In the first series of experiments, we investigated whether the inhibition of serotonergic receptors due to administration of MET would affect the LTP. As a first step, experiments without LTP induction were conducted to test whether prior application of serotonergic receptor inhibitor MET would affect synaptic transmission. In all control recordings, test stimulation without tetanization caused a gradual decrease in the complex EPSPs amplitudes in command neurons (habituation) (Control, n.cutaneus, n = 9, n.intestinalis, n = 9). In another series of experiments, MET was applied during the first 40 min of the experiment and was washed out after. The untetanized inputs (both nerves) (Fig. 2a, b) showed no significant changes in synaptic transmission under MET administration, except a few time points at cutaneal nerve stimulation (Control+MET, n.cutaneus, n = 8, n.intestinalis, n = 9) compared to the Control groups.
In the next series of experiments, we performed test stimulation of cutaneal nerve combined with its tetanization accompanied by the serotonin bath application. The baseline complex EPSPs were recorded for 40 minutes at 10-minute intervals. Five serotonin applications and tetanic stimulations elicited a robust LTP that was constant over 240 minutes (Fig. 2c, group 5x(5-HT+tet), n = 10). Thus, 2 hours after the last tetanization and serotonin application, the EPSP amplitude was 138.1 ± 17.1% of the initial value, while in the Control group, the response attenuated, and at the same time point the EPSP amplitude was 63.1 ± 5.4%, (p < 0.0001). Four hours after tetanization, the amplitude of the EPSP of tetanized inputs also significantly exceeded the response amplitudes in the Control group (5x(5-HT+tet) group, 150.0 ± 23.6%; Control group, 31.6 ± 5.8%, p < 0.0001). Application of MET 40 minutes before tetanization + application of serotonin affected the amplitude of EPSP drastically: it caused markedly declined LTP (Fig. 2c, MET+5x(5-HT+tet) group, n = 10). So, 2 hours after the last tetanization + serotonin, the EPSP amplitude in the MET + 5x (5-HT + tet) group was 59.4 ± 13.1% and significantly differed from the EPSP amplitudes in the 5x (5-HT + tet) (138.1 ± 17.1%, p < 0.005). During the next 2 hours there was a gradual decrease in the amplitude of EPSP in MET+5x(5-HT+tet). So, 4 hours after the last tetanization + application of serotonin the EPSP amplitude in the MET + 5x (5-HT + tet) was 27.7 ± 4.7%, while in the 5x(5-HT+tet) group it was 150.0 ± 23.6% (p < 0.0005) (Fig. 3a).
In the next series of experiments, we performed test stimulation of intestinal nerve combined with its tetanization accompanied by the serotonin bath application. The baseline EPSP was recorded for 40 minutes at 10-minute intervals. Five serotonin application and tetanus stimulation elicited a robust LTP that was persistent over 240 minutes (Fig. 2d, group 5x(5-HT+tet), n = 11). Thus, 2 hours after the last tetanization and serotonin application, the EPSPs amplitude was 139.7 ± 14.4% of the initial value, while in the Control group, the response attenuated, and at the same time point the EPSP amplitude was 56.1 ± 12.1% (p < 0.0005). Four hours after tetanization, the amplitude of the EPSP of tetanized inputs also significantly exceeded the response amplitudes in the Control group (5x(5-HT+tet) group, 124.5 ± 9.8%; Control group, 36.8 ± 10.7%, p < 0.0001). MET application significantly impaired the EPSPs amplitude in MET+5x(5-HT+tet) group (n = 10) in comparison to the control (Fig. 2d) in experiments with stimulation of the intestinal nerve. For example, the levels of LTP, expressed as a percentage baseline and quantified at 2 hours after the tetanus, were 65.9 ± 16.0% in MET+5x(5-HT+tet) and 139.7 ± 14.4% in 5x(5-HT+tet) (p < 0.005). Remarkably, MET significantly influenced only the late phase of LTP. The EPSP amplitude in presence of MET was reduced to 27.9 ± 7.8% vs 124.5 ± 9.8% in 5x(5-HT+tet) at 4 hours after the tetanus (p < 0.0001) (Fig. 3b). It should be noted that MET application didn’t change the EPSPs without the LTP induction. Thus, electrophysiological analysis revealed that repeated serotonin applications and tetanizations facilitated the EPSPs in the premotor interneurons, and this effect was suppressed by pharmacological inhibition of serotonergic receptors with MET.
In the second series of experiments, we investigated whether the increased histone acetylation upon administration of HDACis NaB or TSA would prevent the disruption of LTP by MET. First, we checked for possible effects of the HDACis on basal synaptic transmission. In all recordings, test stimulation without tetanization caused a gradual decrease in the EPSPs amplitudes in command neurons (habituation) (groups Control, n.cutaneus, n = 9, n.intestinalis, n = 9, Fig. 4a, b). In a separate series of experiments, NaB or TSA were applied during the first 40 min of the experiment and were washed out after. The untetanized inputs (both nerves) showed no significant changes in synaptic transmission under HDACis administration (Fig. 4a, b, groups Control+NaB, n.cutaneus, n = 8, n.intestinalis, n = 8; Control+TSA, n.cutaneus, n = 9, n.intestinalis, n = 9) compared to the Control groups.
The next step was to find out whether the HDACis are able to rescue the MET-induced impairment of potentiation (Fig. 4c, d). In the next series of experiments, the cutaneal nerve was chosen for tetanization. The baseline EPSPs were recorded for 40 minutes at 10-minute intervals. Five serotonin applications and tetanic stimulations elicited the robust LTP that lasted over 240 minutes (Fig. 4c, group 5x(5-HT+tet), n = 11). NaB or TSA treatments combined with MET not only rescued the serotonin-evoked LTP, but increased it (Fig. 4c, NaB+MET+5x(5-HT+tet), n = 10, TSA+MET+5x(5-HT+tet) groups, n = 10). For example, the levels of LTP, quantified at 70 minutes after the first tetanus, were 191.7 ± 21.9% in NaB+MET+5x(5-HT+tet) and 196.4 ± 20.1% in TSA+MET+5x(5-HT+tet), these values significantly differed from 5x(5-HT+tet) (133.4 ± 10.7%) at this time point (NaB+MET+5x(5-HT+tet) vs. 5x(5-HT+tet), p < 0.05; TSA+MET+5x(5-HT+tet) vs. 5x(5-HT+tet), p < 0.05). Two hours after the last tetanization and serotonin application, the EPSPs amplitudes were 174.6 ± 23.9% and 177.8 ± 16.5% of the initial value in NaB+MET+5x(5-HT+tet) and TSA+MET+5x(5-HT+tet), respectively, in 5x(5-HT+tet) group the response at the same time point was 138.1 ± 17.1% (p >0.05). Four hours after the tetanus, the EPSPs amplitudes were 143.2 ± 18.1% and 130.6 ± 13.8% of the initial value in NaB+MET+5x(5-HT+tet) and TSA+MET+5x(5-HT+tet), respectively, in 5x(5-HT+tet) group the response at the same time point was 150.0 ± 23.6% (p >0.05) (Fig. 5a). We did not observe any difference between these groups during the late LTP.
In the next series of experiments, the intestinal nerve was chosen for tetanization. The baseline EPSPs were recorded for 40 minutes at 10-minute intervals. Five serotonin applications and tetanic stimulations elicited a robust LTP that was constant over 240 minutes (Fig. 4d, group 5x(5-HT+tet), n = 11). NaB or TSA treatment combined with MET significantly increased the serotonin-evoked LTP (NaB+MET+5x(5-HT+tet), n = 10, TSA+MET+5x(5-HT+tet) groups, n = 11). For example, the levels of LTP, quantified at 70 minutes after the first tetanus, were 190.6 ± 13.8% in NaB+MET+5x(5-HT+tet) and 198.2 ± 13.4% in TSA+MET+5x(5-HT+tet) which were significantly different from that of 5x(5-HT+tet) (149.8 ± 10.3%) at this time point (NaB+MET+5x(5-HT+tet) vs. 5x(5-HT+tet), p < 0.05; TSA+MET+5x(5-HT+tet) vs. 5x(5-HT+tet), p < 0.05). Two hours after the last tetanization and serotonin application, the EPSP amplitude was 173.4 ± 14.8% and 158.0 ± 7.6% of the initial value in NaB+MET+5x(5-HT+tet) and TSA+MET+5x(5-HT+tet) respectively, in 5x(5-HT+tet) group the response at the same time point was 139.7 ± 14.4% (p >0.05). Four hours after the tetanus trains the EPSP amplitude was 118.8 ± 11.8% and 113.1 ± 10.2% of the initial value in NaB+MET+5x(5-HT+tet) and TSA+MET+5x(5-HT+tet) respectively, in 5x(5-HT+tet) group the response at the same time point was 124.5 ± 9.8% (p >0.05) (Fig. 5b). We did not observe any difference between these groups during late LTP.
Thus, in experiments with a strong tetanization protocol, MET application led to a significant decrease in EPSP amplitude. When NaB or TSA was co-applied with MET, the EPSPs remained stably elevated. HDACis-mediated rescue effects were observed for both synaptic inputs (n.cutaneus, n.intestinalis). These results suggest that HDAC blockade during LTP induction could rescue the impaired synaptic potentiation in the MET-treated isolated CNS via upregulation of the histone acetylation.
In an independent series of experiments, we tested whether the histone acetylation blockade can influence synaptic plasticity in experiments with a weak training protocol. There were no long-term effects in all experiments in which the weak training protocols were applied. In groups 5tet, 5tet+1x5-HT, NaB+5tet/TSA+5tet we observed a short-term increase of responses to cutaneal nerve stimulation in the first 90 minutes after the tetanization session (Figs. 6a, c) followed by a decrease in the EPSPs amplitudes below 100%. Similar results were observed for the intestinal nerve (Figs. 6b, d). At the same time, the amplitudes of the EPSPs in these groups during the entire time of the experiment practically did not differ from each other and were significantly less than 5x(5-HT+tet) at the late phase of potentiation. Altogether, the data obtained confirm that multiple 5-HT applications are necessary for LTP in this experimental model. However, applications of HDACis (NaB/ TSA) led to increase of both early and late LTP even in experiments with the weak training protocol with single serotonin application. For example, when the cutaneal nerve was tetanized, the levels of LTP, quantified at 60 minutes after the first tetanus, were 181.6 ± 18.6% in NaB+5tet+1x5-HT which were significantly different from that of 5x(5-HT+tet) (124.5 ± 11.5%) at this time point (p < 0.05). The EPSP amplitude at 2 hours after the last tetanization and serotonin application in the presence of NaB/ TSA was 148.9 ± 16.3% (NaB+5tet+1x5-HT, n = 10) and 134.6 ± 14.3% (TSA+5tet+1x5-HT, n = 10) what was comparable to 5x(5-HT+tet) (138.1 ± 17.1%, n = 10, p > 0.05). Late phase of LTP (4 hours after tetanus) in the presence of NaB/ TSA was comparable to 5x(5-HT+tet) (NaB+5tet+1x5-HT, 149.5 ± 20.3%, TSA+5tet+1x5-HT, 107.0 ± 15.2%, 5x(5-HT+tet), 150.5 ± 23.6%, p > 0.05) (Fig. 7a). When the intestinal nerve was tetanized, the levels of LTP, quantified at 60 minutes after the first tetanus, were 240.3 ± 20.3% in NaB+5tet+1x5-HT, which were significantly different from that of 5x(5-HT+tet) (147.3 ± 13.1%) at this time point (p < 0.001). In the presence of TSA, the levels of LTP, quantified at 60 minutes after the first tetanus, were 205.2 ± 16.7% in TSA+5tet+1x5-HT, which were significantly different from that of 5x(5-HT+tet) (147.3 ± 13.1%) at this time point (p < 0.005). The EPSP amplitude at 2 hours after the last tetanization and serotonin application in the presence of NaB/ TSA was 140.1 ± 23.7% (NaB+5tet+1x5-HT, n = 10) and 181.0 ± 29.3% (TSA+5tet+1x5-HT, n = 10), what was comparable to 5x(5-HT+tet) (139.7 ± 14.4%, n = 10, p > 0.05). Late phase of LTP (4 hours after tetanus trains) in the presence of NaB/ TSA was comparable to 5x(5-HT+tet) (NaB+5tet+1x5-HT, 95.9 ± 15.4%, TSA+5tet+1x5-HT, 125.9 ± 17.6%,5x(5-HT+tet), 124.5 ± 9.8%, p > 0.05) (Fig. 7b). Thus, the EPSPs amplitudes in weak protocol experiments showed the following trend: potentiation was observed during early phase of LTP but the late phase at the end of experiments was absent. It was curious that application of NaB or TSA without serotonin produced potentiation lasting only minutes that gradually declined to the pre-tetanic values. Only in the presence of single pulse of 5-HT and NaB or TSA, the LTP was induced and lasted over 240 minutes which was comparable to the potentiation induced by five pulses of 5-HT.