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.     

The influence of glucose on intracellular and extracellular pH in rat hippocampal slices during and after anoxia

In this study we investigated in rat hippocampal slices (1) how glucose availability affected tissue acidosis during and after anoxia, (2) whether the onset of anoxic depolarization was associated with a specific pH, (3) whether glycolysis was the major source of acidification before and during anoxic depolarization, and (4) whether improved recovery of synaptic function with elevated glucose levels was related to changes in tissue acidosis. Intracellular pH (pH_i) and extracellular pH (pH_o) were measured simultaneously before, during, and after anoxia in hippocampal slices bathed in 0, 5, 10, and 15 mM glucose. Slices exposed to 0 mM glucose were given 20 mM sodium lactate as a metabolic substrate. We found that the pH_i and pH_o at which anoxic depolarization occurred depended upon glucose concentration. We also found that elevated glucose availability increased acidification in both the intracellular and extracellular compartments during anoxia and delayed recovery of pH homeostasis after anoxia. Our results suggest that glycolysis is the primary source of acidosis before the onset of anoxic depolarization, but not during anoxic depolarization. Our results also suggest that moderate increases in acidosis resulting from increased glycolysis are potentially beneficial for anoxic survival.     

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.     

Brevetoxin depresses synaptic transmission in guinea pig hippocampal slices

Extracellular recordings were obtained from area CA1 of guinea pig hippocampal slices. PbTx-3, a brevetoxin fraction isolated from the red tide dinoflagellate Ptychodiscus brevis. was applied by bath perfusion. The toxin produced a concentration-dependent depression of the orthodromically evoked population spike with an EC50 of 37.5 nM. Brevetoxin concentrations below 10 nM were without effect, and concentrations above 100 nM led to total inhibition of evoked responses. PbTx-3 did not produce spontaneous synchronous discharges but did induce afterdischarges following evoked responses in about 50% of the slices tested, particularly at concentrations between 10 nM and 100 nM. Orthodromically evoked responses were more sensitive to PbTx-3 than were those elicited by antidromic stimulation. High-Ca²⁺ solution, 4-aminopyridine, and tetraethylammonium failed to antagonize either orthodromic or antidromic effects of the toxin. Although the precise mechanism by which PbTx-3 depresses evoked responses is not certain, depolarization of the presynaptic nerve terminals leading to failure of transmitter release could explain the toxin's actions. This is the first report of the effects of brevetoxin applied directly to central nervous system tissue.     

Prolonged exposure to high potassium concentration results in irreversible loss of synaptic transmission in hippocampal tissue slices

Since elevation of the concentration of free calcium in the cytoplasm ([Ca2+]i) during hypoxia is believed to cause injury to cells and since during Le?o's spreading depression (LD) excess calcium accumulates in neurons, we asked whether LD of prolonged duration in well oxygenated tissue causes irreversible loss of function. LD-like depolarization of controlled duration was induced by irrigating hippocampal tissue slices with a high-K+ solution for varying time periods. Interstitial potassium concentration ([K+]o) and extracellular potentials were recorded. Following brief LD there was a period of transient hyperexcitability with increased orthodromic population spike amplitude and burst firing, followed by recovery to control levels after 60 min. When depolarization was prolonged beyond 4 or 5 min, the hyperexcitable period was followed by severely depressed transmission. The data are compatible with the hypothesis that prolonged elevation of [Ca2+]i causes neuron injury. Exposure of in vitro preparations to high [K+]o cannot be regarded as the equivalent of a physiological stimulus.     

Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices

Extracellular pH changes were measured in the rat hippocampal slices using the pH-sensitive dye Phenol red. pH changes accompanied artificially evoked synaptic transmission in the dendrite area of dentate gyrus neurones and pyramidal neurones (CA1). Single electrical stimulation of presynaptic pathways produced a rapid acidic pH shift which was followed by a long-lasting alkaline one. The duration (nearly 10 ms) and amplitude of the acidic shift were closely related to the orthodromically evoked population excitatory postsynaptic potential. Population action potential, when elicited antidromically or in conditions of blocked synaptic transmission, did not produce any pH changes which are supposed to be specifically linked to the synaptic transmission.     

Rat hippocampal slices need bicarbonate for the recovery of synaptic transmission after anoxia

The purpose of this study was to see how the nominal removal of bicarbonate (HCO(-)(3)) from the extracellular space of brain tissue influenced recovery of brain tissue from anoxia. Removal of HCO(-)(3) in HEPES-buffered artificial cerebrospinal fluid (aCSF) inhibited almost completely recovery of synaptic transmission in hippocampal slices after anoxia. Altered pH did not contribute to this finding because adjusting intracellular (pH(i)) and extracellular (pH(o)) pH to control levels did not reduce the effect of HCO(-)(3) removal. Our results suggest that HCO(-)(3) levels are important in determining the extent of anoxic or ischemic brain injury.     

α-Bungarotoxin- and methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices

This study demonstrates for the first time that α7 nicotinic receptors (nAChRs) mediate fast synaptic transmission in conventional hippocampal slices. In the presence of antagonists of muscarinic, AMPA, NMDA, GABA_A, ATP, and 5-HT3 receptors, spontaneous and evoked postsynaptic currents (PSCs) recorded from CA1 interneurons were blocked by the α7 nAChR antagonists methyllycaconitine and α-bungarotoxin and by a desensitizing concentration of the α7 nAChR agonist choline. Spontaneous nicotinic PSCs were also accompanied by Na⁺ transients, indicating that α7 nAChR-mediated transmission serves as an excitatory signal to the CA1 interneurons in the hippocampus.     

Synaptic transmission and paired-pulse behaviour of CA1 pyramidal cells in hippocampal slices from a hibernator at low temperature: importance of ionic environment

To investigate the effects of ionic changes possibly associated with hibernation, hippocampal slices prepared from golden hamsters were studied in artificial cerebrospinal fluid (ACSF) of variable composition (K⁺ 3–5 mM, Ca²⁺ 2–4 mM, Mg²⁺ 2–4 mM, pH 7.0–7.7) at temperatures of 15–20°C, just above the temperature below which synaptic transmission is blocked. Population action potentials (population spikes, PSs) of CA1 pyramidal cells were evoked by stimulation of the Schaffer collaterals/commissural fibers with paired pulses (interpulse interval 50 ms, interval pairs 30 s). The responses evoked at given temperatures were investigated as a function of extracellular ion concentrations. In ACSF containing 3 mM K⁺, 2 mM Ca²⁺ and 2 mM Mg²⁺, PSs could be evoked at temperatures of > ∼ 16°C whereas at lower temperatures synaptic transmission was blocked. The threshold temperature was slightly higher for the first (PS1) than for the second PS (PS2) evoked by paired-pulse stimulation. The slices displayed paired-pulse facilitation (PPF) at all temperatures. Elevation of [K⁺]₀ from 3 to 5 mM depressed the amplitudes of both PS1 and PS2, with a stronger effect on PS2. PPF was reduced and, at near-threshold temperatures, turned into paired-pulse depression (PPD). Elevation of [Ca²⁺]₀ from 2 to 4 mM increased the amplitude of PS1. The amplitude of PS2, in contrast, was reduced at near-threshold temperatures. PPF turned into PPD. Elevation of [Mg²⁺]₀ from 2 to 4 mM reduced the amplitudes of both PS1 and PS2, with a stronger effect on PS1. Accordingly, PPF was increased. Acidification by 0.3 pH units strongly depressed the amplitudes of PS1 as well as PS2 and increased PPF. Alkalization by 0.4 pH units had only weak effects in the opposite direction. Changes in the ionic composition comparable to those investigated in the present study presumably occur in the brain interstitium of hamsters during entrance into hibernation. According to our results, such changes depress synaptic transmission at low temperatures in the hamster hippocampus in vitro. This modulation may be important for the regulation of neuronal activity during entrance into hibernation.     

The effect of changing extracellular potassium concentration on synaptic transmission in isolated spinal cords

Hemisected spinal cords of infant mice were exposed in vitro to varying concentrations of K+ in the bathing fluid. Interstitial potassium concentration ([K+]o) as well as dorsal and ventral root (DR and VR) potentials evoked by DR stimulation were recorded. [K+]o in spinal tissue was made to change from its control level of 3.5 mM by superfusing the preparation with modified artificial cerebrospinal fluid (ACSF). [K+]o in tissue followed bath [K+] in the range from 2.0 to 13 mM, but diverged from bath concentration at levels at or below 1.0 mM. Relatively high tissue [K+]o during exposure to low bath [K+] was attributed to leakage of K+ ions from cells. Between 2.0 and 5.0 mM [K+]o a shallow but consistent positive correlation was found between [K+]o and dorsal root reflex (DRR) amplitude, while segmental reflex (VRR) amplitude changed little. Outside the range of 2.0-5.5 mM DR and VR responses were severely depressed in both elevated and lowered [K+]o. In spite of depressed reflex responses, transient stimulus-evoked elevations of [K+]o were strikingly increased when the resting [K+]o fell below 2.0 mM, suggesting enhancement of voltage-dependent K+-current.     

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.     

Basic fibroblast growth factor modulates synaptic transmission in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) is one of the effective growth factors that protect neurons against excitotoxic/ischemic injury and promote neuronal survival. In the present study, we examined the acute modulative effect of bFGF on synaptic transmission by monitoring spontaneous intracellular Ca²⁺ ([Ca²⁺]i]) oscillation, the amplitudes of which reflect excitatory and inhibitory inputs. The hippocampal cells from embryonic day 18 rats were cultured for 11–14 days, and changes in [Ca²⁺]i of single neurons were measured by a microfluometrical technique with fura-2. The amplitude of spontaneous oscillation was decreased by 10 ng/ml bFGF, but not by nerve growth factor (10–1000 ng/ml). Acidic FGF (1000 ng/ml) had a weaker depressant effect. The effect of bFGF was counteracted by suramin. bFGF did not affect the increase in [Ca²⁺]i evoked by glutamate agonists, NMDA or kainate, indicating that glutamate receptors are not involved in the mechanism. This is supported by similar results that kainate-evoked current was not affected by bFGF. On the other hand, bicuculline masked the effect of bFGF on the Ca²⁺ oscillation. But GABA-evoked current was slightly decreased by bFGF. These results suggest the possible role of bFGF in modulating GABAergic rather than glutamatergic neurotransmission.     

Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units/ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 microM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures.     

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.     

Glutamine can enhance synaptic transmission in hippocampal slices

Activity of 40 single antidromically identified supraoptic neurons was recorded and evaluated in response to a combination of tactile, vulvar massage, vaginal distension, and slow intrajugular 1.2 M sodium chloride infusion in unanesthetized, randomly hydrated ewes. Estradiol-implanted Southdown ewes were prepared according to techniques described by Jennings et al. Only 4 spontaneous firing patterns were observed in the supraoptic nuclei. Analysis of evoked activity indicated that each stimulus evoked alterations in mean firing rates or increased numbers of short interspike intervals in some cells. The resultant activity of units to sequential vulvar massage and 1.2 M sodium chloride infusion suggests a possibility of separate mechanisms of release of oxytocin and vasopressin.     

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.     

Hypotonic exposure enhances synaptic transmission and triggers spreading depression in rat hippocampal tissue slices

Low extracellular osmotic pressure (π_o) is known to enhance CNS resposiveneess and the chance of seizures, but the mechanism of the hyperexcitability is not clear. We recorded evoked potentials in st. radiatum and st. pyramidale of CA1. Tissue electrical resistance (R_o) was determined from the voltage drop (V_Ro) evoked by constant current pulses. Lowering of π_o by reducing [NaCl] caused a concentration-dependent increase of amplitude and duration of extracellular excitatory postsynaptic potentials (fEPSPs). fEPSPs increased much more than didV_Ro, but antiddromic population spikes increased in proportion toV_Ro. fEPSP increased also in isosmotic low NaCl (fructose or mannitol substituted) olutions, but not as much as in low π_o. In moderately hypotonic solutions orthodromic population spikes increased as expected from the augmented fEPSP, but in strong hypotonia input-output curves shifted to the left and single stimuli evoked multiple population spikes, indicating lowering of threshold of postsynaptic neurons. Blocking N-methyl-d-aspartate (NMDA) receptors did not diminish the enhancement of fEPSP amplitude. Spreading depression (SD) erupted in most slices in very low π_o, but not in isosmotic low [NaCl] solutions. We conclude that the hypotonic enhancement of EPSPs depends, in part, on the lowering of [Na⁺]_o and/or of [Cl⁻]_o, and it may be augmented by dendritic swelling favoring electrotonic spread of EPSPs from dendrites to somata, and buildup of transmitter concentration due to swelling of perisynaptic glia. SD can be initiated by cell swelling, but the depolarization associated with SD is probably not caused by the opening of stretch-gated ion channels.     

The effect of graded hypertonia on interstitial volume, tissue resistance and synaptic transmission in rat hippocampal tissue slices

Rat hippocampal slices were exposed for 30 min to each of three levels of increased osmolarity (π_o), achieved by adding 25, 50 or 100 mM mannitol to the bathing solution. The interstitial volume (ISV) determined as the relative volume of dilution of the probe ion, tetra-methyl-ammonium (TMA⁺), increased markedly, indicating cell shrinkage. Tissue resistance (R_o) decreased only slightly with increasing π_o. The discrepancy between ISV increase and R_o decrease suggests increased electrical resistance of cell membranes. TMA⁺ dilution appears to be a more reliable measure of ISV than is R_o During recovery from hypertonic treatment the previously expanded ISV frequently shrank, suggesting post-hypertonic cell swelling. Hypertonic treatment significantly depressed orthodromically transmitted population spikes and extracellular synaptic potentials (fEPSPs), and the degree of depression varied with the increase in π_o. Changing recording condition due to reduced R_o could not account for the depression of population spikes and fEPSPs. Following return to normal π_o, orthodromic population spikes frequently overshot initial control amplitude. An isolated episode of spreading depression occurred in about half of the slices following exposure to the most severely hypertonic solution. At the end of 2.5 h recovery, orthodromic spikes did not significantly differ from those of untreated control slices observed for the same length of time. We conclude that synaptic transmission is depressed by elevation of π_o and the depression is concentration dependent and reversible.     

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.     

Action of capsaicin on dorsal root-evoked synaptic transmission to substantia gelatinosa neurons in adult rat spinal cord slices

An action of capsaicin was investigated on dorsal root-evoked synaptic transmission to substantia gelatinosa (SG) neurons in adult rat spinal cord slices by use of the whole-cell voltage-clamp technique. In 79% of neurons examined, superfusing capsaicin (1 μM) for 30 s depressed a C-fiber-evoked excitatory synaptic current in a manner sensitive to a capsaicin-receptor antagonist, capsazepine (10 μM). On the contrary, Aδ-fiber-evoked excitatory and inhibitory synaptic currents were unaffected by capsaicin in all of cells tested. It is concluded that capsaicin specifically acts on C-afferents, resulting in an inhibition of evoked excitatory transmission to the SG; this may contribute to, at least in part, an acute analgesic action of capsaicin.