Effects of quinidine and quinine on the excitability of pyramidal neurons in guinea-pig hippocampal slices

Effects of quinidine (25M–1mM) and its stereoisomer, quinine (1–5 mM), on the excitability of CA3 pyramidal neurons were investigated in guinea-pig hippocampal slices using intracellular recording techniques. At concentrations of quinidine higher than 100 M (and higher than 1 mM for quinine), 1) the resting potential shifted to the depolarizing direction with an increase of the input resistance, 2) the spike duration was prolonged, 3) the spike amplitude was decreased, 4) the late component of the afterhyperpolarization (AHP) (caused by the activity of the Ca²⁺-mediated K conductance) were suppressed, and 5) finally, neurons became inexcitable. The results indicate that the blocking action of quinidine and quinine is not specific to the Ca²⁺-mediated K conductance in mammalian hippocampal neurons, and that this conductance is much less sensitive to the drugs in comparison with other preparations.     

Actions of pentylenetetrazol (PTZ) on CA3 neurons in hippocampal slices of guinea pigs

Summary The actions of the convulsant drug pentylenetetrazol (PTZ) were studied on CA3 neurons of hippocampal slices (300–400 m thick). In 9 out of 109 neurons, epileptic reactions were elicited during a single application of PTZ. After repeated applications of PTZ, 53 neurons showed periodic paroxysmal activity. They developed according to the following sequence: (i) Paroxysmal hyperpolarizations, (ii) burst activity, and (iii) typical paroxysmal depolarization shifts (PDS). The rate of occurrence was about 8/min. Paroxysmal hyper- and depolarizations appeared synchronously in pairs of neurons. The developmental sequence occurred in reverse during washing. After the onset of paroxysmal activity, bursts and PDS persisted if PTZ concentration in tissue ranged between about 2 and 10 mmol/l. When this range was exceeded in either direction, epileptic activity was abolished at a calcium concentration of 2.75 mmol/l. Decreasing and increasing the calcium concentration shifted the epileptogenic concentration range to lower and higher levels, respectively. It is concluded that repetition of PTZ application alters membrane properties of neurons and thus leads to paroxysmal events triggered by synaptic processes.Supported by Deutsche Forschungsgemeinschaft Sp 108/3 and Bi 278/1-3     

Brief anoxic episodes induce long-lasting changes in synaptic properties of rat CA3 hippocampal neurons

The effects of brief anoxic episodes on rat CA3 hippocampal neurons were studied with intracellular and extracellular techniques in the in vitro slice preparation. After repeated (3–7 times), brief (2–6 min duration each) applications of artificial cerebrospinal fluid (ACSF) saturated with 95% N₂ and 5% CO₂, electrical stimulation of various inputs to CA3 neurons, evoked an excitatory postsynaptic potential (EPSP) followed by an all-or-none burst. This response which persisted for several hours after the last anoxic episode, is reminiscent of the bursts induced by various convulsive agents. Post anoxic bursts are generated by a polysynaptic network which converge on the apical distal segment of CA3 neurons. It is concluded that a repetitive impairement of metabolism produces long lasting changes in the synaptic properties of CA3 neurons.     

Action of vasopressin on neurons and microvessels in the rat hippocampal slice

Summary Arginine vasopressin is reported to have an excitatory effect on hippocampal neurons in the slice preparation. However, vasopressin also has a classic vasopressor action on mammalian blood vessels. We used the rat hippocampal slice to examine the effects of this peptide on central neural and vascular targets. The hippocampus is densely vascularized and pyramidal cells are enmeshed in a network of microvessels. Vasopressin increased the excitability of impaled neurons without substantially altering membrane potential or resistance. The peptide also caused pronounced vasoconstriction in penetrating microvessels when applied at micromolar concentrations. The concerted action of vasopressin on neurons and blood vessels and the physical proximity of these cell types suggest mechanisms whereby these responses may be associated.     

海马CA_3区辐射层突触形态的衰老性变化

本文应用电镜体视学和半自动图像分析系统对青年组(3个月),老年组(34个月),SD大白鼠海马CA_3区辐射层的突触进行研究,结果显示老年组突触数密度、面密度、突触平均长度,可见线粒体突触前终未数均比青年组减少,突触切面长度未见明显的代偿性变化,表明海马CA_3区辐射层突触在老年时出现退行性变化。本文并对突触丢失,退变及其意义进行讨论。     

Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker,verapamil

Summary We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculline-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 M) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50–150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.     

Postsynaptic inhibition by adenosine in hippocampal CA3 neurons: Co2+-sensitive activation of an inwardly rectifying K+ conductance

The properties of the current underlying the membrane hyperpolarization evoked by adenosine (50–100 m) were investigated in hippocampal CA3 neurons in vitro using current-clamp and single-electrode voltage-clamp techniques. In voltage-clamp measurements, the adenosine-induced current (I _Ado) was outward at rest and reversed at membrane potentials close to the equilibrium potential of K⁺ (E _K), indicating that I _Ado was carried by K⁺ ions. Determination of I _Ado at several membrane potentials revealed a nonlinear current/voltage (I/V) relationship of the current displaying inward rectification in the hyperpolarizing direction. Similarly, adenosine increased the membrane slope conductance only at membrane potentials negative to rest, whereas the slope of the neuronal I/V curve remained unchanged when determined at potentials positive to rest. Since the electrophysiological properties of I _Ado were very similar to those described for K⁺ conductances activated by other neuroactive substances like serotonin, opioid peptides and -aminobutyric acid B receptor (GABA_B) agonists, we conclude that I _Ado belongs to a family of ligand-operated, inwardly rectifying K⁺ currents which apparently share a common mechanism to reduce postsynaptic excitability. As an additional feature, the postsynaptic adenosine response was reduced by bath application of Co²⁺ or Ni²⁺. The adenosine-induced membrane hyperpolarization was not affected by low-Ca²⁺ or low-Mg²⁺ solutions, nor by buffering of intra-cellular Ca²⁺, but a gradual decline of I _Ado was observed following superfusion with Co²⁺ or Ni²⁺. In contrast, Mn²⁺ caused only a weak attenuation of the adenosine response.     

Action of the hyperpolarization-activated current (I h) blocker ZD 7288 in hippocampal CA1 neurons

 The effects of ZD 7288, a ”bradycardic” agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1–1000 μM) reduced the hyperpolarization-activated current (I _h) in CA1 pyramidal neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 μM) caused membrane hyperpolarization (by 5.9 ± 0.5 mV) and a reduction of membrane conductance (by 17.9 ± 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell’s excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs⁺, another known blocker of I _h, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs⁺-induced inhibition of LTD maintenance is not directly related to block of I _h. Received: 14 February 1997 / Received after revision: 4 July 1997 / Accepted: 21 July 1997     

The long-term suppressive effect of prior activation of synaptic inputs by low-frequency stimulation on induction of long-term potentiation in CA1 neurons of guinea pig hippocampal slices

We have investigated the effects of prior activation of afferent inputs by a train of low-frequency stimulation (LFS) on the induction of long term potentiation (LTP) induced by highfrequency stimulation (tetanus, 100 Hz, 100 pulses) in CA1 neurons of guinea pig hippocampal slices. The parameters of the LFS were altered systematically: the frequency (1 or 5 Hz); the number of pulses (80, 200 or 1000); and the time lag from the LFS to the tetanus (20, 60 or 100 min). Conditioning effects of the LFS on the induction of LTP were evaluated in terms of the slope of the field excitatory postsynaptic potential (S-EPSP) and the amplitude of the population spike (A-PS). LTP could reliably be induced by 100 Hz tetanic stimulation delivered to a naive slice. In contrast, the attempt to induce LTP 60 min after LFS of 200 or 1000 pulses at 1 Hz resulted only in short-term potentiation while the LFS itself produced no significant change in the responses. The suppressive effect on LTP was significantly reduced for 1 Hz LFS with a smaller number of pulses (80 pulses), or a shorter (20 min) or longer (100 min) time lag from the LFS to the tetanus, or with LFS at a higher frequency (5 Hz). When the LFS of 1000 pulses at 1 Hz was delivered in the presence of the n-methyl-d-aspartate (NMDA) receptor antagonist AP5 (d,l-4-amino-5-phosphonovalerate, 50 M), which was washed out after the end of the LFS, the tetanus given 60 min after application of the LFS produced stable LTP, indicating the involvement of NMDA receptor/channels in the mechanisms of this particular form of synaptic plasticity-long-term suppression of LTP.     

Modulatory role of glutamic acid on the electrical activities of pain-related neurons in the hippocampal CA3 region

Glutamic acid (Glu) participates in pain modulation of the central nervous system. The CA3 region of the hippocampal formation has been suggested to be involved in nociceptive perception. However, it is unknown whether Glu could modulate the electrical activities of pain-related neurons in the hippocampal CA3 region. The present study aimed to determine the effects of Glu and its receptor antagonist MK-801 in the pain-evoked response of both pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the hippocampal CA3 region of normal rats. We used a train of electric impulses applied to the sciatic nerve as noxious stimulation. The electrical activities of either PENs or PINs in the hippocampal CA3 region were recorded by a glass microelectrode. The results revealed that intra-CA3 region microinjection of Glu (0.5 μg/1 μl) increased the evoked firing frequency and shortened the firing latency of PEN, while decreased the evoked firing frequency and prolonged the inhibitory duration of PIN in the hippocampal CA3 region of rat evoked by the noxious stimulation. Intra-CA3 region administration of MK-801 (0.25 μg/1 μl) produced the opposite response. These results suggest that Glu and its receptors in hippocampal CA3 region are involved in the modulation of nociceptive information transmission by affecting the electric activities of PENs and PINs.     

The effects of chronic glucocorticoid exposure on dendritic length, synapse numbers and glial volume in animal models: Implications for hippocampal volume reductions in depression

Glucocorticoids (GCs) are hormones secreted by the adrenal glands as an endocrine response to stress. Although the main purpose of GCs is to restore homeostasis when acutely elevated, animal studies indicate that chronic exposure to these hormones can cause damage to the hippocampus. This is indicated by reductions in hippocampal volume, and changes in neuronal morphology (i.e., decreases in dendritic length and number of dendritic branch points) and ultrastructure (e.g., smaller synapse number). Smaller hippocampal volume has been also reported in humans diagnosed with major depressive disorder or Cushing's disorder, conditions in which GCs are endogenously and chronically elevated. Although a number of studies considered neuron loss as the major factor contributing to the volume reduction, recent findings indicated that this is not the case. Instead, alterations in dendritic, synaptic and glial processes have been reported. The focus of this paper is to review the GC effects on the cell number, dendritic morphology and synapses in an effort to better understand how these changes may contribute to reductions in hippocampal volume. Taken together, the data from animal models suggest that hippocampal volumetric reductions represent volume loss in the neuropil, which, in turn, under-represent much larger losses of dendrites and synapses.     

A study of glial cell proliferation in the molecular layer of the dentate gyrus of the rat following interruption of the ventral hippocampal commissure

Summary The proliferation of glial cells in the molecular layer of the dentate gyrus in response to lesions of the ventral hippocampal commissure, has been studied autoradiographically following intraventricular injections of ³H-thymidine. Within 24h of commissurotomy there is an appreciable increase in the number of labeled cells throughout the molecular layer which reaches its peak at approximately 36h. This generalized glial hyperplasia persists for at least 5–6 weeks and there does not appear to be a secondary re-distribution of the newly-generated glial cells as has been reported after entorhinal lesions (Gall et al., 1979). In semi-thin plastic sections most of the proliferating cells more closely resemble the medium-shade oligodendrocytes of Ling et al. (1973) than typical microglia; the reactive astrocytes do not appear to participate in the glial proliferation.This work was supported in part by grants NS-10943 from the NINCDS and DA-00259 from ADAMAH, and was carried out while C.A. was on leave of absence from the Department of Morphology, Autonoma University School of Medicine, Madrid, and was in receipt of a Fogarty International Fellowship (1F05 TWO 2580)     

Bcl-2抑制剂对黄芪注射液降低缺氧缺糖/复氧复糖大鼠海马神经元caspase-3表达的影响

目的:观察Bcl-2抑制剂对黄芪注射液降低缺氧缺糖/复氧复糖大鼠海马神经元caspase-3表达的影响。方法:取体外原代培养8 d的海马神经元,随机分为6组:正常对照组、模型组(缺氧缺糖/复氧复糖组)、黄芪注射液组、黄芪注射液溶剂(无菌去离子水)对照组、Bcl-2抑制剂组和Bcl-2抑制剂+黄芪注射液组。除正常对照组外均进行缺氧缺糖0.5 h再复氧复糖,各组均于复氧复糖后24 h进行指标检测:采用细胞免疫化学染色法观察细胞形态和caspase-3阳性细胞率,Western blotting法检测海马神经元Bcl-2和cleaved caspase-3蛋白的表达,RTPCR法检测海马神经元caspase-3 mRNA的表达。结果:与正常对照组相比,模型组细胞caspase-3阳性率、Bcl-2、cleaved caspase-3蛋白及caspase-3 mRNA表达均明显增强(P0.05);与模型组相比,黄芪注射液组Bcl-2表达明显增加,细胞caspase-3阳性率、cleaved caspase-3蛋白及caspase-3 mRNA表达均明显降低(P0.05);而黄芪注射液溶剂对照组、Bcl-2抑制剂组及Bcl-2抑制剂+黄芪注射液组则无明显差异;黄芪注射液溶剂对照组Bcl-2表达较正常对照组无明显变化,而Bcl-2抑制剂组及Bcl-2抑制剂+黄芪注射液组显著下降(P0.05)。结论:Bcl-2抑制剂可对抗黄芪注射液降低缺氧缺糖/复氧复糖大鼠海马神经元caspase-3表达的作用,黄芪注射液通过Bcl-2发挥对缺氧缺糖/复氧复糖大鼠海马神经元凋亡的抑制作用。     

Control of spontaneous epileptiform discharges by extracellular potassium: an “in vitro” study in the CA1 subfield of the hippocampal slice

Summary 1. The effects evoked by changing [K⁺]_o upon the synchronous epileptiform discharges (SEDs) generated in the presence of GABA antagonists were studied in the in vitro hippocampal slice with extra- and intracellular recordings. [K⁺] in the artificial cerebrospinal fluid (ACSF) was varied in steps of 1 or 2 mM between 3.25 and 10.25 mM. 2. Spontaneous SEDs occurred rarely at [K⁺]_o lower than 5.25 mM. Augmenting [K⁺]_o from 5.25 to 10.25 mM caused a four to five fold increase in the frequency of occurrence of SEDs while the duration of each SED was inversely related to the rate of occurrence. 3. Similar findings were observed when the CA1 subfield had been surgically disconnected from the CA2-CA3 subfields. In these experiments SEDs occurred independently in the two regions, but at any given [K⁺]_o SEDs in the CA3 subfield displayed a frequency two to three times higher than that of SEDs generated in the CA1 area. 4. The intracellular correlate of the SEDs in the CA1 subfield either intact or isolated from the CA2-CA3 ones was a large amplitude depolarization (duration 100–600 ms) associated with a burst of action potentials. This intracellular event, which was similar to the paroxysmal depolarizing shift (PDS) recorded in focal models of epilepsy in vivo, behaved largely like a synaptic phenomenon when the resting membrane potential (Vm) was changed with intracellularly injected current. A long lasting (half-width: 0.3–2 s in 6.25 mM [K⁺]_o) hyperpolarizing potential usually followed the PDS and could be inverted by hyperpolarizing the Vm by 15–25 mV. When [K⁺] in the ACSF was raised from 7.25 to 10.25 mM, pyramidal cells depolarized in a dose related fashion. At the same time the post-PDS hyperpolarization decreased in duration and peaked earlier, thus curtailing the depolarizing envelope of the PDS. Consequently, the effect of increasing [K⁺]_o was that of evoking more frequent, but shorter PDSs. 6. These findings demonstrate that the appearance of spontaneous SEDs in the presence of GABA antagonists is dependent upon [K⁺]_o. The effects of evoked by increasing [K⁺]_o are presumably mediated through: (i) a decreased strength of K⁺ repolarizing conductances; (ii) an increased efficacy of synaptic potentials; (iii) a steady depolarization of the neuronal membrane. The modulation of the frequency of occurrence of SEDs appears to be related to a decreased duration of the hyperpolarization which follows the PDS, a potential which is largely mediated by a K⁺ conductance.Supported by MRC of Canada (grant MA-8109)     

黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元Caspase-3表达

目的 观察黄芪注射液对缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因Caspase-3表达的影响.方法 取原代培养8d的大鼠海马神经元,随机分为正常对照组、缺氧缺糖/复氧复糖组、黄芪注射液溶剂对照组和黄芪注射液组.除正常对照组外均进行缺氧缺糖0.5h再复氧复糖.各组于复氧复糖后0h、0.5h、2h、6h、24h、48h、...     

Effects of carnosine on long-term plasticity of medial perforant pathway/dentate gyrus synapses in urethane-anesthetized rats: an in vivo model

The objective of this study was to examine the effect of carnosine on the hippocampal-dependent learning in perforant pathway/dentate gyrus synapses. The experiments were carried out on adult rats. A bipolar stimulating electrode was placed to the medial perforant path and a double-barrel glass micropipette was placed in the dentate gyrus as the recording electrode. Artificial cerebrospinal fluid (to control group) or carnosine (0.1, 1 μg/μL) was infused into the dentate gyrus via one of the barrels of the glass electrode. Our results showed that the I/O curve of excitatory postsynaptic potential (EPSP) slope or population spike (PS) amplitude was not significantly shifted by carnosine. Although carnosine infused prior to high-frequency stimulation (HFS) decreased the slope of EPSP and amplitude of PS, when infused after HFS, no effect was observed. In the present study, we speculated that carnosine decreased LTP by inhibiting sGC activation. The present experiment provides the first evidence that carnosine may play a role in synaptic plasticity in dentate gyrus in vivo.     

Electrophysiological actions of N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazolamine (R56865) on CA1 neurons of the rat hippocampal slice during hypoxia

The electrophysiological effects of N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazolamine (R56865), a drug which protects heart cells from ischemia-induced arrhythmias, was studied on intracellularly-recorded CA1 neurons of the rat hippocampal slice under normal or hypoxic conditions. On normoxic cells R56865 (1 μM) reduced firing accommodation without changing passive membrane properties, spike characteristics or synaptic transmission. On hypoxic cells R56865 selectively reduced the amplitude of hypoxia-induced membrane depolarization and partly counteracted the depression of synaptic transmission evoked by Schaffers collateral stimulation. Despite its influence on repetitive firing properties, R56865 might be useful to limit the extent of cellular depolarizing responses to hypoxia.     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.