Whole-cell patch-clamp recordings of OXT-mRFP1 neurons were performed in the mPVN of OXT-mRFP1 transgenic rats (Fig. 1A left). The mRFP1 fluorescence was observed in the PVN by fluorescence microscopy (Fig. 1A right). The OXT-mRFP1 neurons were distinguishable from other neurons by DIC fluorescence in the PVN (Fig. 1B a–f).
Electrophysiological response of OXT-mRFP1 neurons in the PVN using whole-cell patch-clamp recordings
We confirmed AI to evaluate the degree of inflammation on the day of the experiment. AI of AA rats was significantly higher than in control rats (control group: 0, n = 6 rats; AA group: 11.83 ± 1.14, n = 6 rats; one-way ANOVA, F1,10 = 108.17, p = 0.0000011, Fig. 2A). We examined the resting membrane potential using whole-cell patch-clamp recordings of neurons in the PVN, and the average value of the resting membrane potential was − 63.10 ± 3.29 mV for controls and − 54.33 ± 2.25 mV for AA rats, and a significant difference was recognized (control group: n = 15 neurons / 7 rats; AA group: n = 19 neurons / 8 rats; one-way ANOVA, F1,32 = 5.13, p = 0.030, Fig. 2B). We show the action potential firing pattern of an OXT-mRFP1 neuron in current-clamp mode by induction of currents (from − 5 pA to + 25 pA with 5 pA) for 400 ms (Fig. 2C) and representative pooled results show neuronal responses to current induction (from − 10 pA to + 40 pA with 5 pA) in control and AA rats. There was no significant difference in the firing rate between the control and AA groups (each group: n = 12 neurons / 6 rats). To examine whether excitatory pre-synaptic transmitter release changed in OXT-ergic neurons after chronic inflammation, we recorded sEPSCs and mEPSCs in OXT-mRFP1 neurons in the mPVN of control and AA rats. We show representative sEPSCs and mEPSCs recorded in the OXT-mRFP1 neurons in slices from control and AA rats at a holding potential of -60 mV, and a cumulative histogram of inter-event interval and amplitude (Fig. 3A, B, D, E). The frequency of mEPSCs and sEPSCs in OXT-mRFP1 neurons in the mPVN significantly increased in the AA group compared with the control group (sEPSC control group: 1.28 ± 0.16 Hz, n = 13 neurons / 6 rats; sEPSC AA group: 3.02 ± 0.48, n = 12 neurons /6 rats; one-way ANOVA, F1,10 = 12.59, p = 0.0017, Fig. 3C left) (mEPSC control group: 0.67 ± 0.082 Hz, n = 34 neurons / 9 rats; mEPSC AA group: 0.88 ± 0.057, n = 36 neurons / 9 rats; one-way ANOVA, F1,68 = 4.53, p = 0.037, Fig. 3F left). The amplitude of mEPSCs and sEPSCs in OXT-mRFP1 neurons in the mPVN were not significantly different between control and AA groups (Fig. 3C and 3F right).
Next, we investigated whether synthesized OXT from AA rats affects the EPSPs in OXT-ergic neurons. We used a bath application of the OXT receptor antagonist L-368,899 (20). The results show representative sEPSCs and mEPSCs recorded in OXT-mRFP1 neurons in slices from control and AA rats at a holding potential of -60 mV (Fig. 4A and D). Bath application of L-368,899 dose-dependently increased the frequency of mEPSCs and sEPSCs in OXT-mRFP1 neurons in AA rats (sEPSC; 10 nM: 111.30 ± 11.68% of baseline, t (10) = 0.96, p = 0.38; 100 nM: 145.58 ± 24.11% of baseline, t (10) = 1.89, p = 0.12; 1 µM: 154.37 ± 14.85% of baseline, t (10) = 3.66, p = 0.014, paired t-test, n = 6 neurons / 3 rats, Fig. 4B right) (mEPSC; 10 nM: 102.66 ± 9.36% of baseline, t (10) = 0.28, p = 0.79; 100 nM: 169.05 ± 16.09% of baseline, t (10) = 4.08, p = 0.015; 1 µM: 235.24 ± 39.14% of baseline, t (10) = 3.46, p = 0.026, paired t-test, n = 6 neurons / 3 rats, Fig. 4E right); however, there was no change in control rats (sEPSC; 10 nM: 107.99 ± 9.87% of baseline, t (10) = 0.81, p = 0.45; 100 nM: 108.23 ± 8.53% of baseline, t (10) = 7.74, p = 0.96; 1 µM: 103.39 ± 6.96% of baseline, t (10) = 0.48, p = 0.64, paired t-test, n = 6 neurons / 3 rats, Fig. 4B left) (mEPSC; 10 nM: 99.01 ± 8.80% of baseline, t (10) = 0.11, p = 0.91; 100 nM: 105.46 ± 13.54% of baseline, t (10) = 0.40, p = 0.69; 1 µM: 101.91 ± 8.77% of baseline, t (10) = 0.22, p = 0.82, paired t-test, n = 6 neurons / 3 rats, Fig. 4E left). Bath application of L-368,899 did not change the amplitude of mEPSCs and sEPSCs in OXT-mRFP1 neurons in control and AA rats (Fig. 4C and F). Therefore, the results suggest that feedback of mEPSCs and sEPSCs from synthesized OXT occurs in OXT-ergic neurons in AA rats.
Examination of retrograde transmitters in the feedback system in OXT-mRFP1 neurons in AA rats
Previous studies have demonstrated that regarding feedback in central neurons, retrograde synaptic transmission via transmitters such as nitric oxide (NO), cannabinoid receptor 1 (CB1), and GABA is known to occur (21–23). Thus, we investigated whether these transmitters were involved in the feedback from synthesized OXT in AA rats. Following bath application of the GABAA receptor antagonist (100 µM picrotoxin) (Fig. 5A and B) and CB1 antagonist (2 µM AM 251) (Fig. 5C and D), bath application of L-368,899 still increased the frequency of mEPSCs (picrotoxin; 100 µM picrotoxin: 125.41 ± 10.7% of baseline, t (10) = 2.52, p = 0.040, 10 nM L-368,899: 129.25 ± 7.20% of baseline, t (10) = 4.06, p = 0.0066; 100 nM L-368,899: 133.87 ± 6.48% of baseline, t (10) = 5.23, p = 0.0034; 1 µM L-368,899: 194.97 ± 20.36% of baseline, t (10) = 4.66, p = 0.00522, paired t-test, n = 6 neurons / 3 rats, Fig. 4B left) (AM 251; 2 µM AM 251: 111.53 ± 8.28% of baseline, t (10) = 1.39, p = 0.23, 10 nM L-368,899: 117.97 ± 13.87% of baseline, t (10) = 1.30, p = 0.26; 100 nM L-368,899: 139.42 ± 6.00% of baseline, t (10) = 6.57, p = 0.0028; 1 µM L-368,899: 215.11 ± 20.70% of baseline, t (10) = 5.56, p = 0.00512, paired t-test, n = 6 neurons / 3 rats, Fig. 4D left), and did not change the amplitude of mEPSCs from baseline (Fig. 4B right and D right). However, following the bath application of the NOS inhibitor (100 µM L-NAME), bath application of L-368,899 did not change the frequency (L-NAME; 100 µM L-NAME: 99.94 ± 4.39% of baseline, t (10) = 0.08, p = 0.94, 10 nM L-368,899: 107.30 ± 5.80% of baseline, t (10) = 1.26, p = 0.26; 100 nM L-368,899: 101.48 ± 11.34% of baseline, t (10) = 0.13, p = 0.90; 1 µM L-368,899: 106.34 ± 8.15% of baseline, t (10) = 0.778, p = 0.48, paired t-test, n = 6 neurons / 3 rats, Fig. 4F left) or amplitude of mEPSCs in AA rats Fig. 4F right). Furthermore, mEPSCs increased significantly from baseline only following the bath application of picrotoxin (paired t-test, p < 0.05). Therefore, this suggests that NOS contributes to the feedback system of synthesized OXT in AA rats.
Confirmation of the effect of OXT and the OXT receptor antagonist L-368,899 in OXT-mRFP1neurons in slices from control rats
We confirmed the effects of OXT and L-368,899 in OXT-mRFP1 neurons. After recording a stable baseline at a holding potential of -60 mV, we recorded mEPSCs following the application of 1 µM OXT and different doses of L368,899 (10 and 100 nM and 1 µM) on mEPSCs in OXT-mRFP1 neurons in control rats. Following bath application of OXT, the frequency of mEPSCs in OXT-mRFP1 neurons was significantly increased (OXT; 1 µM OXT: 233.45 ± 25.99% of baseline, t (10) = 5.1, p = 0.00215, n = 6 neurons / 4 rats, Fig. 6 left), while OXT did not change the amplitude of mEPSCs (Fig. 6 right). After application of L-368,899, the increased frequency of mEPSCs returned to baseline in a dose-dependent manner (10 nM L-368,899: 210.16 ± 18.37% of baseline, t (10) = 5.99, p = 0.00185; 100 nM L-368,899: 125.75 ± 18.81% of baseline, t (10) = 1.37, p = 0.22; 1 µM L-368,899: 98.66 ± 10.17% of baseline, t (10) = 0.13, p = 0.90, paired t-test, n = 6 neurons / 3 rats, Fig. 6 left), but the amplitude of mEPSCs remained unchanged (Fig. 6 right).
We confirmed that OXT affects the pre-synaptic current in OXT-mRFP1 neurons in control rats, and L-368,899 inhibited the effect of OXT.
A hypothetical scheme for the excitatory system and feedback mechanism of OXT-ergic neurons in the hypothalamus of the chronic inflammation rat model is shown in Fig. 7.