To characterize the burst properties of putative dopamine neurons BYL719 manufacturer described above, we used a conventional method in which burst onset is marked by two spikes with an ISI ≤ 80 ms and offset is marked by a subsequent ISI ≥ 160 ms (Grace and Bunney, 1984a). Due to the frequency-dependent

nature of this method, there is the potential for spurious detection of burst activity in cells with higher firing rates; therefore, we performed additional analysis using a modified method of burst detection. We set the threshold for burst initiation to three spikes occurring within the mean of the ISI, allowing us to assess transient rate increases relative to the overall firing rate of the cell. Similar to conventional burst detection, this method also detected a significant increase in burst events and a decrease in the ISI of the first spikes within a burst in hSK3ΔGFP-expressing mice compared to controls (Figures S3). Other burst properties were largely similar to those detected with the conventional method (Figure S3). Increased burst firing and elevated firing rate of putative dopamine neurons is consistent with suppression of SK currents enhancing the excitability http://www.selleckchem.com/products/gdc-0068.html of dopamine neurons (Ji et al., 2009). The mechanism

of this enhanced excitability is not well understood but could involve modulation of glutamatergic synaptic transmission. It is known that SK channel blockade facilitates NMDA-evoked burst firing of dopamine neurons in slice (Seutin et al., 1993 and Johnson

and Seutin, 1997). Additionally, in other brain regions, SK2 channels colocalize with NMDARs in the postsynaptic density (PSD) where they form a calcium-mediated feedback loop (Faber et al., 2005, Ngo-Anh et al., 2005 and Lin et al., 2008); thus, hSK3Δ may enhance excitability of dopamine neurons by potentiating NMDAR-mediated excitatory postsynaptic currents (EPSCs). To determine whether SK3 colocalizes with NMDARs in dopamine neurons, we performed immunoelectron microscopy in the VTA of wild-type mice. Immunogold labeling for the NMDAR subunit NR1 and for SK3 revealed a close about juxtaposition of the two channels within the PSD (Figure 4A). Quantification of the distribution of gold particles within the PSD revealed similar profiles of both NR1 and SK3 (Figure 4B). To test whether suppression of SK channels alters NMDAR-mediated currents in dopamine neurons, we monitored evoked EPSCs in acute slices before and after addition of apamin. Cells were recorded in a zero magnesium solution to relieve the blockade of NMDARs, and NMDAR and AMPAR currents were isolated by bath application of CNQX or AP5, respectively.