In the present study we aimed at

In the present study we aimed at integrating single cell properties and network activity in dissociated cultures of Ts65Dn and Tc1 mouse models and in their respective diploid cultures. We report here that network activity was reduced and that trisomic neurons were less excitable due to an increase in inward rectifying potassium channel expression level (KCNJ6 and KCNJ15) and an increase in HCN currents. In parallel, single neurons exhibit reduced outward K currents compared to diploid neurons, related to changes in A-type, M-type and delayed rectifier channels. A NEURON (Hines and Carnevale, 1997) simulation of a DS cell with changes to these five types of ion channels successfully reproduced our experimental results. The results were usually similar for both models, so that we could characterize the DS cell through changes in several types of potassium currents.
Methods
Results
Discussion The reduced outward potassium currents are evident in a number of measurements. Indirect evidence for this reduction is in the integrin signaling pathway properties, showing that in trisomic cells the amplitude of the action potential is higher, the threshold for excitation is lower and the AHP amplitude is sharply reduced (Fig. 4 and Table 1). Direct measurement giving voltage depolarization steps showed reduced potassium currents at all voltage steps (Fig. 5). The fast potassium currents we measured are known to be mainly A-type currents (Huguenard et al., 1991; Yuan et al., 2005). We also applied 4-AP and observed how these currents were blocked (Fig. 5a and b Inset). The slow potassium currents were shown to be mainly delayed rectifier currents (Huguenard et al., 1991). The reduced excitability is also evident in several measurements, mostly related to network activity. Imaging of [Ca2+]i, we observed that the fluorescence amplitudes and the duration of network bursts in 2D cultures were reduced (Fig. 2). This network bursting activity was suppressed by lower concentrations of baclofen in Ts65Dn networks than in diploid networks, pointing to increased GABAB inhibition (Fig. 2j). The propagation of bursting activity in one-dimensional linear networks is furthermore slower and weaker in Ts65dn and Tc1 cultures versus those of diploid cultures (Fig. 1). Finally, the network activity of Ts65Dn networks measured using electrophysiology, while holding the cells at −60mV, also showed decreased burst duration and reduced overall bursting activity (Fig. 3f and h). At the single cell level we found that neurons in both DS mouse models produced fewer action potentials in a given measured time for repetitive spiking at all current steps in which action potentials appeared (Fig. 4). We also measured increased input conductance in both DS mouse models. The large increase in input conductance causes a large decrease in cell excitability and is a combination of two factors: increased HCN currents and increased inward rectifying currents. These two changes also balance the resting membrane potential: inward rectifiers are known to lower the resting membrane potential (Reimann and Ashcroft, 1999) and HCN channels are known to raise it, because their reversal potential is high (Biel et al., 2009). The overall effect is a slight rise of the resting membrane potential in both DS models and also in the model (Table 1). The reduced excitability can be cell intrinsic, i.e. due to increased inward rectification and increased HCN currents, but it may also be due to weaker synapses: smaller and less frequent mEPSCs were measured in organotypic and acute slices by Hanson et al. (2007) for the Ts65Dn model. On the other hand Best et al. (2008) reported no difference of mEPSCs frequency or amplitude in cultures of Ts65Dn. We measured mEPSCs for Tc1 neurons and did not observe a difference in amplitude. The rate of mEPSCs was reduced, but not significantly. Since we measured reduced excitability also at the cell level (Fig. 4), and since the increase in input conductance we observed is also a major factor in cell excitability, weaker synapses could contribute to reduced excitability, but the intrinsic properties of the cell, increased inward rectification and increased HCN currents, probably play a major role.