Study Of Hyperpolarization-activated Cyclic Nucleotide-gated(HCN) Cation Channel On Synaptic Transmission In Rat Hippocampus

OBJICTIVETo study the contribution of HCN channel to synaptic transmission at perforant pathway (PP)---CA3 region pathway and its regulation on the release of amino acids, and the relationship between them.METHODSEvoked population spike (PS) was recorded in hippocampal CA3 region in vivo, and high-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the contents of glutamate (GLU), aspartate (ASP), glycine (GLY) and gamma-aminobutyric acid (GABA) in hippocampal tissue and in the cultured hippocampal neurons. Electron microscopy was applied to explore ultrastructural pathologic features of the CA3 region of hippocampus. Fluorescence calcium imaging technique was used to observe the change of intracellular calcium in cultured hippocampal neurons. RESULTSPARTⅠEffect of HCN channel on the normal low frequency synaptic transmission at PP --- CA3 region synaptic pathway and release of amino acids in rat hippocampusTo observe the effects of specific HCN cation channel blocker ZD7288 and non-specific blocker CsCl on the synaptic transmission in the patheway from perforant pathway fibers to CA3 region in hippocampus and the contents of amino acid in hippocampal tissue, the experimental rats were divided into different groups as follows: Saline group (receiving low frequency electrical stimulation); ZD7288 group (20,100,200nmol; receiving low frequency electrical stimulation and 1μL different concentration ZD7288); CsCl group(1,5,10μmol; receiving low frequency electrical stimulation and 1μL different concentration CsCl ). After the electrophysiological test, the rats in saline,ZD7288(100 nmol) and CsCl(5μmol) groups were immediately decollated and the experimental isolateral hippocampus were carefully dissociated from the rat's cerebrain, washed with ice cold saline solution, and frozen in–75 oC, then concentrations of the amino acids were determined by HPLC with fluorescence detection. Results are shown as follows:①Specific HCN channels blocker ZD7288(20,100,200 nmol) could decrease the amplitudes of population spikes (PS) in CA3 region in hippocampus evoked by stimulating PP fibers in a dose-dependently matter. The average relative PS amplitude was 69.72%, 45.23% and 28.1%, obviously lower than saline group (P < 0.01), decreased by 30.3%, 54.8% and 71.9% respectively (P < 0.01). This inhibition effect began at 5min and could sustain at least 90min.②Nonspecific HCN channels blocker CsCl 1 and 10μmol could also significantly decrease the amplitudes of PS at hippocampal CA3 region . During the 90 min period after microinjetion of CsCl, the average relative PS amplitude fall to 69.72%,28.1%,decreased by 21.38% and 55.2% respectively than baseline (P < 0.01). Microinjection CsCl 5μmol into CA3 region, at first 30min, the PS amplitude was 58.4 %of baseline and strongly deceased (P < 0.01). The inhibitory effects attenuated with time extension, at the 90min point, were similar to the CsC l 1μmol(P > 0.05).③After the low frequency stimulating PP and administration drug, the contents of glutamate, aspartate, glycine and GABA in hippocampal tissue were measured by HPLC. The concentrations of glutamate,aspartate,glycine and GABA in normal group(without low frequency electrical stimulation) were 129.6±31.7, 117.3±22.6, 123.9±34.9 and 225.3±42.4μmol/g.protein; which in saline group were 138.3±34.3,101.7±15.1,113.4±40.5 and 227.4±41.9μmol/g.protein respectively and showed no significant difference compared with normal group(P > 0.05). In the rats treated with ZD7288 100nmol, the contents of glutamate, aspartate, glycine and GABA in hippocampus were 83.5±15.6, 63.8±11.2, 73.1±28.2 and 124.7±23.6μmol/g.protein respectively, significantly decreased compared with control group (P < 0.01). In the rats treated with CsCl 5μmol, the glutamate, aspartate,glycine and GABA contents were 75.9±10.3,66.6±10.7, 80.4±15.2 and 157.4±26.2μmol/g.protein, markedly lower than control group (all P < 0.01,except for glycine P < 0.05).PARTⅡContribution of HCN channel to long-term potentiation at PP ---CA3 region synaptic pathwayTo study the contribution of HCN channel on the induction and sustain of LTP in PP-CA3 pathway, the effects of administration of ZD7288 and CsCl before/after the tetanic stimulation (Tetanic stimulation, TS; consisted of 50 trains at 0.5 Hz each composed of 4 pulses at 500Hz) on the PS amplitudes of LTP, the onset and peak latency were observed. The synaptic ultrastructure at hippacampal CA3 region and the concentration of amino acids in hippocampus tissues were also observed. The experimental animals were divided randomly into six groups:①control group, i.e saline group (receiving test stimulation, without TS );②LTP induction group (receiving TS and being observed for 90 minutes);③ZD7288 -TS group (local administration of 100nmol ZD7288 in CA3 region at the 5 min point before TS);④CsCl—TS group(local administration of 5μmol CsCl in CA3 region at the 5 min point before TS );⑤TS—ZD7288 group (local administration of 100nmol ZD7288 in CA3 region at the30 min point after TS);⑥TS—CsCl group(local administration of 5μmol CsCl in CA3 region at the 30 min point after TS). The results are as follows:①LTP could been induced in CA3 region by stimulating PP fibers, the PS amplitudes sustain at 281.8±6.6% level of the baseline, which last stably at least 90 minutes and was higher than the control group (97.4±1.8%)(P < 0.01). Local administration of 100nmol ZD7288 and CsCl before TS could inhibit the induction of LTP in CA3 region, the PS amplitudes were 89.5±9.4%,62.9±18.0% of the baseline respectively, which were lower than the LTP group(P < 0.01)and has no significant difference compared with the control group(P > 0.05), but the inhibitory effect of CsCl attenuated after 30 min.②Local administration of ZD7288 and CsCl at the 30min after the induction of LTP could reverse the LTP. The PS amplitudes decreased at the 5 min after the administration of ZD7288 and CsCl. The PS amplitudes sustain at 95.8±6.4%,64.2±17.7% level of the baseline in 1 hour after the administration, the former was similar to the level of the control group(P > 0.05) , the latter was lower than the control group(P < 0.01).With the extension of the time, the inhibition of CsCl was attenuated.③The baseline of onset latency period was 5.154±0.350ms.5 minutes after the TS, the onset latency , began to decrease(4.368±0.254ms, P < 0.01), the average value was 4.386±0.029 ms in 90 min period, which attenuated averagely 0.768 ms. At the different time point, the onset latency was lower than the control group (all P < 0.01). Local administration of ZD7288 100 nmol and CsCl 5μmol before TS could decrease the onset latency, which attenuated 0.332ms,0.267ms.The onset latency at all time points were higher than LTP induction group(P < 0.05); The effects of CsCl was obvious in the period of 20～60 min. After TS, the onset latency of PS in the TS—ZD7288 group rats reduce 0.725ms(P < 0.01) in 30 minutes; Administration of ZD7288 100 nmol at the 30 min could attenuate the decreasing effects on onset latency, and the onset latency reached the baseline level before TS, which was higher than the value before administration. There were obvious effects at 5 min after administration, and sustain at least 60 minutes. The onset latency of PS at all time points were higher than which in LTP induction group (all P < 0.05) and were similar to the control group (P > 0.05). There were similar effects after administration of CsCl after TS at 30 min, and there was an increasing tendency after 30 minutes.④The baseline of peak latency were 7.833±0.585ms in LTP induction group. Five minutes after the TS the peak latency begin to decrease (6.767±0.578ms,P < 0.01),and sustain stably at least 90 minutes. In 90 minutes, the average value of the peak latency period were 6.832±0.107 ms,which decreased 1.001 ms compared with the value before TS; The peak latency of different time points was obviously lower than control (all P < 0.05). Administration of 100 nmol ZD7288 before TS could decrease the peak latency, which reduced 0.579 ms compared with the baseline, the decrease extent was less than the LTP induction group (P < 0.05). Administration of 5μmol CsCl before TS could decrease the peak latency in 30 minutes, there were no significant difference compared with LTP induction group. After 30 minutes the peak latency of PS extended and was similar to or longer than the baseline before TS, which had no significant difference compared with control group (P > 0.05). After TS in TS—ZD7288 group, the peak latency of PS were lower than before TS(7.291±1.602 ms)(P < 0.01),which sustained at the level of 6.415±0.006 ms, reduced averagely 0.876 ms; Administration of 100 nmol ZD7288 at the 30 min after TS could reverse the effect of TS-induced peak latency decurtation, which regained to the level before TS and were longer than the level before administration (P < 0.01). Five minutes later, there were obvious effects (P < 0.01), which sustained stably 60 minutes; Administration of CsCl 5μmol after TS at the 30 min could exert similar effects to the ZD7288, and the effects could increase after 15 minutes.⑤The content of glutamate in control group, LTP induction group,ZD7288-TS group, CsCl—TS group, TS—ZD7288 group and TS—CsCl group were 138.3±34.3,153.7±35.2,112.6±35.6, 99.5±36.6,106.6±30.0 and 132.7±51.9μmol/g.protein, the content of glutamate in LTP induction group was higher than control group, but has no significance difference; The content of aspartate were 96.7±21.0,95.2±31.2,87.3±27.0,70.0±23.0, 69.4±15.6 and 89.2±29.0μmol/g.protein in different group, there were no significance difference between them; The content of glycine were 113.4±40.5,60.1±17.4,105.6±44.5,62.7± 19.8,91.8±39.6 and 88.1±31.7μmol/g.protein in different groups, which in the LTP induction group was lower than the control group (P < 0.01) and in ZD7288—TS group was higher than LTP induction group( P < 0.05),but similar to the control group(P > 0.05); The content of GABA were 206.4±65.4,119.3±30.6,189.6±48.1,176.3±40.0,171.7±10.9 and 200.6±26.7μmol/g.protein, which in LTP induction group was lower than control group(P < 0.01),but in ZD7288—TS group,CsCl—TS group,TS—ZD7288 group and TS—CsCl group which were higher than LTP induction group (P < 0.05 or P < 0.01) .⑥There were abundant flat synapse conjunction and a few convex synapse conjunctions in the control group. The presynaptic, postsynaptic membrane and synapse gap were clear. In the presynaptic end, there were abundant synaptic vesicles and a few presynaptic dense projection and postsynaptic membrance density (PSD). In the LTP induction group, flat synapse conjunction increased and there were many synaptic glomerulus. There were many synaptic vesicles in presynaptic endochylema, which indicated the active axoplasmic transport. PSD was thicker than that in control groups, and the synapse gap was dim; there were many convex synapses and many synaptic vesicles like thyrsiform in the presynaptic end. The synaptic end was dim and the PSD became thicker. The number of the synaptic vesicles in the ZD7288—TS group decreased compared with the LTP induction group. A few flat synapse conjunction and convex synapse conjunction could be observed, but there were no synaptic glomerulus. The PSD became thinner and the number of synaptic vesicles reduced, which indicated that the axoplasmic transport was inhibited. The synapse gap was clear. The synapse constitution in the CsCl—TS group was similar to that in ZD7288—TS group.PARTⅢEffects of HCN channel on release of amino acids and calcium influx of hippocampal neuron by glutamate in vivoTo explore the effects of HCN channel on efflux of amino acid and glutamate induced calcium influx of hippocampal neuron in vivo. Cells were treated with HCN channel specific blocker ZD7228 and agonist cAMP; we measured the content of glutamate, aspatate, glycine and GABA in extracellular fluid, and observed the effects of ZD7228 and cAMP on calcium influx induced by glutamate. Results as follows:①The contents of glutamate in extracellular fluid of control, cAMP 5μM, cAMP 50μM, ZD7288 5μM and ZD7288 50μM were 99.2±17.7, 136.1±25.7, 188.1±47.7, 31.4±7.1 and 4.4±1.0μmol/L individually, the contents of aspartate were 0.181±0.0517, 0.190±0.0884, 0.214±0.1243, 0.203±0.0905 and 0.288±0.0747μmol/L, the contents of glycine were 13.01±4.32, 265.1±37.1, 397.8±50.7, 3.20±1.56 and 1.65±0.94μmol/L, the contents of GABA were 3.256±0.2925, 3.061±0.322, 3.227±0.422, 0.852±0.297 and 0.297±0.052. These data revealed that cAMP could dose-dependently increase the content of glutamate and glycine (P<0.01) and had no significant effect on the content of aspartate and GABA. In addition, ZD7288 could dose-dependently decrease the content of glutamate and GABA (P<0.01), decreased the content of glycine simultaneously, but showed no significant effect on the content of aspartate.②Adding glutamate (50μM) into hipocampal neurons exposed to standard extracellular fluid, we found that the F340/380 of hippocampal neuron were increased significantly, appeared a steep calcium peak, and maintained a high level of calcium during a long period. The increasing rate of calcium was 78.01±10.32%. While adding glutamate (50μM) into extracellular fluid without calcium, the F340/380 was unvaried. Thus, the influx of extracellar calcium by glutamate-induced played an important role in intracellar calcium increasing.③There was no change of the F340/380 of hippocampus neuron pretreated with ZD7288 25μM, 50μM, 100μM for 20min. However, adding glutamate 50μM into standard extracellular fluid pretreated with ZD7288 25μM, 50μM, and 100μM for 20min respectively, we found the F340/380 markedly decreased compared with glutamate 50μM group and the calcium peak was flat. The corresponding increasing rates of calcium of different concentrations were 59.22±8.72, 41.35±7.25, 21.01±4.41% (compared with glutamate 50μM group, P < 0.01), which indicated that ZD7288 could dose-dependently inhibit the calcium influx induced by glutamate. ④Adding glutamate 50μM into standard extracellular fluid pretreated with cAMP 5μM and 50μM for 5min respectively, we found the F340/380 significantly increased compared with glutamate 50μM group (P<0.01), the calcium peak was steep, and maintained a high level of calcium during a long period. The corresponding increasing rates of calcium were 101.33±9.89, 125.36±11.04% (compared with glutamate 50μM group, P < 0.01), which indicated that cAMP could dose-dependently reinforce the glutamate-induced calcium influx. However, incubating cells with ZD7288 50μM for 20 min before adding cAMP 50μM, we found that the increasing argument of the F340/380 and the time and the shape of the calcium peak were nearly the same with glutamate 50μM group. The increasing rate of calcium was 86.23±10.53% (compared with glutamate 50μM group, P > 0.05), which indicated that HCN channel may participate in the regulation of calcium influx by glutamate.CONCLUSIONS1 HCN channel contributed to the process of lower frequency synaptic transmission in PP-CA3 pathway, the mechanism of which may be associated with facilitating the release of amino acids neurotransmitter.2 HCN channel participated in the formation of LTP in PP-CA3 pathway, the mechanism of which was associated with the presynaptic composition and postsynaptic composition. The possible pathways are as follows:①Facilitating the synaptic release of glutamate and inhibiting the release of GABA and glycine;②Facilitating the release of intracellular calcium caused by glutamate;③Altering the presynaptic composition and postsynaptic composition through the mechanism above and other possible mechanisms, and furthering to impact the physiological and biochemistry process in the induction and maintenance of LTP.