Inhibitory Neurotransmitters And Their Transporters Regulate Hippocampal Network Activity And Synaptic Plasticity

Brain function depends on the homeostatic regulation between excitation and inhibition. In the hippocampus, GABAergic system is the main inhibitory system, providing both phasic and tonic inhibition, and has a great impact on neuronal function and network processing. Under certain pathological conditions, GABAergic inhibition is largely reduced, resulting in the disinhibition of neural network, such as that in brain ischemia, epilepsy, pain and spinal cord injury. Furthermore, several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease, as well as mental disorders such as schizophrenia, stress disorder and drug addiction, are associated with an impairment of GABAergic neurotransmission. Thus, increasing GABAergic inhibition is considered to be an important strategy in the treatment of many neurological diseases. Indeed, GABAergic system has already been an important target for drug discovery. On the other hand, some well-known drugs have been shown to be able to modulate GABAergic inhibition. For example, aspirin is such a drug according to our study.Salicylate is the major metabolite and active component of aspirin (acetyl salicylic acid), which is widely used in clinical medicine for treating inflammation, pain syndromes and the cardiovascular disorders. The well-known mechanism underlying salicylate's action mainly involves the inhibition of cyclooxygenase and subsequent decrease in prostaglandin production. Recent evidence suggests that salicylate also affects neuronal function through interaction with specific membrane channels/receptors. However, the effect of salicylate on synaptic and neural network function remains largely unknown. In this study, we investigated the effect of sodium salicylate on the synaptic transmission and neuronal excitation in the hippocampal CA1 area of rats, a key structure for many complex brain functions. With electrophysiological recordings in hippocampal slices, we found that sodium salicylate significantly enhanced neuronal excitation through reducing inhibitory GABAergic transmission without affecting the basal excitatory synaptic transmission. Salicylate significantly inhibited the amplitudes of both evoked and miniature inhibitory postsynaptic currents (IPSCs), and directly reduced GABA_A receptor (GABA_AR)-mediated responses in cultured rat hippocampal neurons. Together, our results suggest that the widely used aspirin might impair hippocampal synaptic and neural network functions through its actions on GABAergic neurotransmission. Given the capability of aspirin to penetrate blood-brain barrier, the present data imply that aspirin intake may cause network hyperactivity and be potentially harmful in susceptible subpopulations.The functional output of principal neurons is largely determined by the temporal and spatial dynamics of inhibition in the network. In most studies, the more "general" pre- and postsynaptic components of GABAergic synapse were studied in synaptic function, such as our study on salicylate. However, little attention has been paid to the role of another component in GABAergic system, GABA uptake. In this study, we took advantage of the GAT-1 (the neuronal isoform of the GAT) knockout (KO) mouse, and were able to study the role GABA uptake in cellular and synaptic mechanisms underlying learning and memory. Here we show that in mouse hippocampal CA3-CA1 synapses, pharmacological blockade or genetic disruption of GAT-1 specifically impaired theta burst stimulation (TBS)-induced long-term potentiation (LTP), but leaving the high frequency stimulation (HFS)-induced LTP and low frequency stimulation (LFS)-induced long-term depression (LTD) intact. During TBS, blockade of GAT-1 robustly prolonged GABAergic inhibition in the interburst interval within a temporal window that correlated with the impairment of LTP induced by multiple bursts delivered at theta frequency (3-7 Hz). The impaired TBS-LTP in GAT-1 KO mice was restored by GABA_AR antagonist picrotoxin, indicating a dominant role of GABA_AR in the increased GABAergic inhibition after GAT-1 blockade. Furthermore, hippocampus-dependent learning and memory was also impaired in the GAT-1 null mice. These data indicate that GABA uptake represents a novel and significant mechanism for regulating theta activity-associated synaptic plasticity underlying learning and memory, and further suggest that LTP induced by physiological theta activity bears more relevance to memory behavior.Blockade of GABA uptake is efficiency in increasing GABAergic inhibition. Indeed, besides GABA_AR, GABA transporter has been another target for antiepileptic drugs. Tiagabine is a GABA transporter inhibitor and has already been used as a clinical antiepileptic drug. Our study have shown that GAT-1-mediated GABA uptake is essential for theta activity-associated LTP and memory. Thus, it is important to know whether tiagabine has cognitive side effect besides its antiepileptic action. We found that in rat hippocampal slices, tiagabine at 50μM significantly suppressed TBS-induced LTP. Meanwhile, 20 mg/kg tiagabine impaired hippocampal-dependent learning and memory of rats. These results suggest a possible cognitive side effect of tiagabine.In the hippocampus, besides GABA_AR, accumulating evidence shows the presence of non-synaptic glycine receptors (GlyRs) containing at leastα2 subunit. However, due to the absence of glycinergic synaptic transmission, the study of hippocampal GlyR has been largely ignored. In this study, we found that blockade of glycine transporter 1 (GlyT1) in the hippocampus reduced hippocampal network excitability through the tonic activation of GlyRs by increased extracellular glycine, while intraperitoneal injection of GlyT1 inhibitor sarcosine inhibited the pentylenetetrazol-induced epileptic behavior of rats. Meanwhile, blockade of GlyT1 facilitated NMDAR-dependent hippocampal LTP. Thus, the simultaneous activation of NMDARs and GlyRs by glycine may be a homeostatic regulation mechanism of hippocampal network. Furthermore, consistent with the expression of glial GlyT1 but no expression of neuronal glycine transporter 2 (GlyT2) in the hippocampus, our data supported the idea that glial cells may be the primary source of hippocampal glycine. First, our study showed that the effect of GlyT1 blockade on network excitability was abolished by a glia-specific metabolic inhibitor, fluoroacetate (FAC). Second, in hippocampal cultures, we found that GlyR-mediated tonic currents were much larger in the neuron-glia mixed culture than that in pure neuron culture. Indeed, our preliminary data obtained from sniffer patch suggest that hippocampal astrocyte can release glycine. Furthermore, we showed the pellet-like expression of vesicular inhibitory amino acid transporter (VIAAT) in cultured astrocyte, suggesting a vesicular release mechanism of glial glycine in hippocampus, which need further investigation.In conclusion, our studies focus on the function of inhibitory transmitter GABA and glycine in hippocampal network activity and synaptic plasticity. First, we demonstrate that hippocampal GABAergic system is one of the central targets of aspirin, the most widely prescribed medicine in the world. Second, we demonstrate that GAT-1-mediated GABA uptake represents a novel and essential mechanism for regulating theta activity-associated synaptic plasticity and memory, extending our understanding on the GABAergic modulation of synaptic function. Third, our findings suggest that GABA transporter tiagabine may have cognitive side effect as a clinical used antiepileptic drug. Last, we have performed some preliminary studies of hippocampal glycine and GlyR, which has been largely ignored in the past studies. We propose that glia-derived glycine may mediate a homeostatic regulation of hippocampal network by acting on GlyRs and NMDARs simultaneously.