Long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in α -neurexin KO mice

Schaffer collateral-CA1 synapses show long term increase in the efficacy of synaptic transmission in response to tetanic stimulation (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999; Malenka and Bear, 2004). We examined whether deletion of α-neurexins leads to any change in long-term potentiation (LTP). To measure LTP, baseline responses were evoked every 30 s for 10 minutes and LTP was induced by 4 trains separated by 20 s, each train consisting of 100 Hz stimuli for 1 s. The post-train responses were then measured every 30 s for 60 minutes. The magnitude of LTP was determined as the average between 50-60 minutes of responses after induction. We found that LTP was significantly higher in neurexin 1α/2α double KOs (191.4 ± 19.48 % of baseline; n = 11 slices, 5 mice) as compared to control mice (142.3 ± 12.96 % of baseline; n = 11 slices, 5 mice) (Figure 3.8). In addition, short-term potentiation defined as the maximum potentiation within 1 minute after the trains was also significantly enhanced in neurexin 1α/2α double KOs (299.0 ± 28.69 % of baseline;

n = 11 slices, 5 mice) as compared to control mice (224.9 ± 16.41 % of baseline; n = 11 slices, and plotted against stimulus number. Error bars are shown only for every 10th response, for clarity. (D) The responses for all three genotypes returned to similar values after 300 stimuli as indicated by the ratio of fEPSP slope 300^th stimulus/fEPSP slope 4^th stimulus. (E) Post-tetanic potentiation (PTP) was not significantly different between control and double KO mice. Baseline responses were obtained by stimulating at 1 Hz, and PTP was elicited by a single 100 Hz train for 1 s (at t = 0). Post-tetanic responses were obtained by stimulation at 1 Hz. The experiments were done in the presence of 100 µM DL-APV to prevent the induction of NMDA receptor-dependent short term potentiation (STP).

Figure 3.8 Increased LTP at Schaffer collateral-CA1 synapses in neurexin 1α/2α double KO mice. (A) Example traces. An average of 10 responses before High Frequency Stimulus-HFS (1) and between 55 and 60 min. after HFS (2). (B) Long-term potentiation elicited by HFS ( 4 x 100 Hz for 1s each, 20 s interval). fEPSP slopes were normalized to baseline and plotted against time. Time 0 represents the application of HFS. (C) The magnitude of LTP was determined by averaging responses between 50 and 60 minutes after HFS. Hippocampal slices from neurexin 1α/2α double KO (n = 11 slices, 5 mice) show significantly enhanced LTP as compared to controls (n = 11 slices, 5 mice). (D) Short-term potentiation (STP) measured as the maximum response within 1 minute after HFS is also significantly increased in neurexin 1α/2α double KO mice as compared to controls.

We then investigated the mechanism for enhanced LTP in neurexin 1α/2α double KO mice.

Induction of LTP at Schaffer collateral-CA1 synapses with the experimental conditions that we used is known to be NMDA-receptor-dependent. We wondered whether the enhanced LTP that we observed in neurexin 1α/2α double KO mice might be due to the appearance of an abnormal NMDA-receptor-independent form of LTP. To check this, experiments were performed in the presence of NMDA receptor antagonist DL-APV (100 µM). We found that the slices from neurexin 1α/2α double KO mice failed to develop any LTP in the presence of

DL-APV (Figure 3.9). This ruled out an abnormal NMDA receptor-independent component of LTP in neurexin 1α/2α double KO mice.

Figure 3.9 Enhanced LTP in neurexin 1α/2α double KO is NMDA-dependent. A high frequency stimulus ( 4 x 100 Hz for 1 s each, 20 s interval) was given in the presence of 100 µM DL-APV (NMDA receptor antagonist). Time 0 represents the application of HFS. No LTP could be induced in the presence of 100 µM DL-APV in slices from neurexin 1α/2α double KOs (n = 4 slices, 2 mice). Slices were bathed in 100 µM DL-APV for 25 minutes before recordings were started and DL-APV was present till 5 minutes after the HFS.

Another reason that could underlie enhanced LTP in neurexin 1α/2α double KO mice is the reduction in inhibitory tone in the CA1 region of hippocampus. To examine this possibility, we performed experiments with control mice and neurexin 1α/2α double KO mice with 10 µM bicuculline methiodide (GABAA receptor antagonist). This treatment should remove any influence of GABAergic synaptic transmission on the analysis of LTP between the two genotypes. After measuring baseline responses every 30 s for 10 minutes, LTP was induced

by 2 trains (100 Hz, 1 s duration) seperated by 20 s. 2 trains were used in this protocol (instead of 4) because under blockade of GABA-ergic transmission multiple population spikes might appear and contaminate the rising slope of fEPSP. After induction, the higher is the potentiation the earlier the population spikes appear. Using only 2 trains reduces the potentiation and hence reduces the contamination of fEPSP slope with pop spikes. The post-train responses were then evoked by stimuli every 30 s for 60 minutes. The magnitude of LTP was determined as the average between 50-60 minutes of responses after induction. We found that in the presence of bicuculline methiodide, there was no difference in the magnitude of LTP between control mice (172.5 ± 38.95 % of baseline; n = 6 slices, 3 mice) and neurexin 1α/2α double KO mice (158.9 ± 13.07 % of baseline, n = 5 slices, 4 mice) (Figure 3.10). This showed that it was the reduction in inhibitory GABAergic synaptic transmission that was responsible for enhanced LTP in neurexin 1α/2α double KO mice.

Figure 3.10 Enhanced LTP in neurexin 1α/2α double KO depends on a reduced activity of inhibitory GABAergic synapses. LTP was induced in the presence of 10 µM bicuculline methiodide (GABA-A receptor antagonist) using an HFS (2 x 100 Hz for 1 s each, 20 s interval). Time 0 represents the application of HFS. There was no significant difference in the amount of LTP between control (n = 6 slices, 3 mice) and neurexin 1α/2α double KO mice (5 slices, 4 mice). Slices were bathed in 10 µM bicuculline methiodide for 25 minutes before recordings were started and bicuculline was present throughout the experiment.