Neuroligin-3 Regulates Excitatory Synaptic Transmission and EPSP-Spike Coupling in the Dentate Gyrus In Vivo

Neuroligin-3 (Nlgn3), a neuronal adhesion protein implicated in autism spectrum disorder (ASD), is expressed at excitatory and inhibitory postsynapses and hence may regulate neuronal excitation/inhibition balance. To test this hypothesis, we recorded field excitatory postsynaptic potentials (fEPSPs) in the dentate gyrus of Nlgn3 knockout (KO) and wild-type mice. Synaptic transmission evoked by perforant path stimulation was reduced in KO mice, but coupling of the fEPSP to the population spike was increased, suggesting a compensatory change in granule cell excitability. These findings closely resemble those in neuroligin-1 (Nlgn1) KO mice and could be partially explained by the reduction in Nlgn1 levels we observed in hippocampal synaptosomes from Nlgn3 KO mice. However, unlike Nlgn1, Nlgn3 is not necessary for long-term potentiation. We conclude that while Nlgn1 and Nlgn3 have distinct functions, both are required for intact synaptic transmission in the mouse dentate gyrus. Our results indicate that interactions between neuroligins may play an important role in regulating synaptic transmission and that ASD-related neuroligin mutations may also affect the synaptic availability of other neuroligins. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02663-9.


Fig. S1
Absolute values of the fEPSP slope, but not the population spike amplitude, during LTP measurements were lower in Nlgn3-deficient mice. (a) During the pre-TBS baseline and following strong TBS, (see Methods) Nlgn3 KO (n = 10) mice had lower fEPSP slopes than WT (n = 8) mice. Diagram shows the mean fEPSP slope during the pre-TBS baseline. (b) The population spike amplitudes during the pre-TBS baseline and following strong TBS were similar in both groups. Diagram shows that the mean population spike amplitude during the pre-TBS baseline did not differ significantly between WT and Nlgn3 KO mice. (c) During the pre-TBS baseline and following the combined weak and strong TBS, Nlgn3 KO (n = 5) had lower slopes compared to WT (n = 10) mice. Diagrams show the mean fEPSP slopes during the pre-TBS baseline, after the weak TBS (0-10 min), and after the strong TBS (30-40 min). (d) The population spike amplitudes during the pre-TBS baseline and following the combined weak and strong TBS were similar in WT and Nlgn3 KO mice. Diagram shows that the mean population spike amplitude during the pre-TBS baseline. Asterisks denote statistical significance by unpaired Welch's t-test (*p < 0.05). Data are represented as mean ± SEM

Anesthesia and Surgery
Urethane (Sigma-Aldrich, Munich, Germany) solution (1.25 g of urethane in 10 ml 0.9% NaCl solution) was used to anesthetize the mice with an initial injection (1.2 g/kg body weight) applied intraperitoneally. Supplemental doses (totalling 0.3-1 g/kg) were injected subcutaneously until the proper anesthetic depth was attained. This anesthetic protocol has been previously published [1] and urethane has been shown to affect inhibitory, excitatory, and modulatory neurotransmitter receptors to similar degrees [2]. The body temperature of the animal was constantly monitored through a rectal probe and maintained at 36.5-37.5°C using a heating pad. Additional local anesthesia was provided by a subcutaneous injection of prilocaine hydrochloride with adrenaline 1:200,000 (Xylonest 1%, AstraZeneca, Wedel, Germany) into the scalp. The mouse was then placed into a stereotactic frame (David Kopf Instruments) for the accurate determination of the recording and stimulation locations. After exposing the skull, the location of bregma was determined from the intersection of the sagittal and the coronal sutures.
The holes for the stimulation (coordinates: 3.7 mm posterior to bregma, 2.5 mm lateral to the midline) and recording (coordinates: 1.7 mm posterior to bregma, 1.0 mm lateral to the midline) electrodes were made with a dental drill (Dremel), and the dura mater was carefully removed before inserting the electrodes. The ground electrode was inserted into the neck musculature. First, the granule cell responses to different stimulation intensities (i.e., the input-output relationship) was determined by applying a series of stimuli ranging from 30 to 800 µA (0.1 ms stimulus duration) and collecting the evoked fEPSP. Three responses were collected and averaged for each stimulus intensity. The amplitude of the population spike (defined as the average of the amplitude from the first positive peak to the antipeak and the amplitude from the antipeak to the second positive peak, see Fig. 2a) was used to determine the excitability of the granule cell population. The slope of the fEPSP, which was measured during the early component of the waveform to avoid contamination by the population spike (see Fig. 1a), was used as an indicator of synaptic efficacy. In the analysis relating the fEPSP slope to the spike amplitude (EPSP-spike plot) each curve was fitted using a Boltzmann function. Only curves with a goodness of fit value (R 2 ) greater than 0.80 were used for the analysis of the v50 values.

Stimulation protocols and data analysis
Next, paired-pulse facilitation (PPF) of the fEPSP was elicited by two subsequent pulses at an intensity below the population spike threshold (between 20 and 120 µA/0.2 ms), with interpulse intervals (IPIs) varying from 15 to 100 ms. A total of three paired-pulse responses at each IPI were collected and averaged, and the facilitation factor for each IPI was calculated by dividing the amplitude of the second fEPSP by that of the first fEPSP.
In order to examine feedback and feedforward inhibition in the dentate network, pairedpulse inhibition (PPI) and disinhibition (PPDI) of the population spike were elicited at weak stimulation intensities (evoking approximately 1 mV population spikes) and at maximal stimulation intensities for IPIs ranging from 15 to 1,000 ms (0.2 ms pulse duration/three repetitions per IPI). The relative inhibition of the population spike amplitude was calculated by dividing the spike amplitude following the second pulse by the spike amplitude following the first pulse. After fitting the PPI/PPDI curves with a Boltzmann function, the IPI at which both responses would be equal was determined.
Finally, LTP was induced by theta-burst stimulation (TBS), a stimulation protocol patterned after the hippocampal theta rhythm [3]. In some mice, a strong TBS protocol (i.e. six series of six trains of six pulses at 400 Hz, with 0.2 s between trains and 20 s between series) was applied [4]. In other mice, an initial weak TBS protocol (i.e., only three series of six trains of six pulses at 400 Hz, 0.2 s between trains and 20 s between series) was followed by the strong TBS protocol 30 min later [1]. In both groups, the perforant path was stimulated with 0.1 ms pulses at 0.1 Hz with a stimulation intensity set to elicit a population spike in the range of 1-3 mV before LTP induction. Both the pulse width and the stimulus intensity during TBS were doubled in comparison to the baseline. The maximum allowable baseline stimulus intensity was 400 µA. The potentiation of the fEPSP and the population spike following TBS were expressed as percentages relative to the pre-TBS mean values.

Preparation of hippocampal synaptosomal fractions and immunoblot analysis
Mice aged 8-12 weeks were sacrificed by rapid decapitation and the brains were removed. The hippocampi were dissected out on ice, and all subsequent steps were carried out at 4 °C. Hippocampi were homogenized in solution A (0.32 M sucrose, 1 mM MgCl2, 0.5 mM CaCl2, 1 mM HEPES, pH 7.4, containing freshly added protease inhibitor cocktail) with 12