Neuroligin-1 is required for normal expression of LTP and associative fear memory in the amygdala of adult animals

Abstract

Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.

Fig. 1.

Neuroligin expression at developmental stages and RNAi-mediated depletion of endogenous neuroligin-1 in the LA. (A) Immunoblots with pan-neuroligin antibody show that neuroligins are persistently expressed in the amygdala of rat brain at various developmental stages. (B) Immunohistochemistry images of the amygdala section (30 μm thick) 3 days after the infusion of shNL1. The outline of the LA is depicted as a dotted line. Virus-infected neurons of the LA were identified by immuno-staining for eGFP (green) and a neuronal marker NeuN (red). More than 18% of the LA neurons were successfully infected by shNL1. The proportion of infected neurons from the total LA neurons was calculated as a percentage of GFP⁺ NeuN⁺/NeuN⁺ cells in the LA. (Scale bar: 300 μm.) (C) Immunoblots exhibiting the time course of shNL1 effect after virus infusion. The level of neuoligin-1 in the shNL1-infected areas of LA started to decrease from 2 days after the virus infusion. Control or shNL1-infused animals (n > 6 per group; n > 4 per each time point) were killed at the indicated time after virus infusion. (D) Summary histograms of neuroligin-1, -2, and -3 immunoreactivity normalized to β-actin levels. The neuroligin-1 protein levels measured 2 days after virus infusion; control group, 100 ± 11.6% vs. shNL1 group, 58.8 ± 3.7% (*, P < 0.05), and 3 days after virus infusion; control group, 100 ± 8.5% vs. shNL1 group, 47.7 ± 6.8% (***, P < 0.001). The protein levels of neuroligin-2 and -3 were not significantly different between control and shNL1-infused groups at any time points (P > 0.2 for all comparisons).

Fig. 2.

Selective reduction of NMDAR-mediated currents by acute suppression of neuroligin-1. (A) DIC image of the amygdala slice (Upper). Positions of stimulation (S) and recording (R) electrodes are shown. CE (central division of the amygdala), EC (external capsule), and bundles of thalamo-amygdala fibers in the ventral striatum (IC, internal capsule) are also indicated. Magnified images of an LA principal neuron (Lower Left) and its eGFP fluorescence (Lower Right) were presented. (Scale bars: Upper, 300 μm; Lower, 20 μm.) (B) Representative traces of EPSCs used to obtain ratio of NMDAR/AMPAR-mediated currents for control- and shNL1-infected neurons are shown (Left). Stimulus artifacts were omitted for clarity. AMPAR- (large red circles) and NMDAR-dependent EPSCs (small red circles) were measured. A summary histogram for NMDAR/AMPAR ratio for each group (Right); control group, 1.25 ± 0.06, n = 9 vs. shNL1 group, 0.59 ± 0.02, n = 10 (***, P < 0.001). (C) Summary histograms for AMPAR- and NMDAR-dependent EPSCs. The mean amplitudes of AMPAR-dependent EPSCs were not significantly different among groups (Left). The mean amplitudes of NMDAR-dependent EPSCs significantly differed from each other (Right); control group, 365.83 ± 57.8 pA vs. shNL1 group, 170.6 ± 32.2 pA, without AP-5; control group, 60.6 ± 13.7 pA vs. shNL1 group, 31.2 ± 5.7 pA with AP-5 (*, P < 0.05; ***, P < 0.001).

Fig. 3.

Unaffected synaptic connections and basal synaptic transmission at thalamo-amygdala synapses. (A) Representative images of a principal neuron expressing shNL1 (Left). The dendritic region outlined with a dotted line was magnified to delineate spines (Right). (Scale bars: Left, 50 μm; Right, 10 μm.) (B) Summary histogram of spine densities (number of spines per 10 μm dendrite segment) for control and shNL1-infected neurons (P > 0.3 for both). (C) Sample traces of mEPSCs recorded from control and shNL1-infected neurons. (D) Cumulative amplitude (Upper) and interevent interval (Lower) plots of mEPSCs for control (n = 17) versus shNL1- (n = 20) infected neurons. Kolmogorov–Smirnov test was used for the comparison (P > 0.1 for both). (E) Summary histograms of mEPSC parameters. The mean peak amplitudes (P > 0.3) and the mean frequencies (P > 0.2) were not significantly different between two groups.

Fig. 4.

LTP impaired by depletion of neuroligin-1 or partial blockade of NMDARs at thalamo-amygdala pathway. (A) Mean EPSC amplitudes before and after the pairing (an arrow) were indicated after the normalization to prepairing levels. LTP was abolished in shNL1-infected neurons (solid circles), whereas the pairing protocol induced LTP in control neurons (open circles). (Inset) Representative traces of EPSCs from control (Left) and shNL1- (Right) infected neurons were indicated before (black) and 30 min after (red) the pairing. Stimulus artifacts were omitted for clarity. (B) A summary histogram of normalized EPSC amplitudes (30 min after the pairing) was depicted (***, P < 0.001); control group, 195.1 ± 53.9% for (n = 9) vs. shNL1 group, 97.0 ± 18.9% (n = 13). (C) Mean EPSC amplitudes before and after the pairing (an arrow) in untreated control slices (open circles) and slices treated with 8 μM AP-5 (solid circles). LTP was also abolished in principal neurons from the LA slices that were pretreated with AP-5 whereas the same pairing induced LTP in control neurons from untreated slices. (Inset) Representative EPSC traces from control (Left) and AP-5- (Right) treated neurons were indicated before (black) and 30 min after (red) the pairing. Stimulus artifacts were omitted for clarity. (D) A summary histogram of normalized EPSC amplitudes (30 min after the pairing) was presented for control and AP-5-treated neurons (**, P < 0.01); control group, 185.6 ± 14.9% (n = 6) vs. AP-5-treated group, 89.9 ± 16.5% (n = 7).

Fig. 5.

Requirement of neuroligin-1 for storage of associative fear memory. (A) Schematized experimental procedures to test effects of virus infusion on fear conditioning. (B) No statistical difference in freezing levels appeared between control and shNL1 groups during the habituation period (n = 11 rats for control group vs. n = 9 rats for shNL1 group). Before the first auditory cue was given, animals of both groups showed almost no freezing in a recording chamber (context) and they exhibited similar baseline freezing responses to repeated auditory cues during habituation (P > 0.6). (C) There was no statistical difference in freezing level to the cues between the two groups (P > 0.2) during the cue-shock paired fear conditioning. In the contextual fear conditioning test, shNL1 group exhibited significantly less freezing in the conditioned context compared with the control group 24 h after training (***, P < 0.001); control group, 93.5 ± 3.6% vs. shNL1 group, 49.0 ± 7.8%. In the cued fear conditioning test, shNL1 group also exhibited significant reduction of freezing behavior to the shock-associated cues compared with the control group 24 h after training (**, P < 0.01); control group, 88.3 ± 6.1% vs. shNL1 group, 43.9 ± 8.6%.