The physiological activity of dopaminergic midbrain (DA) neurons is important for movement, cognition, and reward. Altered activity of DA neurons is a key finding in schizophrenia, but the cellular mechanisms have not been identified. Recently, KCNN3, a gene that encodes a member (SK3) of the smallconductance, calcium-activated potassium (SK) channels, has been proposed as a candidate gene for schizophrenia. However, the functional role of SK3 channels in DA neurons is unclear. We combined patch-clamp recordings with single-cell RT-PCR and confocal immunohistochemistry in mouse midbrain slices to study the function of molecularly defined SK channels in DA neurons. Biophysical and pharmacological analysis, single-cell mRNA, and protein expression profiling strongly suggest that SK3 channels mediate the calciumdependent afterhyperpolarization in DA neurons. Perforated patch recordings of DA neurons in the substantia nigra (SN) demonstrated that SK3 channels dynamically control the frequency of spontaneous firing. In addition, SK3 channel activity was essential to maintain the high precision of the intrinsic pacemaker of DA SN neurons. In contrast, in the ventral tegmental area, DA neurons displayed significantly smaller SK currents and lower SK3 protein expression. In these DA neurons, SK3 channels were not involved in pacemaker control. Accordingly, they discharged in a more irregular manner compared with DA SN neurons. Thus, our study shows that differential SK3 channel expression is a critical molecular mechanism in DA neurons to control neuronal activity. This provides a cellular framework to understand the functional consequences of altered SK3 expression, a candidate disease mechanism for schizophrenia.

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 Kv4a and Kv4b subunit transcript numbers is suf®-cient to explain the full spectrum of spontaneous pacemaker frequencies in identi®ed DA SN neurons. This linear coupling between Kv4a as well as Kv4b mRNA abundance, A-type channel density and pacemaker frequency suggests a surprisingly simple molecular mechanism for how DA SN neurons tune their variable ®ring rates by transcriptional control of ion channel genes. Keywords: Kv4/KChip/pacemaker activity/dopaminergic neurons/quantitative real-time TaqMan PCR Introduction Dopaminergic (DA) midbrain neurons are essential for important brain functions such as voluntary movement, working memory and reward (Kitai et al., 1999;Spanagel and Weiss, 1999;Goldman-Rakic et al., 2000). They are also closely involved in the aetiology of neuropsychiatric disorders including schizophrenia, drug abuse and Parkinson's disease (Spanagel and Weiss, 1999;Abi-Dargham et al., 2000;Obeso et al., 2000). Thus, it is of great interest to de®ne the molecular mechanisms that control electrical activity of DA midbrain neurons and consequently dopamine release. Best studied are the DA neurons in the substantia nigra (SN, A9) that release dopamine in their striatal target areas (Onn et al., 2000;Smith and Kieval, 2000). In vivo, these classical striatonigral DA neurons discharge in a pacemaker or irregular single spike mode and less frequently show burst activity (Grace and Bunney, 1984a,b;Kitai et al., 1999). In brain slice preparations, the regular pacemaker mode is retained even during inhibition of synaptic transmission (Grace and Onn, 1989;Lacey et al., 1989), indicating that the pacemaker activity of DA SN neurons is autonomously generated. Spontaneous electrical activity is believed to originate from intrinsic calcium-dependent oscillations of the membrane potential (Grace, 1991). The tuning of this basic oscillator is mediated by ion channels that operate in the subthreshold range and thus determine the frequency of this cellular pacemaker. Little is known about the molecular identity of the ion channels that control this neuronal pacemaker. Ever since the classic work of Connor and Stevens (1971a,b) in invertebrate neurons a general role for A-type potassium channels in frequency control has been assumed (Rudy, 1988;Grace, 1991;Coetzee et al.,...

Hyperpolarization-activated currents (Ih) are key players in shaping rhythmic neuronal activity. Although candidate genes for Ih channels have been cloned (HCN1-HCN4), the subunit composition of different native Ih channels is unknown. We used a combined patch-clamp and qualitative single-cell reverse transcription multiplex polymerase chain reaction (RT-mPCR) approach to analyse HCN1-4 coexpression profiles in four neuronal populations in mouse CNS. Coexpression of HCN2, HCN3 and HCN4 mRNA was detected in single neurons of all four neuronal cell types analysed. In contrast, HCN1 mRNA was detected in neocortical and hippocampal pyramidal neurons but not in dopaminergic midbrain and thalamocortical neurons. HCN1 expression was correlated with significantly faster activation kinetics on the level of individual neurons. Semiquantitative single-cell RT-mPCR analysis demonstrated that HCN1 mRNA expression is at least eightfold higher in cortical neurons than subcortical neurons. We show that single neurons possess complex coexpression patterns of Ih candidate genes. Alternative expression of HCN1 is likely to be an important molecular determinant to generate the different neuronal Ih channel species adapted to tune either subcortical or cortical network activity.