Effects of acidosis on the post-hypoxic recovery of synaptic transmission in gerbil hippocampal slices

We investigated the effects of acidosis on the hypoxic neuronal damage using gerbil hippocampal slices. Acidosis has delayed the onset of harmful hypoxic depolarization, resulting in a decrease in the total hypoxic period and the hypoxic depolarization. This effect has been considered to be protective. However, the synaptic recovery after reoxygenation was attenuated when acidosis (pH: 6.2-6.9) was sustained. Conversely, the synaptic recovery was potentiated when the acidosis was restored to the physiological milieu during the reoxygenation period. These results suggest that acidosis plays a protective effect against the hypoxic neuronal damage only when rapid appreciable pH recovery is achieved during reoxygenation.     

Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]

Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage-dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained.     

Periodic fluctations in synaptic transmission and enhancement of transmission in hippocampal slices

The properties of the synchronously activated radiatum fiber-CA1 synaptic population were examined with the in vitro hippocampal slice preparation. Periodic fluctuations in synaptic transmission and in the enhancement of synaptic transmission were observed with periods ranging from 8 to 20 s. Such periodic fluctuations did not arise from fluctuations in afferent radiatum fiber activity. The period and amplitude of the cyclic variations in the enhancement of synaptic transmission were found to be altered with repeated electrical stimulation of the radiatum fibers. These results reflect cooperative synaptic actions which must be taken into consideration in the delineation of the mechanisms of potentiation.     

Periodic fluctuations in synaptic transmission and enhancement of transmission in hippocampal slices

The properties of the synchronously activated radiatum fiber-CA1 synaptic population were examined with the in vitro hippocampal slice preparation. Periodic fluctuations in synaptic transmission and in the enhancement of synaptic transmission were observed with periods ranging from 8 to 20 s. Such periodic fluctuations did not arise from fluctuations in afferent radiatum fiber activity. The period and amplitude of the cyclic variations in the enhancement of synaptic transmission were found to be altered with repeated electrical stimulation of the radiatum fibers. These results reflect cooperative synaptic actions which must be taken into consideration in the delineation of the mechanisms of potentiation.     

The effects of temperature on synaptic transmission in hippocampal tissue slices

Fully submerged rat hippocampal tissue slices were exposed to temperature changes, and the effects on CA1 pyramidal cell electrophysiology studied. Raising the temperature from 29 to 33 or 37 degrees C simultaneously increased the focal-excitatory postsynaptic potentials and decreased the population spikes. These changes were largely reversible for slices warmed to 33 degrees C, but not for slices warmed to 37 degrees C. During warming transiently increased excitatory transmission was observed; the degree of increased transmission was related to the rate of temperature rise. It is postulated that neuronal membrane hyperpolarization with warming is responsible for several of the effects seen.     

Effect of 4-aminopyridine on synaptic transmission in rat hippocampal slices

Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in area CA1 of rat hippocampal slices in vitro. The responses evoked by spontaneously released glutamate and GABA were recorded from area CA1 pyramidal neurons in rat hippocampal slices in whole-cell mode. The glutamate and GABA receptor-associated ligand-gated currents were obtained from dissociated single hippocampal pyramidal cells. The results showed that 4-aminopyridine (4-AP) had obvious effects on both presynaptic and postsynaptic events. Applications of 4-AP in micromolar concentration resulted in persistent enhancement of the initial slope of fEPSPs with the half-maximal enhancement concentration (EC(50)) of 46.7+/-2.68 microM. At the concentration of 200 microM, 4-AP increased the initial slopes of the total fEPSPs, NMDA- and AMPA-mediated fEPSPs components to 225.6+/-23.8%, 177.4+/-20.1% and 142.3+/-18.9%, respectively, but had no effect on the fiber volley. The half-maximal stimulus intensity to induce responses was reduced from 5.14+/-0.27 to 3.58+/-0.23 V. The frequencies of mEPSCs and mIPSCs were increased to 324.2+/-25.4% and 287.3+/-36.3% by 200 microM 4-AP. The amplitude histograms of mEPSCs and mIPSCs were fitted with Gaussian distributions. After 200 microM 4-AP application, the first and second peaks in Gaussian distributions of mEPSCs were shifted from 8.73+/-0.94 and 17.78+/-2.13pA to 10.48+/-0.82 and 21.14+/-2.45 pA, while those of mIPSCs were shifted from 13.65+/-0.96 and 25.51+/-2.95 pA to 11.21+/-1.04 and 23.08+/-2.37 pA. At 200 microM, 4-AP reduced paired-pulse facilitation and accelerated synaptic fatigue induced by stimulation at 10 Hz (for 1 s) and the ratio of fEPSPs(10)/fEPSPs(1) was decreased from 1.62+/-0.16 to 0.61+/-0.15. At 200 microM, 4-AP inhibited postsynaptic GABA currents induced by 5 microM GABA to 68.2+/-15.5%: by countering the effect of enhanced release of GABA from presynaptic terminals, this could depress the inhibitory pathway. Also at 200 microM, 4-AP increased NMDA currents to 155.3+/-17.8%, but had no significant effect on AMPA currents (94.2+/-15.6%). Our experimental results thus show that 4-AP-induced changes of synaptic transmission in area CA1 of rat hippocampus may be attributed to 4-AP's effects on both presynaptic terminals and postsynaptic receptors.     

Actions of MPTP and MPP+ on synaptic transmission in guinea-pig hippocampal slices

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinson's disease-like syndrome in man, monkeys, and mice. We studied the effects of MPTP and its metabolite, MPP+, on neuronal properties and synaptic transmission in isolated slices of guinea-pig hippocampus using intra- and extracellular recording methods. Addition of MPTP to the superfusate (50 to 100 microM) produced the following effects: Excitatory postsynaptic potentials and extracellularly recorded population spikes, evoked by stimulation of the Schaffer collaterals were increased in amplitude during the application period (30 min). Within 30 min of washing in normal solution, synaptic transmission was blocked, although axonal population action potentials could still be elicited. The block of synaptic transmission was prevented by prior incubation in pargyline, an inhibitor of monoamine oxidase. The membrane potential and resistance of single pyramidal neurons were virtually unaffected; action potentials elicited by depolarizing intracellular current pulses were also unchanged. MPP+ (50 microM) blocked synaptic transmission during the application period by a pargyline-in-sensitive mechanism. These results suggest that MPP+ blocks synaptic transmission in the hippocampus at a presynaptic site. This effect may be relevant for the acute action of MPTP and may provide some insight into its chronic action on nigrostriatal neurons.     

Mechanisms underlying H(2)O(2)-mediated inhibition of synaptic transmission in rat hippocampal slices

Hydrogen peroxide (H(2)O(2)) inhibits the population spike (PS) evoked by Schaffer collateral stimulation in hippocampal slices. Proposed mechanisms underlying this effect include generation of hydroxyl radicals (.OH) and inhibition of presynaptic Ca(2+) entry. We have examined these possible mechanisms in rat hippocampal slices. Inhibition of the evoked PS by H(2)O(2) was sharply concentration-dependent: 1.2 mM H(2)O(2) had no effect, whereas 1.5 and 2.0 mM H(2)O(2) reversibly depressed PS amplitude by roughly 80%. The iron chelator, deferoxamine (1 mM), and the endogenous.OH scavenger, ascorbate (400 microM), prevented PS inhibition, confirming.OH involvement. Isoascorbate (400 microM), which unlike ascorbate is not taken up by brain cells, also prevented PS inhibition, indicating an extracellular site of.OH generation or action. We then investigated whether H(2)O(2)-induced PS depression could be overcome by prolonged stimulation, which enhances Ca(2+) entry. During 5-s, 10-Hz trains under control conditions, PS amplitude increased to over 200% during the first three-four pulses, then stabilized. In the presence of H(2)O(2), PS amplitude was initially depressed, but began to recover after 2.5 s of stimulation, finally reaching 80% of the control maximum. In companion experiments, we assessed the effect of H(2)O(2) on presynaptic Ca(2+) entry by monitoring extracellular Ca(2+) concentration ([Ca(2+)](o)) during train stimulation in the presence of postsynaptic receptor blockers. Evoked [Ca(2+)](o) shifts were apparently unaltered by H(2)O(2), suggesting a lack of effect on Ca(2+) entry. Taken together, these findings suggest new ways in which reactive oxygen species (ROS) might act as signaling agents, specifically as modulators of synaptic transmission.     

Glycolysis and recovery of pottasium ion homeostasis and synaptic transmission in hippocampal slices after anoxia or stimulated pottasium release

The present study was undertaken to determine whether glycolytic energy production was critical to the survival of brain tissue subjected to metabolic stress. Specifically, the contributions of glycolysis (1) to recovery of ion homeostasis after anoxia or high frequency electrical stimulation, and (2) to recovery of synaptic transmission after anoxia, were examined. Energy metabolism in rat hippocampal slices was manipulated by varying glucose concentrations, and by substituting lactate for glucose. Ion transport was slower and recovery of synaptic transmission after anoxia was greatly impeded in the absence of glycolysis. These results support the hypothesis that glycolytic ATP production is tied directly or indirectly to ion transport. The results also suggest that recovery of synaptic transmission following anoxia requires glycolytic ATP.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

We studied the effects of hypertonia on voltage-gated currents of freshly isolated hippocampal CA1 neurons, using open pipette whole-cell as well as gramicidin-perforated patch-clamp recording. Extracellular osmolarity (π_o) was raised by adding mannitol (50 or 100 mmol/l) to the bathing solution. Hypertonia depressed voltage-gated sodium, potassium and calcium currents in all trials. The threshold activation voltage of the currents did not change during hypertonic depression, but maximal activation of Ca²⁺ current shifted to a more negative potential, suggesting stronger depression of high- compared to low-voltage activated currents. During 30 min high π_o treatment (recorded with open pipette), the depression reached maximum in 10–15 min of exposure. The depression of the computed transient component of the K⁺ current recorded by open pipette was statistically not significant. Following hypertonic treatment recovery of the I_Na, the sustained I_K and sustained I_Ca were incomplete compared to control cells maintained in normal solution for an equal length of time. In hippocampal tissue slices hypertonia (+25, +50 and +100 mmol/l fructose) reversibly depressed excitatory postsynaptic currents (EPSCs). We conclude that the shutdown of membrane ion currents by elevated π_o is not selective, but the degree of the suppression varies among current types. Raising π_o in human patients, possibly combined with mild artificial acidosis, may be useful in the prevention and treatment of acute crises associated with excessive excitation or depolarization of neurons.     

Voltage-dependent blockade by bupivacaine of cardiac sodium channels expressed in Xenopus oocytes

Bupivacaine ranks as the most potent and efficient drug among class I local anesthetics, but its high potential for toxic reactions severely limits its clinical use. Although bupivacaine-induced toxicity is mainly caused by substantial blockade of voltage-gated sodium channels （VGSCs）, how these hydrophobic molecules interact with the receptor sites to which they bind remains unclear. Navl.5 is the dominant isoform of VGSCs expressed in cardiac myocytes, and its dysfunction may be the cause of bupivacaine- triggered arrhythmia. Here, we investigated the effect of bupivacaine on Navl.5 within the clinical concentration range. The electrophysiological measurements on Navl.5 expressed in Xenopus oocytes showed that bupivacaine induced a voltage- and concentration-dependent blockade on the peak of/Na and the half-maximal inhibitory dose was 4.51 pmol/L. Consistent with other local anesthetics, bupivacaine also induced a use-dependent blockade on Navl.5 currents. The underlying mechanisms of this blockade may contribute to the fact that bupivacaine not only dose-dependently affected the gating kinetics of Nay1.5 but also accelerated the development of its open-state slow inactivation. These results extend our knowledge of the action of bupivacaine on cardiac sodium channels, and therefore contribute to the safer and more efficient clinical use of bupivacaine.     

Effects of changes in glucose concentration on synaptic plasticity in hippocampal slices

The effects of a low or high concentration of glucose in the perfusion medium on synaptic activity and plasticity were studied in hippocampal slices from rats. Low-glucose medium depressed the field excitatory post-synaptic potentials (fEPSP) significantly, whereas high-glucose medium had little effect on the fEPSP. Tetanization of the afferent fibres elicited significant potentiation (LTP) of synaptic activity irrespective of the glucose concentration in the medium. This may indicate that LTP induction does not depend on optimal neural transmission. Paired-pulse facilitation (PPF) experiments showed that the medium glucose concentration did not significantly influence potentiation of the second response.     

Effects of anticonvulsants on spontaneous epileptiform activity which develops in the absence of chemical synaptic transmission in hippocampal slices

Spontaneous epileptiform activity (SEA) develops in area CA1 of hippocampal slices, when the Ca2+ concentration in the perfusate is lowered to 0.2 mM, at which level evoked chemical synaptic transmission is blocked. We investigated the effects of different anticonvulsants on this autonomous activity, in order to determine whether the antiepileptic effect can be ascribed to an influence on neuronal excitability. Carbamazepine was the most effective to block SEA at concentrations of 1-15 microM. Phenobarbital and phenytoin depressed SEA at concentrations of 25 microM. Valproate was effective at concentrations of 2-5 mM. Midazolam, a water-soluble benzo-diazepine agonist and the N-methyl-D-aspartate antagonists, DL-alpha-aminoadipic acid and 2-amino-7-phosphonoheptanoic acid were ineffective in blocking SEA suggesting that they exert their antiepileptic action by interference with synaptic mechanisms.     

Effects of stobadine, melatonin, and other antioxidants on hypoxia/reoxygenation-induced synaptic transmission failure in rat hippocampal slices

In vitro reversible ischemia was simulated with rat hippocampal slices in order to test the neuroprotective activity of selected antioxidants with emphasis on the pyridoindole stobadine. Slices were exposed to hypoxia (HYP) combined with lowered D-glucose concentration to induce synaptic transmission (ST) failure, which turned out to be irreversible in approximately 80%-100% of slices during reoxygenation (ROX). The amplitude of population spikes (PoS) evoked trans-synaptically by electrical stimulation of Sch?ffer collaterals and recorded in CA1 neurons was the parameter of ST. Pretreatment of slices with stobadine dissolved in slice superfusion media (1 to 100 microM) improved ST recovery after 20-min tissue ROX. Stobadine decreased the number of irreversibly damaged slices and increased the average amplitude of PoS during tissue ROX. The concentration-response relationship of protective activity was bell-shaped, with maximum at 3-30 microM. Moreover, the half-time of PoS decay (t1/2) during HYP was significantly delayed in stobadine treated groups (10 to 100 microM). The neurohormone melatonin (30 to 100 microM) and 21-aminosteroid U-74389G (10 microM) revealed similar protective activity on ST recovery and on t1/2 during HYP. Trolox (200 microM) improved the PoS recovery, yet it had no effect on t1/2. The iron chelator deferoxamine (250 and 500 microM) had no protective effects at all. alpha-Tocopherol administered to animals orally (200 mg/kg for 10 days) only marginally improved the PoS recovery. Comparing the protective effect of compounds tested on PoS recovery, we assume the following rank order of potency: U-74389G > stobadine > melatonin > trolox. Our findings suggest that stobadine as well as trolox, U-74389G and melatonin, antioxidants with remarkably different chemical structures, exerted neuroprotective activity, probably determined by antioxidative properties of these compounds. Moreover, stobadine, U-74389G, and melatonin were able to delay the early ST decay during HYP, which might indicate improved energetic state of neurons in the treated tissue. The study supports the notion about the neuroprotective activity of certain antioxidants.     

Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices

Hippocampal slices exposed to brief anoxia combined with elevated glucose exhibit greater postanoxic recovery of synaptic transmission. Glucose may have improved recovery of synaptic transmission by enhancing the production of metabolic energy during and after anoxia. This enhancement should provide more ATP for energy-requiring ion transport processes, and lead (1) to a delayed onset of complete depolarization of CA1 pyramidal cells during anoxia (anoxic depolarization) and (2) to greater ion transport activity following anoxia. A delay in anoxic depolarization would protect neurons from damage if the duration of anoxic depolarization was shortened. Greater postanoxic ion transport would allow the re-establishment of ion gradients supportive of neuronal and synaptic excitability. The effects of glucose and anoxia on ion homeostasis and synaptic transmission were examined in rat hippocampal slices exposed to different glucose concentrations (5–20 mM). The duration of anoxic depolarization was held constant so that postanoxic damage related to this duration was controlled. We found that K⁺ transport and recovery of synaptic transmission after anoxia in hippocampal slices improved as glucose concentration increased. Also, anoxic depolarization was delayed as glucose concentration increased. Thus, added glucose may improve postanoxic recovery of synaptic transmission by better supporting ion transport.     

Lithium reduced synaptic transmission and increased neuronal excitability without altering endogenous serotonin, norepinephrine and dopamine in rat hippocampal slices in vitro.

1. Extracellular field potentials were recorded in the CA1 pyramidal cell layer following stimulation of stratum radiatum in rat hippocampal slices during superfusion with different concentrations (1, 2, 5, 10, 20, and 30 mM) of lithium (Li+). Control slices were exposed similarly to choline (Ch+) or sodium (Na+). 2. At high concentrations (greater than or equal to 10 mM), Li+, Ch+ and Na+ reduced the amplitude of the field excitatory postsynaptic potential (EPSP). However, Li+ increased, whereas Ch+ and Na+ reduced the population spike amplitude. Thus, Li+ specifically enhanced the excitability of CA1 pyramidal cells. 3. Electrophysiologically monitored slices, plus an additional group exposed to Li+, Ch+ or Na+ without concomitant field potential recordings, were processed for measurement of endogenous levels of serotonin (5-HT), norepinephrine (NE) and dopamine (DA). The mean endogenous levels of 5-HT and NE were not significantly different in 1-30 mM Li+, Ch+ and Na+. Dopamine contents were unchanged after exposure to Li+ and Na+, but were reduced by Ch+. 4. The non-specific effects of Li+ on synaptic transmission and its specific effects on neuronal excitability appeared independent of changes in endogenous 5-HT, NE and DA levels.     

Age-related modifications of potassium homeostasis and synaptic transmission during and after anoxia in rat hippocampal slices

Age-related changes in the capacity of the brain to survive short anoxic episodes were studied in stratum pyramidale (region CA1) of hippocampal slices from control (6-7 months) and aged (26-27 months) rats. Our primary interest was in how aging affected the ability of slices to maintain or to recover extracellular potassium ion (K+o) homeostasis and orthodromically-stimulated field potentials during and after anoxia. During anoxia, K+o homeostasis was lost faster in slices from aged rats. Following anoxia, K+o homeostasis recovered more slowly, and synaptic transmission recovered less completely, in aged slices. These studies provide what is believed to be the first demonstration that aging diminishes the capacity of brain tissue to maintain K+o during anoxia and to recover K+o homeostasis and synaptic transmission following anoxia, and support suggestions that the aged brain is more vulnerable to anoxia.     

Effects of pentetrazol on neuronal activity and on extracellular calcium concentration in rat hippocampal slices

Effects of pentetrazol (PTZ) were studied on neuronal responses in dentate granule cells and area CA1 hippocampal pyramidal cells with intra- and extracellular recording techniques. PTZ induced spontaneous epileptiform field potential transients in areas CA3 and CA1, but not in the dentate gyrus. The concentration optimum for induction of spontaneous epileptiform activity was 2 mM. The epileptiform activity compared in many respects to that induced by GABA antagonists such as picrotoxin, bicuculline and penicillin. Paired pulse stimulus induced responses were affected by concentrations of 0.5 mM. In the concentration range 0.5-2 mM mostly disinhibitory effects were noted. Stimulus induced Ca2+ concentration changes were found to be maximally augmented at concentrations of 2-5 mM. In this range, intracellular studies revealed a block of frequency habituation and an increase in input resistance. The convulsant action of PTZ decreased at concentrations above 5 mM, probably due to a decrease of inward currents. We suggest that the action of PTZ in screening studies for anticonvulsants is mostly due to a decrease of GABAA-receptor mediated IPSPs.     

Calcium-sensitive recovery of extracellular potassium and synaptic transmission in rat hippocampal slices exposed to brief anoxia

We examined the possibility that Ca2+-sensitive inhibition of synaptic transmission following anoxia involves compromise of ion transport activity. Rat hippocampal slices were superfused with artificial cerebrospinal fluids containing different concentrations of CaCl2, and subjected to short anoxia. Durations of anoxia were sufficient to provoke anoxic depolarization, indicated by a sudden rise in extracellular K+ (K+o). Following anoxia, apparent K+ transport was assessed by measuring the magnitude of subnormal K+o (the K+o undershoot) in hippocampal region CA1. Recovery of synaptic transmission 1 h after anoxia was determined by evaluation of the magnitudes of the orthodromically stimulated population spike recorded from CA1 pyramidal cells. K+o undershoots and recovery of synaptic transmission decreased as CaCl2 or the duration of anoxic depolarization increased. These data suggest: (1) that increased artificial cerebrospinal fluid CaCl2 compromised K+ reaccumulation after anoxia; and (2) that ion transport dysfunction may inhibit recovery of synaptic transmission.