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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

Compensatory up-regulation of Cav3.1 T-type Ca2+ channel subunits and of NCS-1 mRNA in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overview of WT (left) and Cav1.3 KO mouse (middle) coronal midbrain sections after UV-laser-microdissection (UV-LMD) of 10 individual SN DA neurons each. Scale bars: 250 μm. Inserts: photograph of the reaction-tube-cap for inspection of collection of all 10 neurons after UV-LMD, prior to cell lysis and reverse transcription. Scale bars: 500 μm. Lower left/middle: individual SN DA neurons before and after UV-LMD. Scale bars: 10 μm. Right: Multiplex nested PCR results (2% agarose-gel-electrophoresis). All analysed SN DA cDNA pools were PCR-positive for tyrosine hydroxylase (TH) and negative for calbindind28k (CB), for GABAergic markers (GAD65/67) and for astroglial marker (GFAP). (b) Cell-specific quantitative RT-PCR data of the L-type Ca2+ channel subunit Cav1.2, of the T-type Ca2+ channel subunits Cav3.1, Cav3.2, and Cav3.3, and of the neuronal calcium sensor NCS-1, for SN DA neurons from juvenile WT and Cav1.3 KO mice (n numbers given in bars). Data given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse midbrain tissue. All data given as the mean ± SEM. WT data given in black, KO data in green. Note significantly higher mRNA-levels of Cav3.1 and NCS-1 in SN DA from Cav1.3 KO. (c) Cartoon summarising the postulated molecular mechanism of altered dopamine NCS-1/D2-AR/GIRK2 channel signalling in SN DA neurons from juvenile Cav1.3 KO mice: Chronic loss of Cav1.3 L-type-Ca2+ -channels (with its regulatory β- and α2δ subunits) that sensitises D2-ARs in response to extracellular dopamine via the neuronal calcium sensor NCS-1 is functionally compensated in SN DA neurons by Cav3.1 T-type-Ca2+ -channels, boosting calcium-dependent D2-AR/NCS-1 interaction, that prevent GRK2-mediated D2-AR phosphorylation and thus β-arrestin-mediated receptor internalisation and desensitisation—resulting in enhanced GIRK2- mediated SN DA pacemaker-activity inhibition. Abbreviations: D2-AR: D2-autoreceptor, GIRK2: G-protein-coupled, inwardly rectifying K+ channel 2, GRK2: G-protein coupled kinase 2, RGS: regulator of G-protein signalling; for details see text.
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f6: Compensatory up-regulation of Cav3.1 T-type Ca2+ channel subunits and of NCS-1 mRNA in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overview of WT (left) and Cav1.3 KO mouse (middle) coronal midbrain sections after UV-laser-microdissection (UV-LMD) of 10 individual SN DA neurons each. Scale bars: 250 μm. Inserts: photograph of the reaction-tube-cap for inspection of collection of all 10 neurons after UV-LMD, prior to cell lysis and reverse transcription. Scale bars: 500 μm. Lower left/middle: individual SN DA neurons before and after UV-LMD. Scale bars: 10 μm. Right: Multiplex nested PCR results (2% agarose-gel-electrophoresis). All analysed SN DA cDNA pools were PCR-positive for tyrosine hydroxylase (TH) and negative for calbindind28k (CB), for GABAergic markers (GAD65/67) and for astroglial marker (GFAP). (b) Cell-specific quantitative RT-PCR data of the L-type Ca2+ channel subunit Cav1.2, of the T-type Ca2+ channel subunits Cav3.1, Cav3.2, and Cav3.3, and of the neuronal calcium sensor NCS-1, for SN DA neurons from juvenile WT and Cav1.3 KO mice (n numbers given in bars). Data given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse midbrain tissue. All data given as the mean ± SEM. WT data given in black, KO data in green. Note significantly higher mRNA-levels of Cav3.1 and NCS-1 in SN DA from Cav1.3 KO. (c) Cartoon summarising the postulated molecular mechanism of altered dopamine NCS-1/D2-AR/GIRK2 channel signalling in SN DA neurons from juvenile Cav1.3 KO mice: Chronic loss of Cav1.3 L-type-Ca2+ -channels (with its regulatory β- and α2δ subunits) that sensitises D2-ARs in response to extracellular dopamine via the neuronal calcium sensor NCS-1 is functionally compensated in SN DA neurons by Cav3.1 T-type-Ca2+ -channels, boosting calcium-dependent D2-AR/NCS-1 interaction, that prevent GRK2-mediated D2-AR phosphorylation and thus β-arrestin-mediated receptor internalisation and desensitisation—resulting in enhanced GIRK2- mediated SN DA pacemaker-activity inhibition. Abbreviations: D2-AR: D2-autoreceptor, GIRK2: G-protein-coupled, inwardly rectifying K+ channel 2, GRK2: G-protein coupled kinase 2, RGS: regulator of G-protein signalling; for details see text.

Mentions: To address a possible upregulated expression of TTCC subunits, and to molecularly define the underlying nature of the compensatory functional coupling of TTCCs with D2-ARs via NCS-1, we quantified mRNA levels of all three TTCC subtypes (Cav3.1, Cav3.2 and Cav3.3 α1-subunits), as well as of Cav1.2 and NCS-1 in juvenile SN DA neurons from WT and Cav1.3 KO mice, by combining UV-LMD and RT-PCR approaches (Fig. 6). All three TTCC subunits were expressed in mouse SN DA neurons, although Cav3.1 was the by far most abundant TTCC subunit, while Cav3.2 mRNA-levels were about ~8-fold lower, and Cav3.3 mRNA was not detected in most analysed SN DA neurons (Fig. 6b). More importantly, mRNA-levels of only the Cav3.1 subtype were significantly increased (by ~50%) in SN DA neurons of juvenile Cav1.3 KO mice compared to those of WT (WT Cav3.1: 159.4 ± 19.8 pg/cell, n = 31; Cav1.3 KO Cav3.1: 241.2 ± 21.4 pg/cell, n = 29; WMWU = 267, p = 0.006; Fig. 6b). In contrast, mRNA levels of Cav3.2 and Cav3.3 were not elevated (WT Cav3.2: 30.06 ± 5.7 pg/cell, n = 30; Cav1.3 KO Cav3.2: 33.28 ± 3.7 pg/cell, n = 29; WMWU = 351, p = 0.2; WT Cav3.3: 4.9 ± 1.1 pg/cell, n = 31, with only 18 cell-pools giving a PCR positive signal; Cav1.3 KO Cav3.3: 2.5 ± 0.8 pg/cell, n = 30 with only 9 cell-pools giving a PCR positive signal; WMWU = 331.5, p = 0.03). Similarly, mRNA-levels of Cav1.2 were not changed (WT Cav1.2: 134.1 ± 10.2 pg/cell, n = 27; Cav1.3 KO Cav1.2: 134.6 ± 17 pg/cell, n = 27; WMWU = 347, p = 0.8.). These data identify Cav3.1 as the prominent TTCC channel in WT SN DA neurons and its mRNA upregulation in SN DA neurons from Cav1.3 KO mice, likely mediating the compensatory functionally coupled TTCC/D2-AR phenotype, accompanied by significantly elevated fast-inactivating low-voltage-activated TTCC mediated currents in SN DA neurons from 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)

Compensatory up-regulation of Cav3.1 T-type Ca2+ channel subunits and of NCS-1 mRNA in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overview of WT (left) and Cav1.3 KO mouse (middle) coronal midbrain sections after UV-laser-microdissection (UV-LMD) of 10 individual SN DA neurons each. Scale bars: 250 μm. Inserts: photograph of the reaction-tube-cap for inspection of collection of all 10 neurons after UV-LMD, prior to cell lysis and reverse transcription. Scale bars: 500 μm. Lower left/middle: individual SN DA neurons before and after UV-LMD. Scale bars: 10 μm. Right: Multiplex nested PCR results (2% agarose-gel-electrophoresis). All analysed SN DA cDNA pools were PCR-positive for tyrosine hydroxylase (TH) and negative for calbindind28k (CB), for GABAergic markers (GAD65/67) and for astroglial marker (GFAP). (b) Cell-specific quantitative RT-PCR data of the L-type Ca2+ channel subunit Cav1.2, of the T-type Ca2+ channel subunits Cav3.1, Cav3.2, and Cav3.3, and of the neuronal calcium sensor NCS-1, for SN DA neurons from juvenile WT and Cav1.3 KO mice (n numbers given in bars). Data given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse midbrain tissue. All data given as the mean ± SEM. WT data given in black, KO data in green. Note significantly higher mRNA-levels of Cav3.1 and NCS-1 in SN DA from Cav1.3 KO. (c) Cartoon summarising the postulated molecular mechanism of altered dopamine NCS-1/D2-AR/GIRK2 channel signalling in SN DA neurons from juvenile Cav1.3 KO mice: Chronic loss of Cav1.3 L-type-Ca2+ -channels (with its regulatory β- and α2δ subunits) that sensitises D2-ARs in response to extracellular dopamine via the neuronal calcium sensor NCS-1 is functionally compensated in SN DA neurons by Cav3.1 T-type-Ca2+ -channels, boosting calcium-dependent D2-AR/NCS-1 interaction, that prevent GRK2-mediated D2-AR phosphorylation and thus β-arrestin-mediated receptor internalisation and desensitisation—resulting in enhanced GIRK2- mediated SN DA pacemaker-activity inhibition. Abbreviations: D2-AR: D2-autoreceptor, GIRK2: G-protein-coupled, inwardly rectifying K+ channel 2, GRK2: G-protein coupled kinase 2, RGS: regulator of G-protein signalling; for details see text.
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f6: Compensatory up-regulation of Cav3.1 T-type Ca2+ channel subunits and of NCS-1 mRNA in SN DA neurons from juvenile Cav1.3 KO mice.(a) Overview of WT (left) and Cav1.3 KO mouse (middle) coronal midbrain sections after UV-laser-microdissection (UV-LMD) of 10 individual SN DA neurons each. Scale bars: 250 μm. Inserts: photograph of the reaction-tube-cap for inspection of collection of all 10 neurons after UV-LMD, prior to cell lysis and reverse transcription. Scale bars: 500 μm. Lower left/middle: individual SN DA neurons before and after UV-LMD. Scale bars: 10 μm. Right: Multiplex nested PCR results (2% agarose-gel-electrophoresis). All analysed SN DA cDNA pools were PCR-positive for tyrosine hydroxylase (TH) and negative for calbindind28k (CB), for GABAergic markers (GAD65/67) and for astroglial marker (GFAP). (b) Cell-specific quantitative RT-PCR data of the L-type Ca2+ channel subunit Cav1.2, of the T-type Ca2+ channel subunits Cav3.1, Cav3.2, and Cav3.3, and of the neuronal calcium sensor NCS-1, for SN DA neurons from juvenile WT and Cav1.3 KO mice (n numbers given in bars). Data given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse midbrain tissue. All data given as the mean ± SEM. WT data given in black, KO data in green. Note significantly higher mRNA-levels of Cav3.1 and NCS-1 in SN DA from Cav1.3 KO. (c) Cartoon summarising the postulated molecular mechanism of altered dopamine NCS-1/D2-AR/GIRK2 channel signalling in SN DA neurons from juvenile Cav1.3 KO mice: Chronic loss of Cav1.3 L-type-Ca2+ -channels (with its regulatory β- and α2δ subunits) that sensitises D2-ARs in response to extracellular dopamine via the neuronal calcium sensor NCS-1 is functionally compensated in SN DA neurons by Cav3.1 T-type-Ca2+ -channels, boosting calcium-dependent D2-AR/NCS-1 interaction, that prevent GRK2-mediated D2-AR phosphorylation and thus β-arrestin-mediated receptor internalisation and desensitisation—resulting in enhanced GIRK2- mediated SN DA pacemaker-activity inhibition. Abbreviations: D2-AR: D2-autoreceptor, GIRK2: G-protein-coupled, inwardly rectifying K+ channel 2, GRK2: G-protein coupled kinase 2, RGS: regulator of G-protein signalling; for details see text.
Mentions: To address a possible upregulated expression of TTCC subunits, and to molecularly define the underlying nature of the compensatory functional coupling of TTCCs with D2-ARs via NCS-1, we quantified mRNA levels of all three TTCC subtypes (Cav3.1, Cav3.2 and Cav3.3 α1-subunits), as well as of Cav1.2 and NCS-1 in juvenile SN DA neurons from WT and Cav1.3 KO mice, by combining UV-LMD and RT-PCR approaches (Fig. 6). All three TTCC subunits were expressed in mouse SN DA neurons, although Cav3.1 was the by far most abundant TTCC subunit, while Cav3.2 mRNA-levels were about ~8-fold lower, and Cav3.3 mRNA was not detected in most analysed SN DA neurons (Fig. 6b). More importantly, mRNA-levels of only the Cav3.1 subtype were significantly increased (by ~50%) in SN DA neurons of juvenile Cav1.3 KO mice compared to those of WT (WT Cav3.1: 159.4 ± 19.8 pg/cell, n = 31; Cav1.3 KO Cav3.1: 241.2 ± 21.4 pg/cell, n = 29; WMWU = 267, p = 0.006; Fig. 6b). In contrast, mRNA levels of Cav3.2 and Cav3.3 were not elevated (WT Cav3.2: 30.06 ± 5.7 pg/cell, n = 30; Cav1.3 KO Cav3.2: 33.28 ± 3.7 pg/cell, n = 29; WMWU = 351, p = 0.2; WT Cav3.3: 4.9 ± 1.1 pg/cell, n = 31, with only 18 cell-pools giving a PCR positive signal; Cav1.3 KO Cav3.3: 2.5 ± 0.8 pg/cell, n = 30 with only 9 cell-pools giving a PCR positive signal; WMWU = 331.5, p = 0.03). Similarly, mRNA-levels of Cav1.2 were not changed (WT Cav1.2: 134.1 ± 10.2 pg/cell, n = 27; Cav1.3 KO Cav1.2: 134.6 ± 17 pg/cell, n = 27; WMWU = 347, p = 0.8.). These data identify Cav3.1 as the prominent TTCC channel in WT SN DA neurons and its mRNA upregulation in SN DA neurons from Cav1.3 KO mice, likely mediating the compensatory functionally coupled TTCC/D2-AR phenotype, accompanied by significantly elevated fast-inactivating low-voltage-activated TTCC mediated currents in SN DA neurons from 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