cav1.3 KO mice. Plot represents the ratio of conductance (G) to the maximal conductance (Gmax) and has been fitted with a single Boltzmann equation to identify the voltage for half-maximal activation (V50) and the slope (s) of the steady-state activation curve. Note similar steady-state activation of T-type currents in WT and KO. (f) Kinetic properties (time to peak and inactivation time constants τfast and τslow) of T-type currents at a test voltage of −30 mV are similar in SN DA neurons from juvenile WT and Cav1.3 KO mice. All data are shown as mean ± SEM, WT data in black and KO data in green. Significant differences are marked by asterisks.
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Related In: Results  -  Collection

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f5: Larger amplitudes of fast-inactivating low-voltage activated, T-type calcium channel currents in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overlay of representative currents recorded from a juvenile WT SN DA neuron in response to a single step to −30 mV from a LVA/HVA composite protocol (holding current of −100 mV, black trace) and from a HVA protocol (holding current of −60 mV, gray trace), to discriminate between low and high voltage activated currents (LVA/HVA). The red trace represents the subtracted current. Currents were evaluated for their time to peak. The subtracted LVA currents and the LVA/HVA composite current were consistently fitted with a two exponential decay (tau fast [τfast] and tau slow [τslow]). Scale bar 100 pA/1s. (b/c) Representative traces of subtracted, fast-inactivating T-type calcium channel blocker Z944-sensitive barium currents in juvenile SN DA neurons from a WT and a Cav1.3 KO mouse (response to 10 mV incremental depolarising pulses to 0 mV from a holding potential of −100 mV). Dotted boxes indicates the expanded view of the left hand traces shown on the right. Currents exhibited a voltage-dependent fast-activation and voltage-dependent fast-inactivation. Scale bars: left traces: 200 pA/1 s; right traces: 200 pA/500ms. (d) Maximal current amplitude of T-type barium currents in juvenile WT and Cav1.3 KO SN DA neurons at a test voltage of −30 mV. Note that the peak amplitude is significantly (about 3-fold) larger in Cav1.3 KO, suggesting elevated T-type currents (WMWU = 0, p = 0.02). (e) Steady-state activation curves for putative T-type barium currents in WT and Cav1.3 KO mice. Plot represents the ratio of conductance (G) to the maximal conductance (Gmax) and has been fitted with a single Boltzmann equation to identify the voltage for half-maximal activation (V50) and the slope (s) of the steady-state activation curve. Note similar steady-state activation of T-type currents in WT and KO. (f) Kinetic properties (time to peak and inactivation time constants τfast and τslow) of T-type currents at a test voltage of −30 mV are similar in SN DA neurons from juvenile WT and Cav1.3 KO mice. All data are shown as mean ± SEM, WT data in black and KO data in green. Significant differences are marked by asterisks.
Mentions: To compare TTCC currents in SN DA neurons from juvenile WT and Cav1.3 KO mice, we performed whole-cell voltage-clamp recordings. To isolate fast-inactivating, T-type Ca2+ currents we exploited current subtraction between two voltage-clamp protocols used to discriminate between low- and high-voltage-activated currents (HVA and LVA), together with specific intracellular/extracellular solutions that block sodium, potassium and synaptic conductances, as described previously31. To reduce the amount of inactivation seen when isolating Ca2+ currents in SN DA neurons under voltage-clamp protocols (e.g.32 and for easier discrimination between fast-inactivating and persistent currents), we used barium ions instead of calcium ions as the charge carrier33. Neurons were held for 5 seconds at either −100 mV (LVA/HVA composite current protocol) or −60 mV (HVA protocol), and were depolarised in 10 mV increments (for 2 s, every 6 s) up to +20 mV to construct full current-voltage (I–V) curves. Current subtraction was used to compute the peak amplitude of the fast-inactivating LVA currents recruited with the LVA/HVA composite protocol by subtracting the slowly-activating currents recorded with the HVA protocol (Fig. 5). Subtraction of currents resulted in isolation of fast activating, fast-inactivating conductances in SN DA neurons from WT mice (Fig. 5a). The subtracted current exhibited voltage-dependence in its activation, becoming faster at more positive voltages (time to peak at −50 mV: 62.1 ms ± 16.2, n = 5; at 0 mV, 26.0 ms ± 6.4, n = 5, p = 0.049, paired t-test, data not shown). The inactivation phase was consistently better fitted with the sum of two exponentials (τfast and τslow) that contributed on average 50–70% and 30–50% of the total current amplitudes. The fast inactivation time constant exhibited some voltage-dependency in most cells becoming faster throughout the −50 to 0 mV voltage range, the slow inactivation constant did not exhibit changes in the range studied (τfast and τslow at −50 mV: 152.1 ms ± 46.4 and 646.6 ms ± 162.5 respectively; at 0 mV: 75.1 ms ± 6.4 and 676.0 ms ± 67.6 respectively; τfast p = 0.2, τslow p = 0.9 with paired t-tests for inter-voltage τfast and τslow comparisons, n = 5, data not shown). Since we pharmacologically occluded fast-activating sodium and potassium conductances, and since biophysical properties of the fast-inactivating subtracted current are very similar to the T-type calcium currents recorded previously in SN DA neurons in brain slices32, and are pharmacologically inhibited by 10 µM Z944 (n = 14, data not shown), we refer to them henceforth as T-type (barium) currents. Comparing maximal T-type (barium) current amplitudes obtained at −30 mV from juvenile SN DA neurons from WT (n = 5 neurons from 4 mice, representative traces in Fig. 5b) and Cav1.3 KO mice (n = 4 neurons from 3 mice, representative traces in Fig. 5c) revealed that Cav1.3 KO mice displayed significantly larger peak currents (compare Fig. 5d; current amplitude at −30 mV: juvenile WT: −294.4 p ± 77.1, n = 5; juvenile Cav1.3 KO: −686.1 pA ± 118.4, n = 4; WMWU = 0, p = 0.02). SN DA T-type currents from juvenile WT and Cav1.3 KO mice however exhibited similar parameters for the steady-state activation (WT and Cav1.3 KO: voltage for half-maximal activation (V50): −40.1 mV ± 0.8, n = 5 and −42.3 mV ± 1.3, n = 4, WMWU = 6, p = 0.4; slope (s): 3.6 ± 1.0, n = 5 and 3.2 ± 1.2, n = 4, WMWU = 5, p = 0.3, see Fig. 5e) and activation/inactivation kinetics (WT and Cav1.3 KO at −30 mV: time to peak: 39.1 ms ± 11.3 and 18.6 ms ± 2.3, WMWU = 3, p = 0.1; τfast: 86.3 ms ± 15.9 and 52.5 ms ± 6.2, WMWU = 2, p = 0.06; τslow: 827.2 ms ± 95.9 and 604.9 ms ± 123.8, WMWU = 4, p = 0.2; n = 5 and 4 respectively, Fig. 5f). These data suggest that the steady state biophysical and kinetic properties of SN DA T-type (barium) currents are not per se affected in the Cav1.3 KO mice, but the peak current amplitude is about 3-fold larger. This would be in line with an elevated expression of TTCCs in SN DA neurons from juvenile Cav1.3 KO mice.

Bottom Line: This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA.Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults.This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Physiology, University of Ulm, 89081 Ulm, Germany.

ABSTRACT
The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

No MeSH data available.


Related in: MedlinePlus