GluN2D-containing NMDA receptors in cholinergic interneurons control dopamine release in the mouse striatum

Xiaoqun Zhang and Karima Chergui Department of Physiology and , Section of Molecular Neurophysiology, Karolinska Institute, Stockholm, Sweden

Introduction and aim of the study Methods

Dysfunctions of and neurotransmission play central roles in the - Male C57Bl6 mice were decapitated, their brains were rapidly removed and pathophysiology of several neurological and psychiatric disorders. Because current sagittal slices containing the striatum (400 µm thick) were prepared with a therapeutic approaches are associated with severe side effects, it is essential to develop microslicer. Slices were incubated for at least 1 h at 32°C in oxygenated artificial other tools for the treatment of these diseases. The GluN2 subunits of the N-Methyl-D- cerebro-spinal fluid (aCSF), before they were transferred to a recording chamber Aspartate (NMDA) are attractive targets for therapeutic intervention in where they were continuously perfused with aCSF at 28°C. Parkinson’s disease and drug addiction. However, the functional roles of the different GluN2 - We monitored evoked-dopamine release through the use of carbon fiber subunits, particularly in the regulation of dopamine release, is not clearly identified. We electrodes combined with continuous amperometry. Single stimulation pulses previously found that activation of NMDA receptors inhibits evoked, action potential- were applied through a glass electrode filled with aCSF placed on the surface of dependent, dopamine release in the mouse dorsal striatum and that this effect is not the slice. Stimulation evoked a response corresponding to oxidation of dopamine mediated by GluN2B-containing receptors. Given that dopaminergic neurons also express at the surface of the carbon fiber electrode which was held at +500 mV. GluN2D, our aim was to examine whether GluN2D-containing NMDA - were applied in the perfusion solution in known concentrations. receptors modulate dopamine release in the striatum.

Results III – Effect of the muscarinic receptor

To determine whether Cholinergic interneurons in the striatum, which also express I – NMDA depresses evoked-dopamine GluR2D, contribute to the depressant action of NMDA, we tested the effect of release in the dorsal striatum muscarinic and antagonists on dopamine release. A B A B 125 Oxotremorine 100 nM 150 125 NMDA 20 µM 1. baseline Oxotremorine 300 nM 100 100 100 1. 75 75 20 pA 1. 50 ms 50 50 50 2. (%of baseline) (%of baseline) Peakamplitude Peakamplitude (%of baseline) Peakamplitude 25 25 2. 2. NMDA 0 0 0 -10 0 10 20 30 40 -10 0 10 20 30 40 50 -10 0 10 20 30 40 50 Time (min) Time (min) Time (min)

A. Graph shows that NMDA (20 µM), applied in the perfusion C 1. baseline Oxotremorine dose-dependently inhibits evoked- solution for 3 minutes, depresses the peak amplitude of the dopamine release. A and B show the effect of evoked-dopamine release in the dorsal striatum in control slices two different concentrations of oxotremorine (A: (n=8). B. Example of recording obtained in one slice before and 20 pA 100 nM, n=9; B: 300 nM, n=8) applied for 15 after bath application of NMDA. 50 ms minutes on the evoked-dopamine release. C: Example of recordings obtained before and during 300 nM oxotremorine in a slice at the time II – Effect of GluN2D antagonists on the 2. Oxotremorine 300 nM points indicated on the graph B. depressant action of NMDA A B IV – Effect of muscarinic 125 125 NMDA 20 µM on the depressant action of NMDA. 100 100 A B C 75 75 125 NMDA 20 µM 125 NMDA 20 µM 50 50 100 100 1. baseline (%of baseline) (%of baseline) Peakamplitude 25 Peakamplitude 25 1. 75 75 UBP141 3-6 µM UBP141 3-6 µM 2. 20 pA 0 0 50 50 -10 0 10 20 -10 0 10 20 30 40 50 ms (% of baseline) (% of baseline) Peak amplitude Time (min) Time (min) Peak amplitude 25 25 1 µM AF-DX116 0.1 µM 2. NMDA C D 0 0 -10 0 10 20 30 40 -10 0 10 20 30 40 Time (min) Time (min) 120 125 NMDA 20 µM

100 100 75 D E F 80 ** ** 120 50 125 NMDA 20 µM 125 NMDA 20 µM (%of baseline)

Peakamplitude 60 (%of baseline) Peakamplitude 25 100 100 100 PPDA 0.5 µM 75 75 0 40 80 * * -10 0 10 20 30 40 * 50 50 PPDA (% of baseline)

Peak amplitude 60 Time (min) control UBP141 (% of baseline) (% of baseline) Peak amplitude 25 Peak amplitude 25 J104129 10 nM PD102807 0.5 µM 40 0 0 -10 0 10 20 30 40 -10 0 10 20 30 40 The GluN2D antagonist, UBP 141 (3-6 µM, n=4), has no effect on control J104129 Time (min) Time (min) AF-DX116 PD102807 evoked dopamine release by itself (A), but it reduces the inhibitory Pirenzepine effect of NMDA on evoked-dopamine release (B, n=8). The same Antagonists for M1 (A, Pirenzepine, n=11), M2 (B, AF-DX116, n=9), M3 (D, J104129, n=7) observation was made with another GluN2D antagonist PPDA (0.5 and M4 (E, PD102807, n=9) muscarinic receptors counteracted, to some degree, the effect µM, C, n=8). D: Bar graph shows the averaged maximal effect of of NMDA on evoked-dopamine release. C: Example of recordings obtained before and NMDA measured in individual slices for each treatment. ** : p<0.01 after NMDA in presence of AF-DX 116 at the time points indicated on the graph B. F: Bar graph shows the averaged maximal effect of NMDA for each treatment. * : p<0.05

Conclusion: This study identifies a critical role for cholinergic interneurons and for GluN2D- containing NMDA receptors in the presynaptic control of dopamine release in the striatum. GluN2D might thus constitute an alternative target for the development of novel pharmacological treatments.

Contact information: [email protected], [email protected] This study was supported by the Swedish Research Council , The Swedish Society for Von Eulers väg 8, 171 77 Stockholm, Sweden. Medical Research (SSMF), Tore Nilssons stiftelse för medicinsk forskning, Stiftelsen Lars Hiertas Minne, Loo and Hans Ostermans foundation for geriatric research, Swedish Phone: +46 8 524 86882 Parkinson Foundation and SLS Socialstyrelsen