Effects Of Hydrogen Sulfide On Neuronal Discharges And Glutamate-induced Injury In Rat Hippocampal Slices

Hydrogen sulfide (H₂S) is now known as a novel neuronal regulator. A large body of evidence indicates that H₂S may exert diverse biological effect in central nervous system (CNS). Physiological concentration of H₂S may increase the sensitivity of N-methyl-D-aspartate (NMDA) receptor and facilitate the induction of long-term potentiation (LTP), a synaptic model in learning and memory. It was also found that H₂S may protect the cultured neurons in vitro from oxidative stress by increasing glutathione levels, stimulating KATP channel, Cl- channel and increasing the uptake of glutamate. Moreover, H₂S plays anti-neuroinflammatory, anti-oxidant and anti-apoptotic effects in neuron and glial cells. The previous studies in our lab demonstrated that H₂S facilitates carotid sinus baroreflex and baroreceptor activity in anesthetized male rats. However, the effect of H₂S on the electrical activity of neurons in the rat hippocampal slices has not been reported. The aim of the present study is to observe the effects of H₂S on neuron spontaneous discharge and glutamate-induced injury in rat hippocampal slices with extracellular microelectrode recording technique.1 Effects of H₂S on neuron discharges in rat hippocampal slicesObjective: To study the effects of NaHS (H₂S donor) on the electrical activity of rat hippocampal neurons and the mechanism involved.Methods: All experiments were performed on CA1 pyramidal cells of hippocampal slices prepared from male Sprague-Dawley rats. Then the effects of H₂S on the discharges of neurons in CA1 area of rat hippocampal slices were examined by using extracellular recording technique.Results: (1) Application of NaHS (50, 100, 200μmol/L, n=46) into the perfusate for 2 min, the spontaneous discharge rates (SDR) of 39/46 (84.78%) neurons were significantly decreased in a dose-dependent manner (P<0.01). (2) Pretreatment with glibenclamide (Gli, 10μmol/L, a specific K_ATP channel blocker), the SDR of 9/10 (90%) neurons were not significantly changed (P>0.05), and the discharges were not influenced by the NaHS (100μmol/L) applied in 9/9 (100%) slices (P>0.05). (3) Pretreatment with bicuculline (BIC, 50μmol/L, a specific GABAA receptor antagonist), led to a marked increase in the SDR of 7/8 (87.5%) neurons. The increased discharges of 7/7 (100%) neurons were not significantly changed after NaHS (100μmol/L) was applied into the perfusate for 2 min (P>0.05). (4) Pretreatment with picrotoxin (PIC, 50μmol/L), a selective blocker of Cl- channel, led to an increase in the SDR of all 7/7 (100%) neurons. The increased discharges of 6/7 (85.71%) neurons were not influenced by the NaHS (100μmol/L) adminstration (P>0.05). (5)Application of nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 50μmol/L) into the perfusate for 2 min also significantly augmented the SDR of 6/8 (75%) neurons, then NaHS (100μmol/L) applied into the perfusate reduced the increased SDR of all neurons (P<0.01). (6) In response to the application of HA (20 mmol/L, n=11) into the perfusate for 2 min, the spontaneous discharge rates (SDR) of 9/11 (81.82%) neurons were significantly increased (P<0.01). (7) Pretreatment with L-Glu (200μmol/L, an excitatory amino acids), led to a marked increase in the SDR of 7/7 (100%) neurons in an epileptiform pattern. The increased discharges were significantly eliminated after NaHS (100μmol/L) was applied into the perfusate for 2 min (P<0.01).Conclusion: H₂S decrease spontaneous discharge and L-Glu-induced discharge most probably by activating K_ATP channels and GABAA coupled Cl- channels, while NO is not involved. 2 Effects of hydrogen sulfide on glutamate-induced injury in rat hipocampal slicesObjective: To study the effects and mechanism of NaHS on hippocampal slice injury induced by L-Glu.Methods: The hippocampal slices were incubated with L-Glu (1 mmol/L) for 30 min to induce the injury. The effect of NaHS on glutamate-induced injury were determined by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, organic solvent extraction and spectrophotometric colorimetry in hipocampal slices with NaHS (50, 100, 200μmol/L) supplementation. .The Morphology changes of neurons in CA1 were observed by electron microscopy.Results: 30-min L-Glu (1 mmol/L) application with the following 2-h normal incubation reduced the tissue activity by approximately 62% (i.e. % tissue injury) (P<0.01). NaHS (50, 100μmol/L) protected hippocampal slices from L-Glu injury, decreased % tissue injury to 29% and 19%. While NaHS (200μmol/L) reduced the tissue activity by 64%, which shows no significant difference compared with L-Glu group (P>0.05). Pretreatment with Glibenclamide (10μmol/L), bicuculline (50μmol/L) and pcrotoxin (50μmol/L) respectively did not significantly aggravate the severity of the tissue damage induced by L-Glu (P>0.05), but significantly reversed the protective effects of NaHS (100μmol/L) group, the % tissue injury in hippocampal slices co-treated with glibenclamide or bicuculline or pcrotoxin respectively in the presence of NaHS (100μmol/L) were decreased to approximately 49%, 48% and 51%, which have significant differences compared with NaHS (100μmol/L) group (P<0.01). Cotreated hippocampal slices with three inhibitors above completely blocked the effect of NaHS with 59% tissue injury, which have no significantly difference with L-Glu group (P>0.05).The ultrastructure of CA1 region in hippocampal slice was aggravated after L-Glu injury. NaHS (100μmol/L) attenuated this aggravation. Pretreatment with 100μmol/L Gli or PIC respectively in the presence of 100μmol/L NaHS decreased the protective effect of H₂S. Conclusion: NaHS can protect hippocampal slices from injury induced by L-Glu, and this neuroprotective effects may be related to the activation of K_ATP channels and GABA_A coupled Cl- channels.