Figure 6.

Dendritic neurexin expression reduces vGlut1 clustering and neurexin binding in dissociated hippocampal neurons. A1, A2, Dissociated hippocampal neurons were cotransfected with expression constructs for EGFP and HA–NRX1β4(−) at 14–15 DIV and analyzed by immunohistochemistry at 17 DIV. Cells were triple immunostained with anti-GFP (green), anti-HA (red), and anti-EEA1 antibodies (A1, blue) or anti-PSD95 antibodies (A2, blue). Some punctate structures that are colabeled for the tagged neurexin and EEA1 or PSD95 are marked by arrows. Images shown are projected confocal image stacks, but colocalization was also observed when single optical sections were examined at high magnification (data not shown). Scale bar, 5 μm. B1, B2, Hippocampal neurons at 12 DIV were cotransfected with EGFP and with the different NRX isoforms and analyzed 2 d later. Before permeabilization, fixed cells were stained with anti-HA antibodies to detect cell surface distributions of HA-tagged NRX isoforms NRX1β4(−), NRX1β4(+), and NRX1α4(−) (shown in B1). Subsequently, cells were permeabilized and glutamatergic presynaptic sites were labeled with antibodies to vGlut1 (red), and transfected cells were visualized by EGFP (green) that had been cotransfected with the NRX isoforms (shown in B2). Scale bar, 5 μm. C, Hypothetical model for postsynaptic β-neurexin function. Postsynaptic NL1 interacts trans-synaptically with NRX1 β. A pool of postsynaptic NL1 associates with NRX1β in the postsynaptic membrane in cis and therefore is not available for trans-synaptic binding. In dendrites, additional NRX1β molecules are present in early endosomal structures (EEA1-positive) from where they may recycle over the cell surface. D, Quantification of vGlut1 cluster density on transfected cells (expressed as percentage of control cells transfected with EGFP alone; n = 10; *p < 0.05, **p < 0.01).