Effects of K252a and K252b on CIH induced Changes in mEPSCs from PVN‐projecting MnPO.

R4328 --> 896.2 --> Chronic intermittent hypoxia (CIH) has been shown to alter the response of neurons in Median Preoptic nucleus (MnPO) to activation of cardiovascular inputs. Our previous results indicate that CIH enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. In other systems, BNDF/TrkB signaling has been shown to facilitate excitatory neurotransmission, so we tested its role in the increased MnPO excitation triggered by CIH. Data were collected from PVN‐projecting MnPO neurons which play an important role in CIH‐induced hypertension. Adult 250‐350g male rats were injected bilaterally in the PVN with 200 nL retrograde adeno‐associated virus (pAAV‐CAG‐tdTomato). Three weeks after the injections, the rats were exposed to a 7‐day intermittent hypoxia protocol (6 min cycles; 3 minutes of 21% oxygen, 3 min of 10% oxygen maintenance at 10% oxygen for 8 h during the light phase). Normoxic controls were exposed to room air. At the end of the protocol, the rats were anesthetized with isoflurane (2‐3%) and horizontal brain slices of MnPO were prepared. Miniature Excitatory Postsynaptic Currents (mEPSCs) were recorded using whole cell voltage clamp configuration from PVN‐projecting MnPO neurons. The mEPSCs were recorded with the holding potential of ‐60 mV in the presence of a GABAa receptor antagonist (SR 95531 hydrobromide, 25 µM), tetrodotoxin (1µM), and a glycine receptor antagonist (strychnine, 3 µM) in aCSF. Data were analyzed off‐line using Synaptosoft software. Differences in mEPSCs properties, such as peak amplitude, frequency, decay time, area, and cell capacitance were analyzed by ANOVA and Tukey multiple comparison tests. The results showed that CIH significantly increased mEPSCs frequency (Hz: CIH 1.32 ± 0.09 vs Norm 0.83 ± 0.09, p=5E‐04), decay time (ms: CIH 8.01 ± 0.28 vs Norm 6.64 ± 0.36, p=0.004), area (pA.ms: CIH 63.85± 3.65 vs Norm 50.27 ± 3.6, p=0.01), and cell capacitance (pF: CIH 31.55 ± 1.8 vs Norm 25.34 ± 1.95, p=0.02). The contribution of BDNF/TrkB signaling to these CIH effects were tested using K252a and K252b, two structurally related kinase receptor inhibitors. MnPO slices were pre‐incubated with either K252a or K252b (200nM) for 30 min before recording. Neither K252a nor K252b influenced mEPSCs of MnPO neurons from normoxic slices. Both K252a and K252b significantly affected CIH‐induced changes mEPSCs but at different magnitudes. The K252a significantly reduced the effects of CIH on frequency (Hz: K252a 0.46 ± 0.05, p=1E‐08), decay time (ms: K252a 6.07 ± 0.34, p=1E‐04), area (pA.ms: K252a 43.26 ± 3.55, p=5E‐04), and cell capacitance (pF: K252a 19.59 ± 1.61, p=1E‐04;). In addition, K252a also significantly decreased mEPSCs peak amplitude in CIH neurons (pA: CIH 14.87 ± 0.41 vs K252a 13.27 ± 0.39, p=0.03). While K252b pretreatment also affected the same parameters, the changes produced by K252a were significantly greater. These studies suggest a role for BDNF/TrkB signaling in CIH induced changes in MnPO but additional experiments will be required to validate the specificity of our results. [ABSTRACT FROM AUTHOR]