Dopaminergic (DA) midbrain neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are involved in various brain functions such as voluntary movement and reward and are targets in disorders such as Parkinson9s disease and schizophrenia. To study the functional properties of identified DA neurons in mouse midbrain slices, we combined patch-clamp recordings with either neurobiotin cell-filling and triple labeling confocal immunohistochemistry, or single-cell RT-PCR. We discriminated four DA subpopulations based on anatomical and neurochemical differences: two calbindin D₂₈-k (CB)-expressing DA populations in the substantia nigra (SN/CB+) or ventral tegmental area (VTA/CB+), and respectively, two calbindin D₂₈-k negative DA populations (SN/CB−, VTA/CB−). VTA/CB+ DA neurons displayed significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with other DA neurons. In contrast, all four DA populations possessed significant differences inI_h channel densities andI_h channel-mediated functional properties like sag amplitudes and rebound delays in the following order: SN/CB− → VTA/CB− → SN/CB+ → VTA/CB+. Single-cell RT-multiplex PCR experiments demonstrated that differential calbindin but not calretinin expression is associated with differentialI_h channel densities. Only in SN/CB− DA neurons, however, I_h channels were actively involved in pacemaker frequency control. In conclusion, diversity within the DA system is not restricted to distinct axonal projections and differences in synaptic connectivity, but also involves differences in postsynaptic conductances between neurochemically and topographically distinct DA neurons.

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC₅₀) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3–5 mg/kg) and Cmpd-21 (0.4–1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

The activity of dopaminergic (DA) substantia nigra (SN) neurons is essential for voluntary movement control. An intrinsic pacemaker in DA SN neurons generates their tonic spontaneous activity, which triggers dopamine release. We show here, by combining multiplex and quantitative real‐time single‐cell RT–PCR with slice patch–clamp electrophysiology, that an A‐type potassium channel mediated by Kv4.3 and KChip3 subunits has a key role in pacemaker control. The number of active A‐type potassium channels is not only tightly associated with the pacemaker frequency of individual DA SN neurons, but is also highly correlated with their number of Kv4.3L (long splice variant) and KChip3.1 (long splice variant) mRNA molecules. Consequently, the variation of Kv4α and Kv4β subunit transcript numbers is sufficient to explain the full spectrum of spontaneous pacemaker frequencies in identified DA SN neurons. This linear coupling between Kv4α as well as Kv4β mRNA abundance, A‐type channel density and pacemaker frequency suggests a surprisingly simple molecular mechanism for how DA SN neurons tune their variable firing rates by transcriptional control of ion channel genes.