Roles Of Nitric Oxide In Hippocampal Dentate Gyrus In Learning And Memory

Learning and memory are major functions of the higher levels of the brain, and the long-term potentiation (LTP) is a potential neurophysiological mechanism for learning and memory. Hippocampus is a critical structure for learning and memory processes, and it is mainly divided into the CA1, CA3, and dentate gyrus (DG). As the entry point of information into the hippocampus, the hippocampal dentate gyrus (DG) encodes and processes new information and plays an essential role in learning and memory. In the hippocampus, LTP can be achieved via high frequency afferent stimulation, nay, it can be induced by learning and memory behavior, we consider latter as learning-dependent LTP (LD-LTP). The hippocampal LTP is typically dependent on the activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, a consequent increase in Ca2+ influx into the postsynaptic cells, and a number of Ca2+-activated biochemical processes in the postsynaptic neurons, including nitric oxide (NO) formation. NO is an important neuronal messenger in the central nervous system, and it is involved in hippocampal LTP as an intercellular retrograde messenger. However, because these experiments were conducted in in vitro hippocampal slices, the results may not faithfully reflect the natural functioning of the organism. In addition, despite behavioral studies have revealed that NO has modulatory effects on learning and memory processes, but these studies did not consider the possibility of using both neurotransmitter release and synaptic efficiency as read-outs to elucidate the role of NO in conscious, freely moving animals. Although it has been demonstrated that nNOS is highly concentrated within the hippocampal DG region, the role of NO within the DG in the LD-LTP is rarely studied. Therefore, in part I of present study, we microinject NG-Methyl-L-arginine acetate salt (L-NMMA, an inhibitor of NO synthase) or sodium nitroprusside (SNP, a NO donor) directly into the DG region, then measure both glutamate (Glu) release and synaptic efficiency in the DG, during the acquisition and extinction of active-avoidance behavior in conscious, freely-moving rats.Alzheimer’s disease (AD) is the most common form of dementia in the elderly, and its core symptom is progressive cognitive dysfunction. Extracellular deposits of neurotoxic amyloid peptides (AP) and neurofibrillary tangles are hallmark pathological lesions found in the brains of AD. Several lines of evidence indicate that dysfunctions of glutamatergic system (mainly including NMDA and AMPA receptors) and NO are involved in learning and memory deficits of AD. Our previous studies indicate that NO is involved in the LD-LTP via enhancing the presynaptic release of Asp and Glu in the DG region, and NMDA and AMPA receptors in the DG also play an important roles in the LD-LTP. However, the roles of NO and glutamatergic system in the LD-LTP of the DG region in AD rats are rarely studied. Therefore, in part II of present study, base on these results, we investigate neurochemical mechanisms of effects of NO on learning and memory-related synaptic plasticity in the DG in a rat model of AD.Six parts of experiment were carried out:(Part Ⅰ)1. The effects of L-NMMA (NOS inhibitor) and SNP (NO donor) into the hippocampal DG in the extracellular concentrations of Glu and synaptic efficiency (field excitatory postsynaptic potential, fEPSP) were measured.2. The effects of L-NMMA and SNP on extracellular Glu concentrations and amplitudes of fEPSP were measured in the DG region during the acquisition and extinction of active-avoidance behavior in freely-moving conscious rats.(Part Ⅱ)3. AD model rats prepared with ovariectomy (OVX) combined with intraperitoneal injection of D-gal, and then 1) the extracellular concentrations of amino acids, including asparate (Asp), glutamate (Glu) and glutamine (Gln) were examined by in vivo microdialysis and HPLC methods; 2) the expression of Aβ,NMDA receptors and AMPA receptors in the DG were measured by immunohistochemistry.4. The extracellular concentrations of amino acids (Asp, Glu, Gin) and fEPSP amplitude in the DG in AD rats were examined during Morris water maze (MWM) test.5. The effects of microinjection of L-NMMA into the DG in the synaptic efficiency, learning and memory, and LD-LTP were demonstrated in AD rats.6. The effects of microinjection of MK-801(an antagonist of NMD A receptors) and CNQX (an antagonist of AMPA receptors) into the DG in the synaptic efficiency, learning and memory, and LD-LTP were demonstrated in AD rats.The experimental results are as follows:(PartⅠ)1. In the non-training groups, the microinjection of L-NMMA into the DG elicited significant decrease in the extracellular concentrations of Glu and fEPSP amplitude, whereas the microinjection of SNP into the DG elicited increase in the Glu concentrations and fEPSP amplitude (P< 0.05, respectively). The changes of Glu in the DG were paralleled with changes in fEPSP amplitude.2. In the control group, extracellular levels of Glu and fEPSP amplitudes in the DG region were significantly increased (P<0.05, respectively) during the acquisition of the active avoidance behavior and gradually returned following extinction training. The changes of Glu in the DG were accompanied by changes in fEPSP, and that both changes correspond roughly to the avoidance rate.3. In L-NMMA group, rats did not acquire the conditioned reflex, and Glu levels and fEPSP amplitude in the DG were reduced and then returned to the baseline levels.4. Microinjection of SNP into the DG facilitated the acquisition and suppressed the extinction of active-avoidance behavior, furthermore, these behavioral changes were associated with alterations in Glu levels that paralleled with LD-LTP.(Part Ⅱ)5. In the hippocampal DG of AD rats appeared the following changes:1) Aβ was significantly deposited; 2) the extracellular concentrations of Glu and Asp were significantly increased, but the extracellular concentrations of Gin was significantly reduced(P< 0.05, respectively); 3) the number of NMD A receptors and AMPA receptors immuno-positive cells in the DG were significantly reduced (P< 0.05, respectively).6. In MWM test, the escape latency was significantly longer and the number of platform crossings was markedly lessened in AD group than sham-operated group (P < 0.05, respectively), suggesting the impairment of spatial learning and memory.7. In sham-operated group, fEPSP amplitude in the DG were increased during MWM test (P< 0.05), indicating the LD-LTP formation; however, the LD-LTP in the DG of AD rats was significantly inhibited.8. In sham-operated group, the extracellular concentrations of Glu and Gin were significantly increased on the 2nd and 3 th days of MWM test, and then returned to pre-training levels; in AD group, Glu concentration was significantly increased, whereas the Gln concentration was significantly reduced during MWM test (P< 0.05, respectively).9. During MWM test, the extracellular concentration of Asp did not changed, but it was significantly reduced in AD group (P< 0.05, respectively).10. In AD rats, microinjection of L-NMMA and MK-801, but not CNQX, enhanced the synaptic efficiency in the DG region.11. In AD rats, microinjection of L-NMMA and MK-801 into the DG improved the impairment of spatial learning and memory as well as the inhibition of LD-LTP in the DG; microinjection of CNQX into the DG improved the impairment of spatial learning but not memory, and delayed the LD-LTP formation in the DG.Conclusion:1. NO in the hippocampal DG facilitates learning and memory via enhancements of glutamate levels and synaptic efficiency, and it is involved in LD-LTP as an intercellular retrograde messenger.2. Impairment of learning and memory in AD rats is related to deposits of AP, increase in Glu and Asp concentrations, over-activation of NMDA and AMPA receptors in the hippocampal DG3. In AD rats, expression of NMDA and AMPA receptors in the DG is down-regulated, it is likely to be a compensatory mechanism against over-activation of glutamatergic systems.4. NO and NMDA receptor, but not AMPA receptor, in the DG suppress the LD-LTP in the AD rats.5. Role of NO of the hippocampal DG in LD-LTP in AD rats is contrary to that in normal rats.