Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

doi:10.1093/brain/awu131

. 2014 Aug;137(Pt 8):2287-302.

doi: 10.1093/brain/awu131. Epub 2014 Jun 16.

Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

Affiliations

¹ 1 Institute of Applied Physiology, University of Ulm, Ulm, Germany.
² 2 Nancy Pritzker Laboratory, Department of Psychiatry and Behavioural Sciences, Stanford University School of Medicine, Palo Alto, USA.
³ 3 Institute of Neurophysiology, Goethe University Frankfurt, Germany.
⁴ 4 Rosalind Franklin University of Medicine and Science, North Chicago, USA.
⁵ 5 Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.
⁶ 6 Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Universidad Castilla-La Mancha, Albacete, Spain.
⁷ 7 Institute of Pharmacy, Department of Pharmacology and Toxicology, Centre of Molecular Biosciences, University of Innsbruck, Austria.
⁸ 1 Institute of Applied Physiology, University of Ulm, Ulm, Germany birgit.liss@uni-ulm.de.

PMID: 24934288
PMCID: PMC4107734
DOI: 10.1093/brain/awu131

Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

Elena Dragicevic et al. Brain. 2014 Aug.

. 2014 Aug;137(Pt 8):2287-302.

doi: 10.1093/brain/awu131. Epub 2014 Jun 16.

Authors

Affiliations

¹ 1 Institute of Applied Physiology, University of Ulm, Ulm, Germany.
² 2 Nancy Pritzker Laboratory, Department of Psychiatry and Behavioural Sciences, Stanford University School of Medicine, Palo Alto, USA.
³ 3 Institute of Neurophysiology, Goethe University Frankfurt, Germany.
⁴ 4 Rosalind Franklin University of Medicine and Science, North Chicago, USA.
⁵ 5 Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.
⁶ 6 Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Universidad Castilla-La Mancha, Albacete, Spain.
⁷ 7 Institute of Pharmacy, Department of Pharmacology and Toxicology, Centre of Molecular Biosciences, University of Innsbruck, Austria.
⁸ 1 Institute of Applied Physiology, University of Ulm, Ulm, Germany birgit.liss@uni-ulm.de.

PMID: 24934288
PMCID: PMC4107734
DOI: 10.1093/brain/awu131

Abstract

Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.

Keywords: D2-autoreceptor; Parkinsons disease; cocaine; isradipine; l-DOPA.

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Figures

**Figure 1**
Elevated D2-autoreceptor (AR) and GIRK2 messenger RNA levels in remaining human SN DA neurons from patients with Parkinson’s disease (PD). (A) Neuromelanin-positive human SN DA neurons (SN DA) (before and after UV laser microdissection) from *post mortem* Parkinson’s disease and control midbrain sections (for details, see Supplementary Table 1). (B) Real-time quanititative PCR results identified significantly higher relative messenger RNA levels for D2-autoreceptors and *GIRK2* in human SN DA neurons from Parkinson’s disease brains compared to controls (ctr) (D2-autoreceptors: controls, *n =* 26, 1 ± 0.19; Parkinson’s disease, *n =* 39, 4.25 ± 0.73; ****P < 0.0001; Mann-Whitney test; *GIRK2*: controls, *n =* 25, 1 ± 0.25; Parkinson’s disease, *n =* 34, 6.31 ± 1.12; ****P < 0.0001; Mann-Whitney test). (C) D2/GIRK2 ratios were not different in SN DA neurons from controls and Parkinson’s disease patients. All data are shown as the mean ± SEM.

**Figure 2**
*In vivo* transient high-dopamine states (induced by l-DOPA or cocaine) cause adult-like non-desensitizing dopamine D2-autoreceptor responses in juvenile SN DA neurons (SN DA). (A) Representative SN DA neuron pacemaker activity recordings from *in vitro* brain slices of adult and juvenile wild-type mice, pretreated (4 or 3 days, respectively) *in vivo* with saline, l-DOPA, or cocaine (perforated/cell-attached patch-clamp). Dopamine bath application is indicated by red bars. (B) Normalized frequencies plotted against time for all analysed SN DA neurons revealed pronounced desensitization of D2-autoreceptor responses in juveniles (n = 14) compared to adults (n = 10), as well as adult-like, non-desensitizing D2-autoreceptor responses in juvenile SN DA neurons after *in vivo* l-DOPA (n = 15) or *in vivo* cocaine (coc; n = 12), compared to controls (ctr; saline, *n =* 14). (C) Bar graphs show no change in mean frequencies before and after sulpiride application in SN DA neurons from controls, as well as after saline, l-DOPA, or cocaine *in vivo* injections, before dopamine application. (D) Pacemaker precision [given as coefficient of variation of interspike interval values, (CV ISI)] of juvenile and adult SN DA neurons was also not altered after sulpiride application, as well as after saline, l-DOPA, or cocaine *in vivo* injections. (E) D2-autoreceptor responses of mouse substantia nigra and ventral tegmental area dopaminergic neurons represented as mean activity of respective dopaminergic neurons at the last minute of dopamine application (15 min). Data are shown as the mean ± SEM. Data values detailed in Supplementary Tables 3, 5 and 6.

**Figure 3**
Functional expression of D2-autoreceptors (AR) [D2 short/long variants (s/l), D3, D4] and GIRK2 is not altered in juvenile SN DA neurons after *in vivo* induction of a transient high-dopamine state. (A, *top*) Agarose gel electrophoresis of multiplex nested reverse transcritpion PCR products identified D2 receptor (both splice variants) and GIRK2 as the molecular components of the D2-autoreceptors in SN DA neurons. Note the absence of D3 and D4 receptor, as well as GIRK1. Bottom: Quantitative analysis (reverse transcritpion quantitative PCR) of laser-microdissected (LMD) juvenile SN DA neurons showed no change in the ratio of D2 long and D2 short splice variant expression levels, after cocaine in single SN DA neurons (saline, *n =* 15, 1 ± 0.12; cocaine, *n =* 15, 0.85 ± 0.07; P = 0.29; Mann-Whitney test). Further, no changes in D2-autoreceptors (relative messenger RNA levels normalized to saline controls: saline, *n =* 18, 1 ± 0.14; cocaine, *n =* 19, 0.90 ± 0.08; P = 0.73; Mann-Whitney test), D3 or D4 receptor messenger RNA (data not shown, as no signal was detected. D3 receptor: saline, *n =* 6; cocaine: *n =* 6; D4 receptor: saline, *n =* 6; cocaine, *n =* 6) were detected. *GIRK2* messenger RNA levels were also decreased rather than increased after *in vivo* cocaine (saline, *n =* 19, 1 ± 0.15; cocaine, *n =* 20, 0.62 ± 0.07; *P = 0.04; Mann-Whitney test; note the significantly altered D2-autoreceptor/GIRK2 ratios after cocaine due to decreased GIRK2-levels; saline, *n =* 18, 1 ± 0.08; cocaine, *n =* 19, 1.57 ± 0.12; **P = 0.002; Mann-Whitney test). All data given as mean ± SEM normalized to controls. (B, *left*) Pre-embedding double-immunolabelling at electron microscopy level for D2-autoreceptors and GIRK2 in TH-positive SN DA neurons 3 days after saline/cocaine *in vivo* injection of juvenile wild-type mice (n = 3 animals each; arrows point to immunogold labelling at extrasynaptic plasma membrane and crossed arrows at intracellular sites). *Right*: No significant differences in immunoreactivity or subcellular localization of D2-autoreceptor protein (n = 3 animals each; saline, 1107.3 ± 8.7; cocaine, 1034.3 ± 33.5; P = 0.2; Mann-Whitney test), and GIRK2 protein (n = 3 animals each; saline, 1408.3 ± 9.1; cocaine, 1396.3 ± 7.1; P = 0.4; Mann-Whitney test). (C, *left*) Representative traces of dopamine evoked GIRK currents in juvenile SN DA neurons recorded in whole-cell voltage-clamp *in vitro* in the presence of 100 µM tolbutamide and 20 µM ZD7288 to block ATP-sensitive potassium channels (K-ATP) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. *Right*: Mean dopamine-evoked GIRK current amplitudes were not increased, but significantly reduced after *in vivo* cocaine in juvenile SN DA neurons (saline, *n =* 12, 101.48 ± 7.69 pA; cocaine, *n =* 10, 75.88 ± 12.74 pA; *P = 0.03; Mann-Whitney test). (D) *DAT*/*VMAT2* messenger RNA ratios in juvenile SN DA neurons, defining their individual dopamine-release capacity, were not altered after *in vivo* cocaine (normalized ratios: saline, *n =* 8, 1 ± 0.14; cocaine, *n =* 8, 1.23 ± 0.13; P = 0.29; Mann–Whitney test). Data are given as mean ± SEM.

**Figure 4**
Ca²⁺and Ca_v1.3 L-type-Ca²⁺ channels are crucial for adult-like, non-desensitizing D2-autoreceptor responses, induced by a transient high-dopamine state *in vivo,* in juvenile SN DA neurons. (A) Mean activity of substantia nigra dopaminergic neurons at the last minute of dopamine application [15 min, recorded in perforated patch (pp) juvenile: saline, *n =* 14; cocaine, *n =* 12] compared to those recorded in whole cell (wc) configuration with 0.1 mM EGTA (whole cell: juvenile: saline, *n =* 6; cocaine, *n =* 7) to buffer free [Ca²⁺]_i in juvenile SN DA neurons. Note the absence of cocaine-induced stabilization of D2-autoreceptor responses in whole cell compared to unperturbed perforated patch recordings. (B) Experiments as in Fig. 1 but in the presence of the L-type-Ca²⁺ channel blocker isradipine (300 nM, represented with blue bar). Isradipine completely blocked changes in kinetics of D2-autoreceptor desensitization of SN DA neurons, after transient high-dopamine state induced by cocaine, in wild-type mice (saline, *n =* 7; cocaine, *n =* 7). (C) Similar experiments as in B, but using Ca_v1.2 DHP^−/− mice (with Ca_v1.2 L-type-Ca²⁺ channels, insensitive to isradipine; saline, *n =* 6; cocaine, *n =* 9). Application of 300 nM isradipine still abolished cocaine D2-autoreceptor response potentiation, pointing to the crucial role of Ca_v1.3 L-type-Ca²⁺ channels in this process. (D and E) Isradipine (300 nM) did not reduce basal pacemaker frequencies or activity pattern (coefficient of variation of interspike interval) of juvenile SN DA neurons. (F) Activity of SN DA neurons at the last minute of dopamine application. Data values detailed in Supplementary Tables 3 and 5. (G) Quantitative analysis (reverse transcription quantitative PCR) of laser-microdissected (LMD) mouse and human SN DA neurons detected no change in messenger RNA levels of Ca_v1.2 or Ca_v1.3, the pore-forming alpha subunit of L-type-Ca²⁺ channels in the brain, after *in vivo* cocaine (mice, *left*) or in Parkinson’s disease compared to controls (humans, *right*). Normalized expression levels: mouse: Ca_v1.2: saline, *n =* 18, 1 ± 0.01; cocaine, *n =* 18, 1.01 ± 0.02; P = 0.92; Ca_v1.3: saline, *n =* 17, 1 ± 0.17; cocaine, *n =* 18, 0.81 ± 0.16; P = 0.37; Mann-Whitney test. Human: Ca_v1.2: control, *n =* 4, 1 ± 0.01; Parkinson’s disease, *n =* 5, 0.90 ± 0.11; P = 0.43; Ca_v1.3: control, *n =* 16, 1 ± 0.16; Parkinson’s disease, *n =* 33, 1.14 ± 0.14; P = 0.64; Mann-Whitney test). Data are shown as mean ± SEM.

**Figure 5**
Adult-like, non-desensitizing D2-autoreceptor responses in SN DA neurons are mediated by the neuronal calcium sensor 1 (NCS-1). (A, *left*) Normalized frequency plots show that non-desensitizing D2-autoreceptor responses of juvenile SN DA neurons after *in vivo* cocaine were completely blocked by 10 µM DNIP (cell-permeant D2R/NCS-1 interfering peptide, *n =* 8), whereas the scrambled version had no effect (10 µM srDNIP, *n =* 6). (*Right*) Respective analysis (as in A) of a general NCS-1 knockout mouse (NCS-1^−/−) demonstrated that NCS-1 is crucial for non-desensitizing D2-autoreceptor responses of juvenile SN DA neurons. (B) Activity of SN DA neurons from (A) at the last minute (15 min) of dopamine application (note the significantly pronounced D2-autoreceptor desensitization in NCS-1^−/− already under control conditions). Data values detailed in Supplementary Table 3. (C) Reverse transcrition quantitative PCR analysis of mouse and human SN DA neurons identified 2-fold higher *Ncs-1* messenger RNA levels after cocaine (saline, *n =* 36, 1 ± 0.08; cocaine, *n =* 32, 1.94 ± 0.15; ***P = 0.0001; Mann-Whitney test), as well as in Parkinson’s disease, compared with controls (control, *n =* 27, 1 ± 0.20; Parkinson’s disease, *n =* 33, 2.60 ± 0.27; ****P < 0.0001; Mann-Whitney test). (D) Cartoon depicting molecular components of a novel Ca_v1.3/NCS-1/D2-autoreceptor/GIRK2 signalling network in SN DA neurons, which allows homeostatic adaptation of their inhibitory dopamine D2 responses, and thus their pacemaker activity.

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Calcium currents regulate dopamine autoreceptors.
Borgkvist A, Mosharov EV, Sulzer D. Borgkvist A, et al. Brain. 2014 Aug;137(Pt 8):2113-5. doi: 10.1093/brain/awu150. Brain. 2014. PMID: 25057130 Free PMC article.

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References

1. Aguado C, Colon J, Ciruela F, Schlaudraff F, Cabanero MJ, Perry C, et al. Cell type-specific subunit composition of G protein-gated potassium channels in the cerebellum. J Neurochem. 2008;105:497–511. - PubMed
1. Anzalone A, Lizardi-Ortiz JE, Ramos M, De Mei C, Hopf FW, Iaccarino C, et al. Dual control of dopamine synthesis and release by presynaptic and postsynaptic dopamine D2 receptors. J Neurosci. 2012;32:9023–34. - PMC - PubMed
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1. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev. 2011;63:182–217. - PubMed
1. Beckstead MJ, Ford CP, Phillips PE, Williams JT. Presynaptic regulation of dendrodendritic dopamine transmission. Eur J Neurosci. 2007;26:1479–88. - PMC - PubMed

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[1] Aguado C, Colon J, Ciruela F, Schlaudraff F, Cabanero MJ, Perry C, et al. Cell type-specific subunit composition of G protein-gated potassium channels in the cerebellum. J Neurochem. 2008;105:497–511. - PubMed

[2] Aguado C, Colon J, Ciruela F, Schlaudraff F, Cabanero MJ, Perry C, et al. Cell type-specific subunit composition of G protein-gated potassium channels in the cerebellum. J Neurochem. 2008;105:497–511. - PubMed

[3] Anzalone A, Lizardi-Ortiz JE, Ramos M, De Mei C, Hopf FW, Iaccarino C, et al. Dual control of dopamine synthesis and release by presynaptic and postsynaptic dopamine D2 receptors. J Neurosci. 2012;32:9023–34. - PMC - PubMed

[4] Anzalone A, Lizardi-Ortiz JE, Ramos M, De Mei C, Hopf FW, Iaccarino C, et al. Dual control of dopamine synthesis and release by presynaptic and postsynaptic dopamine D2 receptors. J Neurosci. 2012;32:9023–34. - PMC - PubMed

[5] Bean BP. Neurophysiology: stressful pacemaking. Nature. 2007;447:1059–60. - PubMed

[6] Bean BP. Neurophysiology: stressful pacemaking. Nature. 2007;447:1059–60. - PubMed

[7] Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev. 2011;63:182–217. - PubMed

[8] Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev. 2011;63:182–217. - PubMed

[9] Beckstead MJ, Ford CP, Phillips PE, Williams JT. Presynaptic regulation of dendrodendritic dopamine transmission. Eur J Neurosci. 2007;26:1479–88. - PMC - PubMed

[10] Beckstead MJ, Ford CP, Phillips PE, Williams JT. Presynaptic regulation of dendrodendritic dopamine transmission. Eur J Neurosci. 2007;26:1479–88. - PMC - PubMed

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Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

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Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons

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