A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin

Abstract

Factors that control differentiation of presynaptic and postsynaptic elements into excitatory or inhibitory synapses are poorly defined. Here we show that the postsynaptic density (PSD) proteins PSD-95 and neuroligin-1 (NLG) are critical for dictating the ratio of excitatory-to-inhibitory synaptic contacts. Exogenous NLG increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory synaptic currents. In contrast, PSD-95 overexpression enhanced excitatory synapse size and miniature frequency, but reduced the number of inhibitory synaptic contacts. Introduction of PSD-95 with NLG augmented synaptic clustering of NLG and abolished NLG effects on inhibitory synapses. Interfering with endogenous PSD-95 expression alone was sufficient to reduce the ratio of excitatory-to-inhibitory synapses. These findings elucidate a mechanism by which the amounts of specific elements critical for synapse formation control the ratio of excitatory-to-inhibitory synaptic input.

Fig. 1.

NLG induces excitatory and inhibitory presynaptic contact formation. Hippocampal neurons were transfected with HA-tagged NLG (HA-NLG) or GFP and stained for synaptophysin (Syn), PSD-95, VGAT, or VGLUT. (A) Size and number of Syn-positive terminals (white arrowheads; n = 10 and 11 cells) were enhanced on dendrites from neurons expressing HA-NLG when compared with GFP-expressing cells (B)(n = 12 and 9 cells). (C and D) A modest increase in the number of PSD-95 puncta was observed. (E) Similar changes in Syn cluster size and number were seen at both DIV 12 and 15. (F) Summary of changes in PSD-95 cluster number and size in cells expressing HA-NLG. (G–I) Size and number of VGAT-positive (n = 11 cells) and VGLUT-positive (n = 8 cells) contacts (white arrowheads) increased on dendrites from HA-NLG-expressing cells when compared with GFP-expressing cells (n = 12 cells, 242 terminals). (J) Summary of changes in VGAT and VGLUT cluster size and number in HA-NLG-expressing cells. In A–D and G–I, black arrowheads depict synaptic puncta from neighboring untransfected cells. *, P < 0.05; ***, P < 0.001 (Mann–Whitney U test). (Scale bars: 10 μm, A, C, and G; 1 μm, A Inset, B Inset, D, H, and I.)

Fig. 2.

PSD-95 regulates NLG clustering and presynaptic maturation. Hippocampal neurons were transfected with either PSD-95 GFP or a mutant form of PSD-95 containing PDZ domains 1 and 2 (PSD-95 GFP 1-PDZ2). (A–C) Cells were stained with antibodies specific to NLG1 and NLG3 and the presynaptic marker synaptophysin (Syn). Dendritic clusters of endogenous NLG were compared between cells expressing PSD-95 GFP (A) (white arrowheads; n = 11 cells) and neighboring untransfected cells (B) or cells expressing PSD-95 GFP 1-PDZ2 (C). (D) Summary of changes of NLG and Syn in these neurons. PSD-95 GFP expression increased NLG and Syn cluster size. No significant change (n.s.) in NLG and Syn cluster size was observed in neurons expressing PSD-95 GFP 1-PDZ2. (E–H) Expression of PSD-95 GFP with HA-NLG enhanced the size of NLG clusters but reduced Syn-positive contact number when compared with neurons expressing HA-NLG alone. (E and F Right) Higher magnification micrographs of boxed regions overview images are shown. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (Scale bars: 10 μm, A Left, C Left, E Left, and F Left; 1 μm, A Right, B, C Right, E Right, and F Right.)

Fig. 3.

PSD-95 restricts NLG localization to excitatory synapses. Cells transfected with HA-NLG and PSD-95 GFP and stained with VGAT or VGLUT. (A Right) Magnifications of Inset Left.(A and B) PSD-95 GFP recruited HA-NLG to sites positive for VGLUT (n = 7 cells) but not VGAT (n = 10 cells). (C) Graph summarizing these results. (D and E) GluR1 cluster size was enhanced in dendrites from cells expressing PSD-95 GFP alone (n = 9 cells) or HA-NLG (n = 8 cells; white arrowheads) when compared with GluR1 clusters from untransfected controls (n = 10 cells). Expression of HA-NLG alone (n = 8 cells) did not enhance GluR1 clustering. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (Scale bars: 10 μm, A Left; 1 μm, A Right, B, and D.)

Fig. 4.

PDZ-dependent interactions regulate the morphology of synapses induced by PSD-95 and NLG. (A and B) Neurons were transfected with PSD-95 GFP and either WT HA-NLG or a mutant HA-NLG lacking the PDZ-binding site (HA-NLGΔPDZb) and immunostained as indicated. (A) Colocalization (white arrowheads) of HA-NLG with PSD-95 GFP and apposed synaptophysin (Syn)-labeled presynaptic terminals. (A1 and A2) Localization of HA-NLG clusters and apposed presynaptic terminal to PSD-95 GFP. (B) HA-NLGΔPDZb is not recruited to PSD-95 GFP clusters (white arrowheads). (C) The correlation between the size of PSD-95 GFP, HA-NLG, and Syn clusters at single synaptic sites (linear fit; red line) was stronger in cells coexpressing PSD-95 GFP and WT HA-NLG (P < 0.0001; n = 10 cells, 219 clusters) when compared to cells expressing PSD-95 GFP and HA-NLGΔPDZb (P < 0.001; n = 10 cells, 225 clusters). (Scale bars: 10 μm, A and B;1 μm, A1, A2, B1, and B2.)

Fig. 5.

Manipulation of NLG and PSD-95 expression alters the ratio of excitatory to inhibitory synaptic contacts. (A Upper) The number and size of VGAT-labeled contacts significantly decreased in cells coexpressing HA-NLG and PSD-95 GFP (n = 10 cells, 701 terminals) compared with cells expressing HA-NLG alone (n = 11 cells, 2,145 terminals). (A Lower) No change in number and size of VGLUT-labeled excitatory presynaptic contacts in cells coexpressing HA-NLG and PSD-95 GFP (n = 7 cells, 572 terminals) compared with cells expressing HA-NLG alone (n = 8 cells, 705 terminals). (B and C) Electrophysiological recordings from hippocampal neurons transfected with HA-NLG and GFP (to visualize cells) (n = 26), PSD-95 GFP (n = 8), HA-NLG and PSD-95 GFP (n = 9), or GFP alone (controls, n = 16). Spontaneous mEPSCs (B) and mIPSCs (C) were measured at holding potentials of –60 mV and +10mV, respectively. (B) Enhanced mEPSC frequency and amplitude in cells expressing PSD-95 GFP and HA-NLG with PSD-95 GFP. Enhancement of mEPSC frequency, but not of amplitude, in cells expressing HA-NLG with GFP is shown. (C) mIPSC frequency was enhanced in cells expressing HA-NLG with GFP, reduced in cells expressing PSD-95 GFP, and unchanged in cells expressing HA-NLG with PSD-95 GFP. No difference in the average mIPSC amplitude between cell groups was found. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test).

Fig. 6.

Altered expression of PSD-95 influences the ratio of excitatory-to-inhibitory presynaptic contacts. (A) Hippocampal cells were transfected with either GFP (controls, Left) or PSD-95 GFP (Center) and immunostained at DIV 12 for GFP, synaptophysin (Syn), and the GABA inhibitory presynaptic marker VGAT. PSD-95 GFP expression resulted in a significant decrease in the percentage of VGAT-positive inhibitory contacts (Upper Right) but did not alter the number of PSD-95 clusters (Lower Right) (n = 15 cells, 288 VGAT+, 778 VGAT–) compared with controls (n = 12 cells, 242 VGAT+, 272 VGAT–). (B and C) Introduction of GFP with either a scrambled siRNA (control siRNA; Left) or PSD-95 siRNA (Center). (B Right) A reduction in the number of PSD-95-positive puncta in cells cotransfected with GFP and PSD-95 siRNA (n = 11 cells, 301 puncta) compared with controls (n = 7 cells, 406 puncta) was found. (C Right) A significant increase in the percentage of inhibitory contacts (VGAT+) and a concurrent decrease in the percentage of excitatory contacts (VGAT–)(Upper) was found, but there was no change in the total number of synapses in cells expressing GFP and PSD-95 siRNA (n = 11 cells, 322 VGAT+, 282 VGAT–) compared with controls (n = 12 cells, 230 VGAT+, 494 VGAT–)(Lower). **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (Scale bars: = 1 μm.)