Figure 3.

cis-expression of neurexins reduces synaptogenic activity of neuroligin-1 in hippocampal neurons. A, Hippocampal neurons were cotransfected with VSV-epitope-tagged NL1 and different HA-epitope-tagged NRX1 expression constructs at 10 DIV and were analyzed 2 d later. For these experiments, the NL1 splice variant containing A and B splice insertions was used because this is the most abundant variant endogenously expressed in hippocampal neurons. Examples of transfected cells for the following conditions are shown: NL1 alone, NL1 plus NRX1β4(−),NL1 plus NRX1β4(+), NL1 plus NRX1α4(−), and NL1 plus NRXΔLNS. Left column, Immunostaining for the VSV epitope in NL1; middle column, immunostaining for the HA epitope in the NRXs; right column, immunostaining for the synaptic vesicle marker VAMP2/synaptobrevin. The staining for NL and NRX isoforms was performed in nonpermeabilized cells, and images for all conditions were recorded with identical confocal acquisition settings. Scale bar, 50 μm. B, Quantification of VAMP2 immunoreactivity on the transfected cells, normalized to control cells overexpressing only NL1. Similar results were obtained in at least three independent experiments with at least 10 cells measured per condition in each experiment. Data from one experiment are shown (number of cells per condition, n = 10; **p < 0.01, ***p < 0.001). C, Quantification of average NL1 cell surface staining intensity in dendrites of neurons expressing NL1 alone or together with neurexin isoforms (n = 10 cells; **p < 0.01).