| 5-HT, as a neurotransmitter or neuromodulator in the central nervous system, involves in multiple physiological processes and behaviors. The modulation disorder of the serotonergic system can result in depression, anxiety, posttraumatic stress disorder, et al., which are companied by cognitive disorder of learning and memory. The modulation of the serotonergic system affects LTP and long-term depression (LTD), the likely neurophysiologic derivates of learning and memory formation, which has been involved in the treatment of Alzheimer's disease. The Na+-K+ pump (also known as sodium pump or Na+, K+-ATPase) is a member of P-ATPase family and present in all animal cells. Na+-K+ pump is a membrane protein responsible for the active transport of sodium and potassium ions in the nervous system necessary to maintain the ionic gradient for neuronal excitability. Changes in Na+-K+ pump activity have been associated with several abnormalities, including epilepsy, hypertension and diabetes. Additionally, Na+-K+ pump might play a relevant role in activity-dependent synaptic plasticity, such as LTP. Evidence also suggests that LTP in the hippocampus is one important phenomenon for memory processing. Na+-K+ pump inhibition impaired learning and memory in Morris water maze and step-through passive avoidance tasks, showing the main role of this enzyme on learning and memory. The sufficient studies have demonstrated that the molecular mechanism of learning and memory in hippocampus is that the activation of NMDA receptor promotes the generation of LTP, synaptogenesis, synaptic plasticity, and neurogenesis, improving the formation of learning and memory. LTP and its relationship with NMDA receptors may be the most important discovery in neuroscience since the early 1970s. Furthermore, 5-HT and NMDA receptor both involve in the regulation of cognition and emotion, and the interaction is complicated. In the absence of Mg2+, 5-HT reduces NMDA-mediated depolarization; whereas in the presence of Mg2+, 5-HT increases NMDA-mediated depolarization. 5-HT inhibits NMDA receptor- mediated response in pyramidal cells of the rat medial prefrontal cortex, while facilitates the effects of excitatory amino acids and enhances NMDA receptor-mediated response on several motoneurone pools. Additionally, 5-HT and NMDA receptors also appeared to interact, which was particularly critical for the expression or retrieval of hippocampal-dependent long-term spatial memory. The serotonin system and NMDA receptors are both critically involved in the regulation of cognition and emotion under normal and pathological conditions. The Na+-K+ pump inhibition leads to hyperstimulation of Glu receptors, Glu accumulation, due to the decrease of the glutamate uptake and the increase of neurotransmitter release, as well as allosterically decreases [3H] dizocilpine, a non-competitive antagonist of the NMDA receptor, binding to NMDA receptor, and increases the expression of NMDA receptor subunits, localization and clustering in cerebral cortex and hippocampus. The Na+-K+ pump inhibition modulates NMDA receptor not only under normal conditions but also in the short-term period that follows the ischemic status. Moreover, 5-HT activates glial Na+/K+ pump activity in rat cerebral cortex and hippocampus, and inhibits it in kidney and T sensory neurons of the leech. Therefore, the interactive modulation of cognitive learning and memory mediated by Na+-K+ pump, 5-HT and NMDA is complicated. Furthermore, more and more attention was paid to the serotonin system involved in the regulation of cognitive function. However, the mechanisms await to be further explored. In the present study, we used the whole-cell patch-clamp technique to address the following questions: (1) whether Na+-K+ pump and 5-HT can modulate NMDA current in hippocampal CA1 pyramidal neurons; (2) whether there lies a possible modulatory effect of 5-HT on Na+-K+ pump current in hippocampal CA1 pyramidal neurons.Part 1 The regulation of Na+/K+ pump and 5-HT on NMDA current in hippocampal CA1 pyramidal neurons Objective: To observe the modulation of NMDA response by Na+/K+ pump and 5-HT in hippocampal CA1 pyramidal neurons and explore the underlying mechanisms.Methods:Transverse hippocampal slices (300μm thick) were obtained by cutting with a vibroslice MA752 in ice-cold ACSF well-saturated with 95% O2 and 5% CO2, and transferred to oxygenated ACSF at 30-32℃for 1-h incubation. Individual slice was transferred to a perfusion chamber and continuously superfused with oxygenated ACSF at a rate of 3 ml min-1 at room temperature (22-25℃). NMDA current was induced by NMDA (30μM), which was applied consecutively 3 times for 1.5 min each, being separated by superfusion periods of 10-15 min with drug-free ACSF. In the present experimental conditions, 30μM NMDA can evoke reproducible inward currents, which were stable, with no evidence of receptor desensitization. The 3-4 min application of DHO (10-7-10-3 M) respectively, finally we applied DHO accompanied by NMDA (30μM) for 1.5 min. The amplitude of the current induced by the superfusion application of NMDA in control was standardized (100%).Results: (1) DHO (10-5, 10-4, 10-3 M) significantly potentiated INM, which were respectively increased to 146.25±10.34%, 195.78±8.94%, and 154.75±14.18% contrast to the control INM as 100% (P<0.01 and 0.05). (2) Pretreatment of hippocampal CA1 pyromidal neurons with H89, a specific inhibitor of PKA, did not antagonize DHO-induced potentiation of INM, which was changed from 202.28±23.28% to 175.86±26% (P>0.05). Pretreatment of hippocampal CA1 pyromidal neurons with Stau, a specific inhibitor of PKC, did not antagonize DHO-induced potentiation of INM, which was changed from 187.46±27.57% to 178.32±34.3% (P>0.05). Extracellular H89 and Stau alone did not significantly affect the control INM (H89 101.7±11.3%, Stau 108.92±10.77%, P>0.05). (3) In the presence of extracellular PP2, a specific inhibitor of Src, DHO-induded potentiation of INM was significantly reduced from 173±25.66% to 106.8±5.92% (P<0.05). Pretreatment of hippocampal CA1 neurons with PD-98059, a specific inhibitor of MAPK, also partly antagonized DHO-induced potentiation of INM from 210±28.11% to 135.25±15.60% (P<0.05). Extracellular PP2 and PD-98059 alone did not significantly affect the control INM (PP2 94.4±12.58%, PD-98059 120.25±12.09%, P>0.05). (4) 5-HT (0.01, 0.1, 1 mM)-mediated INM is 109.4±4.96% (P>0.05), 146.4±13.48% and 161.83±13.06% (P<0.05) contrast to 100% of the first NMDA response of the control. These data showed that 5-HT (0.1, 1 mM) significantly potentiated the INM.Conclusion: DHO potentiates hippocampal NMDA response primarily via tyrosine kinase Src and MAPK, while 5-HT also potentiates hippocampal NMDA response, which discloses novel mechanisms for the function of Na+/K+ pump and 5-HT in learning and memory.Part 2 The regulation of 5-HT on the Na+/K+ pump current in rat hippocampal CA1 neuronsObjective: 5-HT modulates learning and memory, and inhibition of Na+/K+ pump activity in rat hippocampal CA1 cells causes impairment of learning and memory and amnesia. The aim of this study was to investigate whether 5-HT can modulate hippocampal CA1 neurons Na+/K+ pump and then possibly involve in the modulation of learning and memory.Methods: Transverse 300-μm-thick hippocampal slices containing CA1 neurons were obtained by cutting with a vibroslice MA752 in ice-cold ACSF well-saturated with 95% O2 and 5% CO2 (PH 7.3-7.4), then they were pre-incubated in oxygenated ACSF at room temperature (30-32℃) for 1h. The hippocampal slice containing CA1 neurons was transferred to a submerged recording chamber and continuously superfused with oxygenated ACSF at a rate of 2 ml min-1 at room temperature (22-25℃). Whole-cell patch-clamp technique was performed to record the Na+-K+ pump current (Ip): the Na+-K+ pump exchanges 3 intracellular Na+ for 2 external K+ across the cell membrane during each active transport process. This excess positive charge movement generates a net outward current (Ip). With selected external and pipette solutions, membrane currents through K+ channel, Ca2+ channel, and Na+-Ca2+ exchanger were minimized. Under the experimental conditions, Ip was measured as the 0.5×10-3 M strophanthin (Str)-blocked current.Results: (1) The density of the control Ip was 0.64±0.04 pA/pF. 5-HT 0.1 and 1 mM could significantly inhibit Ip, which densities were 0.39±0.05 and 0.35±0.04 pA/pF respectively (P<0.01), demonstrating a dose-dependent inhibition of Ip in hippocampal CA1 neurons. (2) In the presence of WAY100635 (10μM), an antagonist of 5-HT1AR, 5-HT-induced inhibition of Ip did not change, which densities were 0.39±0.05 pA/pF and 0.38±0.07 pA/pF (P>0.05). (3) In the presence of ondasetron (10μM), an antagonist of 5-HT3R, 5-HT-induced inhibition of Ip was significantly recovered from 0.39±0.05 pA/pF to 0.59±0.05 pA/pF (P<0.05). These data showed that 5-HT-induced inhibition of Ip was antagonized by ondasetron not by WAY100635. (4) 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG) (0.1 mM), a 5-HT3R specific agonist, mimicked the effect of 5-HT (0.1 mM) on Ip, which was decreased from 0.64±0.04 pA/pF to 0.41±0.05 pA/pF (P<0.01), suggesting that 5-HT-induced inhibition of Ip was mediated by 5-HT3R.Conclusion: 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in hippocampal CA1 neurons, which suggests that Na+/K+ pump may involve in 5-HT-mediated modulation of NMDA current, then modulating learning and memory performance.SUMMARY1 DHO potentiates hippocampal NMDA response primarily via tyrosine kinase Src and MAPK, while 5-HT also potentiates hippocampal NMDA response, which discloses novel mechanisms for the function of Na+/K+ pump and 5-HT in learning and memory.2 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in hippocampal CA1 neurons, which suggests that Na+/K+ pump may involve in 5-HT-mediated modulation of NMDA current, then modulating learning and memory performance. |
PDF Full Text Request