1. DA Transporter Currents: A Reason for Excitement Susan L. Ingram WSU-Vancouver, Vancouver WA

2. Neurotransmitter transporters: Transport is electrogenic cytoplasmextracellular space Neurotransmitter 1-3 Na + 1Cl - DA, 5HT, NE, GABA, Glycine Substrate-gated uncoupled conductance

3. Questions Does activation of DAT produce measurable currents in DA neurons? What ions are responsible? Is the current large enough to change excitability of the DA neurons? Does the current play a role in neurotransmitter release?

4. Dopaminergic Synapse Presynaptic cell Postsynaptic cell DA D2 MGluR D2D1 11 GIRK Amph X COC X

5. Whole-cell patch clamp recordings from DA neurons in culture biocytin Raclopride Sulpiride Prazosin TTX

6. Does activation of DAT produce measurable currents?

7. DAT substrates activate an inward current

8. DAT currents are uncoupled from transport Current (pA) 50 10 -9 10 -5 [DA] Uptake (fmol/cell/s) 10 -9 10 -5 [DA] 0.1

9. What ions are responsible?

10. DAT currents are carried by chloride ions * Permeability to nitrate is 10-fold greater than GABA A -mediated chloride conductance (p NO3 /p Cl = 2.5) control nitrate Normalized current Voltage (mV) -100 p NO3 /p Cl = 14

11. Currents are larger using the gramicidin perforated patch technique

12. Is the current large enough to change excitability of the DA neurons?

13. DA stimulates firing by a D2-independent mechanism * Shi, et al. (2000) Dual effects of amphetamine on dopamine neurons mediated by dopamine and nondopamine receptors. J Neurosci 20(9):3504-3511.

14. Low DA concentrations stimulate firing via a DAT-dependent mechanism

15. Muscimol - control V rest Voltage (mV) Normalized current * Gramicidin perforated-patch recordings GABA A -mediated chloride conductance is inhibitory DA - control V rest Voltage (mV) Normalized current DAT-mediated chloride conductance is excitatory

17. Is the DAT-mediated excitation sufficient to stimulate neurotransmitter release?

18. DA neurons make glutamatergic autapses in culture

19. Amphetamine increases the AMPA-mediated EPSC

20. 32 ± 8%

22. Conclusions DAT mediates an uncoupled chloride conductance. DAT currents in neurons are activated at much lower DA concentrations than the D2-receptor or transport. The DAT-mediated chloride conductance excites DA neurons. Activation of DAT-mediated currents is a potential mechanism for stimulating DA release in the SN.

23. Susan Amara Krishna Prasad Spencer Watts Mark Sonders Kevin Poth Ole Mortensen Chialin Cheng Bill Freeman Andreia Fontana Jonathan Morris Alessandra Cecchini Wendy Fairman Barb Leighton Mary Chalmers Debra Peth Jean Yang Bozena Kutyba Nikki Lind