Neuroligin-1 loss is associated with reduced tenacity of excitatory synapses

Abstract

Neuroligins (Nlgns) are postsynaptic, integral membrane cell adhesion molecules that play important roles in the formation, validation, and maturation of synapses in the mammalian central nervous system. Given their prominent roles in the life cycle of synapses, it might be expected that the loss of neuroligin family members would affect the stability of synaptic organization, and ultimately, affect the tenacity and persistence of individual synaptic junctions. Here we examined whether and to what extent the loss of Nlgn-1 affects the dynamics of several key synaptic molecules and the constancy of their contents at individual synapses over time. Fluorescently tagged versions of the postsynaptic scaffold molecule PSD-95, the AMPA-type glutamate receptor subunit GluA2 and the presynaptic vesicle molecule SV2A were expressed in primary cortical cultures from Nlgn-1 KO mice and wild-type (WT) littermates, and live imaging was used to follow the constancy of their contents at individual synapses over periods of 8-12 hours. We found that the loss of Nlgn-1 was associated with larger fluctuations in the synaptic contents of these molecules and a poorer preservation of their contents at individual synapses. Furthermore, rates of synaptic turnover were somewhat greater in neurons from Nlgn-1 knockout mice. Finally, the increased GluA2 redistribution rates observed in neurons from Nlgn-1 knockout mice were negated by suppressing spontaneous network activity. These findings suggest that the loss of Nlgn-1 is associated with some use-dependent destabilization of excitatory synapse organization, and indicate that in the absence of Nlgn-1, the tenacity of excitatory synapses might be somewhat impaired.

Figure 1. A mouse cortical neuron expressing PSD-95:EGFP.

A composite image of a cortical neuron from a Nlgn-1 KO mouse expressing PSD-95 tagged with EGFP. Most bright puncta represent postsynaptic densities of dendritic spines. 17 days in vitro. Bar, 50 µm.

Figure 2. Exchange rates of PSD-95:EGFP at individual synapses.

A) Dendrites of a neuron from a WT mouse expressing PSD-95:EGFP. Two PSD-95:EGFP puncta, one in each rectangular region, were selected for photobleaching. Bar, 10 µm. B) The two puncta (enclosed in squares) were selectively photobleached by high intensity laser light and fluorescence recovery at these sites was subsequently followed by time-lapse imaging. Fluorescence intensities shown in false color. C) Fluorescence recovery time course for the two photobleached puncta shown in A and B. D) Average FRAP curves for synapses from Nlgn-1 KO (13 neurons, 129 synapses) and WT littermates (13 neurons, 140 synapses; Mean ± SEM). Fits to sums of two exponentials (see Results) are shown as thick lines. E) Distribution of fractional recovery values for all photobleached PSD-95:EGFP puncta. The fractional recovery of each punctum was calculated as the average value of the 3 last points of its normalized recovery curve (as indicated by curly bracket in D).

Figure 3. Constancy of PSD-95:EGFP contents at individual synapses.

A) Dendrites of a neuron from a Nlgn-1 KO mouse expressing PSD-95:EGFP (23 days in vitro). Full time lapse sequence of this region is shown in Video S1. Bar, 10 µm. B) Changes over time in the fluorescence intensity of four puncta (enclosed in numbered and color coded rectangles) over >18 hours. Raw data is shown as thin lines whereas thick lines show the same data after smoothing with a 5 point low pass filter. A calculation of the range over mean measure is illustrated for punctum #1: the dashed lines show the minimal and maximal values of the smoothed data and the arrow indicates the mean fluorescence intensity over the imaging period. C) Distribution of range over mean values for all PSD-95:EGFP puncta measured in these experiments (Nlgn-1 KO: 46 neurons, 3465 puncta; WT: 44 neurons, 3367 puncta). D) SI decay rates measured in neurons from Nlgn-1 KO and WT mice (mean ± SEM).

Figure 4. PSD-95:EGFP puncta loss rate.

A) Dendrites of a neuron from a Nlgn-1 KO mouse expressing PSD-95:EGFP (21 days in vitro). B) Time lapse sequence of region enclosed in rectangle in A. Full arrowheads point to PSD-95:EGFP lost over time (empty arrowheads). Fluorescence intensities shown in false color. C) Fractional loss rates (% lost over 12 hour periods) in neurons from Nlgn-1 KO mice and WT littermates.

Figure 5. Constancy of synaptic vesicle contents at individual presynaptic sites.

A) Axons of a neuron from a WT mouse expressing EGFP:SV2A (22 days in vitro). Individual fluorescent puncta represent presynaptic clusters of synaptic vesicles. Bar, 10 µm. B) Distribution of range over mean values for all EGFP:SV2A puncta followed in these experiments (Nlgn-1 KO: 19 neurons, 599 boutons; WT: 21 neurons, 856 boutons). C) SI decay rates measured in neurons from Nlgn-1 KO and WT mice (mean ± SEM).

Figure 6. Constancy of AMPAR contents of individual postsynaptic sites.

A) A composite image of a cortical neuron from a Nlgn-1 KO mouse expressing SEpH:GluA2. Most bright puncta are located on the postsynaptic membrane of dendritic spines. 19 days in vitro. Bar, 50 µm. B) Time lapse sequence of region enclosed in rectangle in A. Only a subset of images is shown here. C) Distribution of range over mean values for all SEpH:GluA2 puncta followed in these experiments (Nlgn-1 KO: 15 neurons, 563 synapses; WT: 14 neurons, 652 synapses). D) SI decay rates measured in neurons from Nlgn-1 KO and WT mice (mean ± SEM).

Figure 7. AMPAR redistribution dynamics of Nlgn-1 deficient neurons in a WT environment.

A) A cortical neuron expressing SEpH:GluA2 from a Nlgn-1 KO mouse plated in a network in which the majority of cells were obtained from a WT littermate. 23 days in vitro. Bar, 10 µm. B) Distribution of range over mean values for all SEpH:GluA2 puncta followed in these experiments (Nlgn-1 KO neurons in WT neuronal networks: 6 neurons, 563 synapses; Nlgn-1 KO networks: 6 neurons, 652 synapses). C) SI decay rates (mean ± SEM).

Figure 8. Activity dependence of AMPAR redistribution dynamics.

A) Distribution of range over mean values for SEpH:GluA2 puncta in the presence of CNQX (10 µM), AP-5 (50 µM) and TTX (1 µM) (Nlgn-1 KO: 10 neurons, 481 synapses; WT: 10 neurons, 514 synapses). B) SI decay rates in the presence of the aforementioned pharmacological agents.