Periodic Reporting for period 1 - SorCSbalance (Role of the sorting receptor SorCS1 in controlling excitation/inhibition balance in neural circuits.)
Reporting period: 2015-05-01 to 2017-04-30
A major aspect raised by the previous findings is to understand whether SorCS1 controls synaptic function in vitro and in vivo (aim 2). To test whether SorCS1 controls synapse number, I electroporated mouse cortical cells from SorCS1flox/flox mice with GFP and Cre recombinase and then I quantified the density of excitatory and inhibitory synapses. I found that loss of SorCS1 does not affect the density of puncta positive for the excitatory synaptic markers (Fig.1 G and H), but decreases the density of puncta positive for the inhibitory synaptic markers (Fig.1 I and J). Then, I tested whether an altered abundance of synaptic receptors and decreased number of inhibitory synapses affects synaptic transmission. We recorded spontaneous miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) from somatosensory layer 5 pyramidal neurons in acute slices from SorCS1flox/flox and Emx1-Cre:SorCS1flox/flox mice to measure basal synaptic transmission (SorCS1flox/flox mice were crossed with Emx1-Cre transgenic mice to specifically decrease SorCS1 expression in principal cortical neurons). The frequency, but not the amplitude, of mEPSCs and mIPSCs was significantly decreased in Emx1-Cre:SorCS1flox/flox cortical neurons comparing with control cortical neurons (Fig. 1 A-C). This result shows that loss of SorCS1 in vivo impairs synaptic transmission.
To further explore this data, namely to understand whether this defect was caused by a pre-synaptic or postsynaptic impairment in neuronal function, we moved to an in vitro system. We recorded mEPSCs and mIPSCs from cortical neurons prepared from SorCS1flox/flox mice and electroporated with GFP and Cre, but also from non-electroporated neurons in Cre-electroporated cultures, to assess whether the effect of SorCS1 loss on spontaneous synaptic transmission is cell-autonomous (postsynaptic) or non-cell-autonomous (presynaptic). Frequency of mEPSCs and mIPSCs, but not amplitude, was decreased in SorCS1flox/flox in Cre-electroporated neurons in comparison with GFP-electroporated cells (Fig.1 D-F). We also found no change in the frequency of non-electroporated in comparison to GFP-electroporated cells (Fig.1 D-F), strongly suggesting that the effect of SorCS1 loss on mPSC frequency is cell-autonomous. The decrease in inhibitory synapse density aforementioned fully explains the reduced mIPSC frequency in SorCS1 KO cells, however the decrease in mEPSC frequency should be caused by another mechanism since the density of excitatory synapses was not altered following loss of SorCS1. Because I found that synaptic and surface levels of AMPA receptors are decreased upon loss of SorCS1, next I investigated whether the number silent synapses was increased in cortical cells without SorCS1 by using immunostaining. Mouse cortical neurons electroporated with Cre and GFP were labeled for surface GluA1 and the NMDA-receptor-subunit GluN1 under non-permeabilizing conditions. Quantification of the density of synaptic surface GluN1 puncta lacking surface GluA1 showed an increased in the number of silent synapses in SorCS1KO neurons (Fig.1 T and U). Altogether, these results show that SorCS1 differentially controls excitatory and inhibitory synapse function: SorCS1 sustains functional excitatory synapses (with AMPARs) and is required to keep normal density of inhibitory synapses. These results were published recently in Neuron (Neuron 87, 764-780; Savas et al., 2015), and Marie Skłodowska-Curie fellowship support is acknowledged in this publication.