Influence of agonist concentration on AMPA and kainate channels in CA1 pyramidal cells in rat hippocampal slices

We have determined the functional properties of single AMPA receptor (AMPAR) and kainate receptor channels present in CA1 cells in hippocampal slices, to shed light on the relationship between single-channel behaviour and synaptic currents in these cells. To derive basic properties of AMPA and kainate channels activated by their excitatory transmitter, we examined outside-out patches exposed to glutamate. The kainate agonist SYM 2081, was used to confirm the presence of kainate receptors. Channels activated by glutamate or SYM 2081 exhibited conductance levels of 2–20 pS. Properties of single channels depended on the glutamate or AMPA concentration used. We observed a marked increase in mean channel conductance (γ) from γ= 6.9, to 11.2 pS, when glutamate was increased from 10 μ m to 10 m m . The kinetic behaviour of AMPAR channels was also influenced by agonist concentration, with an increase in 'bursty' events at higher concentrations. In contrast, kainate channels were characterized by brief openings without bursts. Consistent with the view that 'bursty' events arose from AMPARs, these openings decreased in the presence of the AMPAR blocker GYKI 53655. Furthermore, our experiments revealed a concentration-dependent increase in the number of conductance states during an individual AMPAR opening; AMPAR channels displayed up to four distinct levels. Our results are consistent with the view that the AMPAR channel conductance depends on the number of transmitter molecules bound in CA1 cells. We consider the implications of these findings for the change in EPSC properties during long-term potentiation (LTP).     

PP2, a potent inhibitor of Src family kinases, protects against hippocampal CA1 pyramidal cell death after transient global brain ischemia

It has been indicated that Src family protein tyrosine kinases (SrcPTKs) potentiate N-methyl-D-aspartate (NMDA) receptor function by phosphorylating NR2A subunits and that postsynaptic density protein 95 (PSD-95) facilitates this regulation. In this paper, we define the role of SrcPTKs in delayed neuronal damage following transient brain ischemia and explore the underlying mechanisms involved in this event. Transient global brain ischemia was induced by the four-vessel occlusion method. A specific Src family kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) and a PP2 negative control PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) were infused into rat cerebroventricule 30 min before occlusion. Hematoxylin and eosine staining showed that the number of surviving pyramidal neurons in rat hippocampal CA1 subfield increased markedly in PP2-treated rats comparing to PP3-treated groups after 5 days of reperfusion following ischemia. Additionally, immunoprecipitation and immunoblot analysis revealed that preadministration of PP2, but not PP3, attenuated not only the increased tyrosine phosphorylation of NR2A but also the enhanced interactions among Src, NR2A and PSD-95 induced by ischemia/reperfusion. In conclusion, SrcPTKs promote binding of the kinases and their substrate NR2A attributed to the scaffolding effect of PSD-95 during transient brain ischemia and reperfusion, which are responsible for the elevation of NR2A tyrosine phosphorylation and consequent delayed neuronal cell death.     

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24 h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 μM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption.     

Block of GABAb-activated K+ conductance by kainate and quisqualate in rat CA3 hippocampal pyramidal neurones

Current and voltage-clamp techniques were used to study the effects of kainic (KA) and quisqualic (quis) acids on the slow synaptic inhibition evoked by mossy fibre stimulation in CA3 hippocampal pyramidal neurones in vitro. The K⁺ conductance underlying the slow synaptic inhibition is coupled to a gamma-aminobutyric acid b (GABAb) receptor by a guanosine-triphosphate (GTP)-binding protein. Both KA and quis reduce (after 7–10 min) the slow inhibitory post-synaptic current (IPSC) without changing the reversal potential. They also reduce the cellular response to exogenously applied (±)baclofen and 5-hydroxytryptamine, which are known to activate a similar K⁺ conductance. We conclude that KA and quis block the post-synaptic K⁺ conductance underlying the slow IPSC.     

Alterations of perisomatic GABA synapses on hippocampal CA1 inhibitory interneurons and pyramidal cells in the kainate model of epilepsy.

In the kainate model of epilepsy, electrophysiological and anatomical modifications occur in inhibitory circuits of the CA1 region of the rat hippocampus. Using postembedding GABA immunocytochemistry and electron microscopy, we characterized perisomatic GABA and non-GABA synaptic contacts in CA pyramidal cells, and GABAergic interneurons of stratum oriens/alveus and stratum lacunosum-moleculare, and examined if changes occurred at these synapses at two weeks post-kainate treatment. We found that, in control rats, the number and total length of perisomatic GABA synapses were significantly smaller (approximately 40-50%) in lacunosum-moleculare interneurons than in oriens/alveus interneurons and pyramidal cells. Additionally, the number and total length of perisomatic non-GABA synapses were different among all cell types, with these parameters increasing significantly in the following order: pyramidal cells相似文献   

Galantamine increases excitability of CA1 hippocampal pyramidal neurons

Galantamine is a third generation cholinesterase inhibitor and an allosteric potentiating ligand of nicotinic acetylcholine receptors. It enhances learning in aging rabbits and alleviates cognitive deficits observed in patients with Alzheimer's disease. We examined galantamine's effect on CA1 neurons from hippocampal slices of young and aging rabbits using current-clamp, intracellular recording techniques. Galantamine (10-200 microM) dose-dependently reduced the postburst afterhyperpolarization and the spike-frequency accommodation of CA1 neurons from both young and aging animals. These reductions were partially, but significantly, reversed by the addition of the muscarinic receptor antagonist, atropine (1 microM), to the perfusate. In contrast, the nicotinic acetylcholine receptor antagonist, alpha-bungarotoxin (10 nM), had no effect; i.e. alpha-bungarotoxin did not reverse the afterhyperpolarization and accommodation reductions. The allosteric potentiating ligand effect was examined by stimulating the Schaffer collateral and measuring the excitatory postsynaptic potentials for 30 min during bath application of galantamine. Galantamine (200 microM) significantly enhanced the excitatory postsynaptic potential amplitude and area over time. These effects were blocked by 10 nM alpha-bungarotoxin, supporting a role for galantamine as an allosteric potentiating ligand. We did not observe a facilitation of the excitatory postsynaptic potentials with 1 microM galantamine. However, when the excitatory postsynaptic potential was pharmacologically isolated by adding 10 microM gabazine (GABA(A) receptor antagonist) to the perfusate, 1 microM galantamine potentiated the subthreshold excitatory postsynaptic potentials into action potentials. We propose that the learning enhancement observed in aging animals and the alleviation of cognitive deficits associated with Alzheimer's disease after galantamine treatment may in part be due to the enhanced function of both nicotinic and muscarinic excitatory transmission on hippocampal pyramidal neurons.     

Suppression of postsynaptic density protein 95 by antisense oligonucleotides diminishes postischemic pyramidal cell death in rat hippocampal CA1 subfield

Our previous investigation has shown that postsynaptic density protein 95 (PSD-95) is critical for the Src family kinases-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunit 2A (NR2A) in the postischemic hippocampus. To clarify the roles of PSD-95 in the ischemic brain damage, histological method was performed to examine the effects of PSD-95 antisense oligonucleotides (AS) on the postischemic delayed cell death in rat hippocampus. Transient (15 min) brain ischemia was induced by the four-vessel occlusion method in Sprague-Dawley rats. Five days of reperfusion following brain ischemia (I/R5d) led to hippocampal CA1 pyramidal cell death upward of 90%. Intracerebroventricular infusion of AS (every 24 h for 3 days before ischemia) not only decreased the PSD-95 expression but also increased the number of surviving pyramidal neurons, while missense oligonucleotides (MS) had no effects. To further investigate the mechanisms underlying the neuroprotection of PSD-95 deficiency, the interaction of proline-rich tyrosine kinase 2 (Pyk2) with NR2A as well as autophosphorylation (Tyr402) of Pyk2 were detected. Immunoprecipitation and immunoblot analysis showed that preischemic treatment with AS, but not MS or vehicle, attenuated the I/R6h-induced increases in Pyk2-NR2A association and Pyk2 autophosphorylation. The protein levels of NR2A and Pyk2 had no differences under the above conditions. Our data suggest that the recruitments of ion channels and signaling molecules may be involved in the PSD-95 neurotoxicity in the postischemic hippocampus.     

Caffeine-mediated presynaptic long-term potentiation in hippocampal CA1 pyramidal neurons

We report a new form of long-term potentiation (LTP) in Schaffer collateral (SC)-CA1 pyramidal neuron synapses that originates presynaptically and does not require N-methyl-d-aspartate (NMDA) receptor activation nor increases in postsynaptic-free Ca2+. Using rat hippocampal slices, application of a brief "pulse" of caffeine in the bath evoked a nondecremental LTP (CAFLTP) of SC excitatory postsynaptic currents. An increased probability of transmitter release paralleled the CAFLTP, suggesting that it originated presynaptically. The P1 adenosine receptor antagonist 8-cyclopentyltheophylline and the P2 purinoreceptor antagonists suramin and piridoxal-5'-phosphate-azophenyl 2',4'-disulphonate blocked the CAFLTP. Inhibition of Ca2+ release from caffeine/ryanodine stores by bath-applied ryanodine inhibited the CAFLTP, but ryanodine in the pipette solution was ineffective, suggesting a presynaptic effect of ryanodine. Previous induction of the "classical" LTP did not prevent the CAFLTP, suggesting that the LTP and the CAFLTP have different underlying cellular mechanisms. The CAFLTP is insensitive to the block of NMDA receptors by 2-amino-5-phosphonopentanoic acid and to Ca2+ chelation with intracellular 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, indicating that neither postsynaptic NMDA receptors nor increases in cytosolic-free Ca2+ participate in the CAFLTP. We conclude that the CAFLTP requires the interaction of caffeine with presynaptic P1, P2 purinoreceptors, and ryanodine receptors and is caused by an increased probability of glutamate release at SC terminals.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.     

小鼠海马CA1区锥体神经元树突棘的发育

目的 对小鼠海马CA1区锥体神经元正常发育中树突棘密度及各种形态变化进行分析测定,为深入研究突触发生及突触可塑性提供直接的形态学依据.方法 分别取出生后0、5、10、20及30d 5个年龄段的C57BL/6小鼠各10只,采用基因枪对小鼠海马CA1区锥体神经元树突棘进行亲脂性荧光染料DiI标记,通过激光共焦显微镜对其进行观察分析;同时利用透射电镜技术对树突棘的超微结构进行分析.结果 树突棘的形态、大小及其密度随小鼠发育而变化,成熟树突棘内部存在滑面内质网与棘器,可能参与了突触后膜结合蛋白及其转运体的合成.结论 树突棘的发育过程与突触连接的形成以及突触可塑性密切相关.     

DSP4 treatment worsens hippocampal pyramidal cell damage after transient ischemia.

Recent studies in the rat have suggested that hippocampal norepinephrine can regulate the amount of damage seen after transient forebrain ischemia. We used the gerbil to study the role of norepinephrine in ischemic damage. Using tyrosine hydroxylase immunocytochemistry and chemical measurements of norepinephrine, we determined that the gerbil hippocampus has a similar but topographically different norepinephrine innervation than the rat. Brains from gerbils treated with 100 mg/kg of N-(2-chloroethyl)-N-methyl-2-bromobenzylamine (DSP4) had 60% less norepinephrine than saline-treated controls, similar to the effect of the drug in rats. We administered DSP4 to gerbils two weeks before exposing them to 5 min of bilateral carotid artery occlusion. Animals treated with DSP4 and subjected to ischemia had worse pyramidal cell loss in the CA3 and CA4 regions than saline-treated ischemic controls. CA1 pyramidal cell loss (about 90%) was severe in both the saline- and DSP4-treated animals. These data provide further evidence that norepinephrine can regulate the neuronal death in the hippocampal formation after transient forebrain ischemia. Furthermore, this is the first demonstration of that regulation in the gerbil and suggests that noradrenergic input to the hippocampus may be important in ischemia in other species besides the rat.     

Dendritic mechanisms of phase precession in hippocampal CA1 pyramidal neurons.

Dual whole-cell patch clamp recordings from the soma and dendrites of CA1 pyramidal neurons located in hippocampal slices of adult rats were used to examine the potential mechanisms of phase precession. To mimic phasic synaptic input, 5-Hz sine wave current injections were simultaneously delivered both to the soma and apical dendrites (dendritic current was 180 degrees out-of-phase with soma). Increasing the amplitude of the dendritic current injection caused somatic action potential initiation to advance in time (move forward up to 180 degrees). The exact pattern of phase advancement is dependent on the dendritic location of input, with more distal input causing a more gradual temporal shift in spike initiation and a smaller increase in spike number. Patterned stimulation of Schaffer collateral/perforant path synaptic input can produce phase precession that is very similar to that observed with sine wave current injections. Finally, the exact amount of synaptic input required to produce phase advancement was found to be regulated by dendritic voltage-gated ion channels. Together, these data demonstrate that the summation of primarily proximal inhibition with an increasing amount of out-of-phase, primarily distal excitation can result in a form of phase advancement similar to that seen during theta activity in the intact hippocampus.     

Hyperpolarizing responses to application of glutamate in hippocampal CA1 pyramidal neurons

Micropressure injection of glutamate onto the apical dendrites of hippocampal CA1 pyramidal cells usually produces a fast rising, brief depolarization. However, hyperpolarizing responses with longer durations (300-500 ms) can be produced over a range of drug electrode locations. These hyperpolarizations can be reversed with intracellular injection of hyperpolarizing current. Localized application of glutamate in the stratum radiatum produces a depolarizing response in intracellularly recorded CA1 interneurons. Previous studies have shown that the dendrites of GABA-ergic basket cell interneurons extend into the stratum radiatum and are involved in mediating feedforward inhibition of pyramidal neurons. The glutamate-induced hyperpolarizations observed in pyramidal neurons are probably due to direct excitation of dendrites of interneurons, which in turn produce a synaptic inhibition in pyramidal cells.     

Distribution of slow AHP channels on hippocampal CA1 pyramidal neurons

This work was designed to localize the Ca(2+)-activated K(+) channels underlying the slow afterhyperpolarization (sAHP) in hippocampal CA1 pyramidal cells. Cell-attached patches on the proximal 100 microm of the apical dendrite contained K(+) channels, but not sAHP channels, activated by backpropagating action potentials. Amputation of the apical dendrite approximately 30 microm from the soma, while simultaneously recording the sAHP whole cell current at the soma, depressed the sAHP amplitude by only approximately 30% compared with control. Somatic cell-attached and nucleated patches did not contain sAHP current. Amputation of the axon >/=20 microm from the soma had little effect on the amplitude of the sAHP recorded in cortical pyramidal cells. By this process of elimination, it is suggested that sAHP channels may be concentrated in the basal dendrites of CA1 pyramids.     

The N-methyl-D-aspartate receptor and burst firing of CA1 hippocampal pyramidal neurons

Previous intracellular investigations in the rat hippocampus have demonstrated that N-methyl-D-aspartate, ibotenate and 2,3-pyridine dicarboxylate (quinolinate) all evoke burst firing of CA1 pyramidal neurons, whereas kainate and quisqualate, which are thought to react with different receptors, do not. The purpose of the present study has been to investigate the ability of a series of compounds either to trigger burst firing or to antagonize this pattern of excitation. We report here that N-methyl-L-aspartate, 1,2-benzene dicarboxylate (phthalate) and methylene succinate (itaconate) are also capable of evoking burst firing. The results of this investigation suggest that since both quinolinate and phthalate are rigid planar molecules and only the 2 and 3 positioning of the carboxylates of pyridine was active, a cis configuration of the carboxyls with respect to the 2,3 carbon bond appears to be necessary for excitation. While a nitrogen atom is not necessary for activity (this is absent in phthalate and itaconate) a third functional group, bearing at least a partial positive charge, and in a position alpha to one of the carboxyl groups is required. The requirements for pyridine derivatives to trigger burst firing is similar to that reported as necessary for evoking convulsions and neurotoxicity after intrahippocampal infusion and a correlation between N-methyl-D-aspartate-like burst firing and depolarization and this neuropathology is considered. An important observation has been that the addition of a benzene ring to either quinolinate or phthalate to yield 2,3-quinoline dicarboxylate and 2,3-napthalene dicarboxylate, respectively, converted these excitants into antagonists of burst firing.(ABSTRACT TRUNCATED AT 250 WORDS)