Neuroligin-1 knockdown reduces survival of adult-generated newborn hippocampal neurons

Survival of adult-born hippocampal granule cells is modulated by neural activity, and thought to be enhanced by excitatory synaptic signaling. Here, we report that a reduction in the synaptogenic protein neuroligin-1 in adult-born neurons in vivo decreased their survival, but surprisingly, this effect was independent of changes in excitatory synaptic function. Instead, the decreased survival was associated with unexpected changes in dendrite and spine morphology during granule cell maturation, suggesting a link between cell growth and survival

Neuroligin-1 overexpression in newborn granule cells in vivo.

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons

Neuroligin-1 overexpression does not change granule cell soma or dendrite morphology at 21 days post-mitosis.

<p>A. Low-power images of 21-day-old granule cells labeled with retroviruses encoding GFP only (above) or neuroligin-1 IRES GFP (below). Scale bar: 50 µm. B. Tracings of the same 21-day-old neurons, used for quantification. Scale bar: 50 µm. C, D, E. Summary data for 21 day old neurons showing no difference in cell soma size, total dendritic length, or dendritic process number between conditions (shown ± SEM; control, n = 9 cells; NLG-1, n = 15 cells; n.s. all conditions). F. Dendritic branch (Sholl) analysis demonstrates a similar overall branching pattern in control and neuroligin-1 overexpressing 21-day-old cells.</p

Neuroligin-1 overexpression increases the amplitude and frequency of spontaneous excitatory post-synaptic currents in 3-week-old granule cells.

<p>A. Representative sEPSC recordings from control (GFP only) and neuroligin-1 IRES GFP infected 21-day-old neurons in the absence of TTX. Scale bars: 10 pA, 500 ms. B, C. Summary data for sEPSC amplitude and frequency in 21-day-old adult-born neurons (n = 13 cells in each group, data shown ± SEM; * p<0.05, *** p<0.005). D. sEPSC amplitude distributions skew toward larger event amplitudes in neuroligin-1 overexpressing cells (p<0.0005, K-S test). E. Paired pulse facilitation is not altered in 21-day-old neuroligin-1 overexpressing cells relative to control (GFP only) cells. PPF is represented as the ratio of the peak amplitude of the second current to that of the first (control, n = 13,4,4 cells; NLG-1, n = 18,6,6 for 50, 100, and 250 ms intervals respectively; p>0.2 all groups). F. Representative traces at a 50 ms interval are shown for control and neuroligin-1 overexpressing 21-day-old neurons. Scale bars: 10 pA (Control), 20 pA (Neuroligin-1), 50 ms (both). G. Neuroligin-1 overexpression increases the ratio of AMPAR-mediated currents relative to NMDAR-mediated currents in 21-day-old granule cells (shown ± SEM; control, n = 16 cells; NLG-1, n = 12 cells; ** p<0.01). H. Sample recordings showing AMPAR-mediated currents (downward deflections) and slower combined AMPAR/NMDAR-mediated currents (upward deflections) recorded from single granule cells. Scale bars: 20 pA, 50 ms.</p

Neuroligin-1 overexpression increases the frequency of miniature excitatory post-synaptic currents in 3-week-old granule cells.

<p>A. Representative recordings showing mEPSCs in control (GFP only) and neuroligin-1 IRES GFP infected 21-day-old neurons. An asterisk is placed next to each detected event for clarity. Scale bars: 5 pA, 1 sec. B, C. Summary data for mEPSC amplitude and frequency (shown ± SEM; control n = 15 cells, NLG-1 n = 13 cells; * p<0.05). D. Cumulative mEPSC amplitude distribution for control (GFP only) and neuroligin-1 IRES GFP infected 21-day-old cells. E. Representative average mEPSCs are depicted for control (GFP only) and neuroligin-1 overexpressing cells. Scale bars for single traces: 2 pA, 5 ms. An overlay of peak-scaled responses demonstrates similar rise and decay kinetics between conditions. F, G, H. Neuroligin-1 overexpression does not change inhibitory innervation onto 21-day-old cells. F. Representative mIPSC recordings from control and neuroligin-1 overexpressing 21-day-old granule cells. Scale bars: 20 pA, 2 sec. G, H. Summary data for mIPSC amplitudes and frequencies at 21 dpi (shown ± SEM; control n = 14, NLG-1 n = 9; p>0.6 for each measurement).</p

Retrovirus-mediated neuroligin-1 expression in newborn granule cells.

<p>A. Experimental design schematic illustrating retroviral infection of dentate stem cells in anesthetized mice, allowing <i>in vivo</i> cell maturation, and subsequent <i>ex vivo</i> imaging or recording at defined post-mitotic stages (14 or 21 dpi). B. Retroviral constructs used in this paper. The Ubiquitin promoter drives expression of control proteins (either GFP or mCherry), a neuroligin-1-GFP fusion protein, or neuroligin-1 in conjunction with GFP via an IRES sequence. All neuroligin-1 constructs also carried an extracellular HA tag. C. Newly born granule cells 21 days post-mitosis, infected with HA-neuroligin-1 IRES GFP retrovirus at day 0. Confocal stacks of anti-HA and anti-GFP stained granule cells demonstrate co-expression of exogenous neuroligin-1 and GFP. Scale bar: 20 μm. D. Higher power image of an infected granule cell dendrite stained with anti-HA antibody (red), showing the exogenous HA-neuroligin-1 expression pattern. Scale bar: 5 μm. E. High-power image of a dendritic segment from a double-infected granule cell (one retrovirus encoding mCherry to outline cell morphology, and a separate virus encoding a neuroligin-1-GFP fusion protein). Scale bar, 5 μm.</p

Neuroligin-1 overexpression transiently increases dendritic process number.

<p>A. HA-neuroligin-1 expression pattern in 14-day-old adult-born granule cells, visualized with anti-HA staining. Exogenous neuroligin-1 is expressed throughout the dendritic tree (left; scale bar, 10 µm.) and is concentrated along filopodium-like protrusions from the dendritic shaft (right; scale bar: 5 µm). B, C. Dendritic tree morphology of representative 14-day-old neurons shows an increased number of processes along the dendritic tree of neuroligin-1 overexpressing cells. Scale bar: 25 µm. D, E, F. Summary data for 14 day old neurons shows no difference in cell soma size, but a neuroligin-1 mediated increase in total dendritic length and dendritic process number (shown ± SEM, control, n = 12 cells; NLG-1, n = 11 cells; *** p<0.005). G. Dendritic branch (Sholl) analysis demonstrates increased branching along more distal dendrites in neuroligin-1 overexpressing 14-day-old cells (* p<0. 01, two way repeated measures ANOVA). The shaded areas correspond to the average distance from each soma to the granule cell layer (GCL)-inner molecular layer (IML) border in the two groups (n.s.). H. Overlay of Sholl analyses from 14 and 21 day old neurons. Between 14 and 21 days, granule cell dendrites grow to span the full width of the dentate molecular layer and basal dendrites (10–20 µm from soma) retract.</p

Neuroligin-1 overexpression does not accelerate excitatory synapse formation.

<p>A. Dendritic segments (inner molecular layer) of control and neuroligin-1 overexpressing granule cells rarely contain spines at 14 days post-mitosis. Scale bar: 2 µm. B. Sample mEPSC traces from control (GFP only infected) or neuroligin-1 overexpressing cells showing a lack of excitatory currents in 14-day-old cells. For each example, 10 consecutive sweeps (400 continuous seconds each) are overlaid. Scale bars: 20 pA, 5 sec. C. mEPSC frequencies from 14 day-old cells. The event frequency is shown for each cell, with mean data in red. The mode frequency in each group is zero events, and averaged frequencies are not significantly different (control, n = 13 cells; NLG-1, n = 10 cells, p>0.1). D. mIPSC recordings show no change in mIPSC amplitude or frequency in 14-day-old neuroligin-1 overexpressing cells (control, n = 10 cells; NLG-1, n = 8 cells; p>0.2 for each). E. Representative averaged mIPSCs from single cells, as well as a peak-scaled overlay, demonstrate no difference in mIPSC kinetics between conditions. Scale bars: 50 pA, 50 ms.</p