Hyperexcitability following moderate hypoxia in hippocampal tissue slices

The effects of moderate hypoxia on CA1 pyramidal cells were studied in submerged rat hippocampal tissue slices. Hyperexcitability developed during re-oxygenation following 30-45 min of hypoxia at 29 degrees C. During hypoxia a sustained increase in extracellular potassium was observed. The post-hypoxic hyperexcitability of CA1 neurons may be the result of their inability to fully recover intracellular potassium lost during oxygen deprivation.     

Adaptation of adult brain tissue to anoxia and hypoxia in vitro

The rat hippocampal slice preparation was used in the present study to demonstrate the ability of adult brain tissue to adapt to anoxic and hypoxic conditions. Adaptation was induced by pre-exposure of hippocampal slices to a short (5 min) anoxic episode. The evoked electrical activity of pre-exposed slices recovered from a subsequent, longer anoxic insult, while that of controls (without pre-exposure), receiving the same insult, did not. The adaptation process is time-dependent; an interval of 0.5 h between the pre-exposure and the subsequent anoxic insult allowed slices to resist anoxic periods of 13 +/- 2 min while after an interval of 2 h an anoxic period of 16 +/- 2 min could be tolerated. Evoked electrical activity persisted in adapted slices during exposure to hypoxia while their non-adapted controls exhibited synaptic silence under hypoxic conditions.     

A specific effect of morphine on evoked activity in the rat hippocampal slice

The effect of morphine (0.5-50 microM) was examined on CA1 field potentials in the tranverse hippocampal slice. Morphine consistently produced an augmentation of evoked activity manifest as (i) a decrease in the threshold for generation of a population spike and (ii) generation of an additional population spike(s) whose amplitude was proportional to the position of the sampled response on its input/output curve. Both of these opiate effects were stereospecific and naloxone-reversible. Additional population spikes occurred in opiate medium with either orthodromic or antidromic activation of the pyramidal cells, and the antidromic effect was abolished when synaptic transmission was blocked, suggesting that morphine did not act directly upon the pyramidal cells. Recordings of population EPSPs in the dendrites of the pyramidal cells showed no changes due to opiate exposure near threshold. Opiate effects were mimicked by the gamma-aminobutyric acid (GABA) antagonist picrotoxin, and were partially to fully reversed by GABA itself, suggesting that disinhibition of pyramidal cells might be involved as a mechanism in this opiate effect. The data are evidence for a specific primary effect of morphine within the hippocampus in spite of the low numbers of opiate receptors in this brain region.     

Influences of sinusoidal electric fields on excitability in the rat hippocampal slice

The influence of extracellular sinusoidal electric fields on the amplitude of population spikes evoked by single test pulses in excitatory pathways to CA1 pyramidal neurons was studied in rat hippocampal slices. The fields in the tissue were of the order of EEG gradients. Stimulation at 5 Hz, a frequency representative of hippocampal theta activity, was compared with 60 Hz, which is often used in kindling procedures. Brief stimulation (5-30 s) with both 5 and 60 Hz fields (20-70 mV/cmp-p in the perfusing solution) often produced a long-term increase (longer than 10 min) of the population spike. Fields at 60 Hz, but not at 5 Hz, also induced short-term depression (1-6 min) or transient post-field excitation (15-30 s). Prolonged stimulation (3 min) emphasized this frequency dependent response: fields at 5 Hz induced long-lasting potentiation while fields at 60 Hz always resulted in progressive depression persisting for a few minutes after the end of stimulation. These effects appeared as a global response of CA1 neurons. Antidromic responses studied during blockade of synaptic transmission (0.2 mM Ca2+, 4 mM Mg2+) were depressed during and following 3 min field stimulation at either frequency, which could reflect failing calcium mechanisms in the tissue. The field influence on the potential evoked by synaptic or antidromic stimulation was independent of the phase of the sine wave at which the test pulse was delivered, arguing against a direct polarization of the cell membrane by the fields. The experimental evidence suggests a functional role for EEG-like fields in hippocampal excitability.     

Effects of tissue preincubation and hypoxia on CA3 hippocampal neurons in the in vitro slice preparation

Using the in vitro hippocampal slice preparation, we studied the electrophysiological properties of pyramidal cells in tissue that was 'preincubated' (2-6 h in a large, static volume of oxygenated bathing medium) before being placed in an interface chamber for study. Striking differences were found in 'preincubated' vs 'non-preincubated' CA3 cells. The preincubated cells had more negative resting potentials, higher input resistance, lower threshold for stimulus-evoked burst discharge and larger hyperpolarizing afterpotentials. Cells in the preincubated CA3 region were also more likely to show spontaneous synchronized burst discharge, but were relatively resistant to hypoxia-induced spreading depression. CA1 cells were less dramatically affected by preincubation, showing little difference from their non-preincubated counterparts. Possible mechanisms involved in the CA3 preincubation effect, including glial buffering alterations and changes in Na+, K+-ATPase activity, are discussed.     

Long-term potentiation in the hippocampal slice: Possible involvement of pyruvate dehydrogenase

Tetanic stimulation of fibers in the hippocampal slice preparation produces long-term potentiation (LTP) and also decreases the in vitro incorporation of phosphate into the alpha subunit of pyruvate dehydrogenase (αPDH)¹². This paper describes 6 experiments that were undertaken to replicate this observation. Hippocampal slices were incubated in a specially designed chamber and stimulated with a tungsten wire electrode in the stratum radiatum for 1 s at 100 Hz. Two minutes after the tetanus, the stimulated slices were removed alternately with control (not tetanized) slices and each group was pooled for subcellular fractionation and labeling of the fractions with [³²P]ATP. Proteins were separated by electrophoresis and relative³²P contents of 41K and 50K protein bands were studied. Tetanic stimulation of the stratum radiatum did not affect subsequent phosphorylation of a 50K M_r protein that has been reported to be altered by perforant path activation². Stimulation also had no effect on pyruvate dehydrogenase enzyme activity or on the ratio of active (dephosphorylated) to inactive enzyme. In most cases tetanic stimulation produced no significant change in the in vitro phosphorylation of this enzyme. Only under one set of conditions, labeling with 250 μM [γ-³²P] ATP for 10 s, was a decrease in the in vivo labeling of αPDH shown to be statistically significant. These findings suggest that LTP is not necessarily accompanied by an initial change in PDH phosphorylation level or activity but may be associated with a decrease in the kinase activity directed toward this protein.     

Phorbol ester alters the electrophysiological responses to hypoxia and ischemic-like conditions in the rat hippocampal slice

The effect of incubation with the protein kinase C activator, 4β-phorbol 12,13-dibutyrate (β-PDBu) on the electrophysiological responses to hypoxia and combined hypoxia and hypoglycemia was investigated in the rat hippocampal slice. Preincubation with β-PDBu prevents adenosine-mediated inhibition of synaptic transmission under normoxic, normoglycemic conditions. β-PDBu preincubation also reduces the adenosine-mediated hypoxia-induced depression of synaptic transmission revealing a substantial adenosine-independent hypoxia-induced depression of synaptic transmission. During combined hypoxia and hypoglycemia, slices preincubated in β-PDBu display a significant shortening of the time to anoxic depolarization, an effect of β-PDBu that is not mimicked by application of the adenosine antagonist cyclopentyltheophylline (8-CPT). It is concluded that the state of PKC activation may influence the electrophysiological responses to hypoxia and ischemia.     

Structure-function relationships for kynurenic acid analogues at excitatory pathways in the rat hippocampal slice

Eight kynurenic acid analogues were bath-applied to rat hippocampal slices while recording extracellular synaptic field potentials and the potencies of these analogues for inhibition of these responses were compared to that of kynurenic acid. Quinaldic acid, 4-hydroxyquinoline, 4-hydroxypicolinic acid, L-kynurenine and picolinic acid inhibited evoked field potentials, but were at least 15-fold less potent than kynurenic acid in all pathways tested. Xanthurenic acid was inactive in the pathways tested. Quinolinic acid and dipicolinic acid showed signs of agonist activity with IC50's of approx. 400 microM and 2500 microM, respectively. These studies show that the 2-carboxy group and the 4-hydroxy moiety are essential for the antagonist activity exhibited by kynurenate. They also show that the unsubstituted second aromatic ring greatly enhances the affinity of kynurenate for these receptors and that substitution in at least one position on this aromatic ring abolishes activity.     

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.     

Inhibition of GABA uptake in the rat hippocampal slice

Pharmacological manipulations known to inhibit GABA uptake prolonged GABA-evoked conductance increases in CA1 pyramidal cells in the rat hippocampal slice preparation. Treatments included reduction of extracellular sodium and exposure to cis-4-OH-nipecotic acid, nipecotic acid or L-2,4-diaminobutyric acid (all at 1 mM). These effects contrast with the results obtained with 4-OH-isonipecotic acid, an inactive structural analog of nipecotic acid, which had no effect on the time-course of GABA responses. 4,5,6,7-Tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THPO), an impotent but selective inhibitor of GABA uptake into glia, did not prolong GABA-evoked responses. The effect of sodium reduction depended on the distance between the source of GABA and its receptors, as predicted for an uptake-limited response. GABA-receptor agonists that are poor substrates for GABA uptake (muscimol, thiomuscimol, piperidine-4-sulphonic acid, isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP) evoked very long conductance changes that were not further prolonged by uptake inhibitors. These results demonstrate the presence of a functional GABA uptake system in the hippocampal slice. The accessibility of hippocampal GABAergic synapses and the known susceptibility of the hippocampus to epileptiform events suggest that the hippocampal slice could be a valuable CNS preparation to study the role of GABA uptake in synaptic physiology.     

Effects of serotonin on extracellular potassium concentration in the rat hippocampal slice

The effects of serotonin (5-HT) on extracellular potassium concentration ([K⁺]₀) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2–4 mM rise in [K⁺]₀ in the stratum pyramidale of area CA1. 5-HT caused a 0.6–1.1 mM rise in [K⁺]₀. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K⁺]₀ was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K⁺]₀ changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale.GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K⁺]₀ in stratum pyramidale. Extracellular calcium concentration measured with a Ca²⁺-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K⁺]₀.     

Excitatory effects of low-level lead exposure on action potential firing of pyramidal neurons in CA1 region of rat hippocampal slices

Lead is putatively regarded as an environmental neurotoxicant. Long-term low-level lead exposure causes cognitive deficits, but the mechanism remains to be elucidated. In the present study, the excitatory effects of low-level lead exposure on action potential (AP) firing of pyramidal neurons in CA1 region of rat hippocampal slices and the pathway through which lead induced these effects were studied with conventional whole-cell recording. Low-level lead (0.5 and 5 microM) exposure did not significantly change either voltage threshold or amplitude, duration, rise time, or rising velocity of single AP; conversely, 5 microM lead exposure significantly increased AP firing rates and reduced spike frequency adaptation. These excitatory effects of 5 microM lead were blocked by mibefradil, a selective blocker of T-type voltage-dependent calcium channels (VDCC), but not by verapamil and omega-conotoxin, selective blockers of L-type and N-type VDCC, respectively. Five micromolar lead could not change the excitability of pyramidal neurons when slices were perfused with calcium-free ACSF. In addition, the effects were abolished by inhibitors of two intracellular calcium release channels: 2-APB, an inhibitor of inositol trisphosphate receptors, and dantrolene, an inhibitor of ryanodine receptors, but not by thapsigargin, an inhibitor of endoplasmic reticulum calcium uptake. These results provide evidence for excitatory neurotoxicity of low-level lead exposure, contribution of T-type VDCC in the entrance of lead into neurons, and a possible involvement of calcium flux alteration during APs in this excitatory neurotoxicity.     

Metabolic changes induced in rat hippocampal slices by norepinephrine

The oxidative metabolic activity of restricted regions of hippocampal slices was assessed by a continuous measurement of the fluorescence of intramitochondrial nicotinamide-adenine dinucleotide (NADH). A large increase in NADH fluorescence was triggered by substituting the oxygen supply to the slice by nitrogen gas. A large and transient increase in NADH fluorescence was also produced by superfusion of the the slice with a high (50 mM) potassium-containing medium. Addition of norepinephrine (NE) to the superfusion medium caused a propranolol-inhibited increase in NADH fluorescence. Furthermore, ouabain, which inhibits the Na-K pump, blocked the effects of NE. An analog of cyclic adenosine monophosphate (cAMP), 8-bromo cAMP, mimicked the effect of NE. Finally, effects of NE could still be produced in a kainic acid-treated hippocampus, where most neurons were previously destroyed by the drug. It is suggested that NE activates a Na-K-ATPase, that this effect might be mediated by cAMP, and that these interrelations may underly the physiological action of NE in the brain.     

Perfusion with lithium modifies neurophysiological responses in the CA1 region of the hippocampal slice preparation

The effects of acute lithium exposure on extracellular electrophysiological responses in the CA1 region of the in vitro hippocampus were investigated. Field potentials were assessed while perfusing slices with normal media or media in which LiCl was substituted for NaCl in 30, 20, 10 and 2 mM amounts. Lithium concentration in the slice following 20 min perfusion with 20 mM lithium was determined to be about 14 mM. At the higher concentrations, lithium exposure depressed the presynaptic fiber volley and antidromic population spike. On the other hand, the population EPSP and orthodromic population spike were enhanced. No significant changes were found at 2 mM. The findings are compatible with one action of lithium being on the excitability of axons and synaptic terminals. Comparisons were drawn between previous studies involving chronic lithium exposure and the present results. In this acute preparation lithium effects, as reflected in the population EPSP, were in opposition to those found with chronic lithium exposure. Changes demonstrated in this preparation in fiber volley and antidromic population spike paralleled those found with chronic lithium exposure.     

Magnesium-free medium activates seizure-like events in the rat hippocampal slice

The effect of magnesium-free medium on the electrical activity in CA3 of the rat hippocampal slice was examined. Magnesium removal resulted in the development of spontaneous and triggered interictal-like bursting, followed by spontaneous ictal-like events and finally periodic clustered bursts. The ictal-like events consisted of a tonic firing phase and a phase of clustered burst discharges resembling the tonic and clonic phases of seizures. The return to normal medium resulted in spontaneous and triggered interictal-like bursts.     

Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice

This study examined anatomical correlates of: (1) long-term potentiation (LTP); (2) equivalent low frequency synaptic activity; (3) continuous high frequency synaptic activation which did not produce LTP; and (4) synaptic inactivation by high Mg2+/low Ca2+ incubation in hippocampal subfield CA1 in the in vitro slice, and examined the persistence of changes at 10-15 min, 2 h and 8 h after stimulation. After potentiating stimulation (6 trains at 100 Hz for 1 s or 200 Hz for 0.5 s), compared to an equivalent number of low frequency stimuli (1 Hz for 600 s), there were increases in numbers of shaft and sessile spine synapses (synapses on stubby, headless spines). This suggested an increase in the number of shaft synapses onto inhibitory interneurons and/or an enhancement of synapse formation on pyramidal neurons possibly involving initial formation of shaft synapses and a transition from shaft, to sessile spine, to full grown spine synapses. Postsynaptic spine heads also assumed a rounder shape, as indicated by decreases in spine perimeter to area ratios, contact lengths, and the percentage of 'cup' shaped spines. There was no effect of potentiating stimulation on bouton or spine areas. After continuous high frequency synaptic activation (40 Hz or 100 Hz for 10 min), which produced no apparent LTP, there were no changes in synapse numbers or spine head shape parameters. However, in contrast to effects of LTP, there was an increase in bouton mitochondrial area and a marginal increase in bouton area compared to the low frequency condition. Inactivation did not affect any of these measures. LTP-associated increases in numbers of shaft and sessile spine synapses persisted over an 8 h incubation period, while the effect on spine shape disappeared after 2 h. Physiologically-demonstrable LTP persisted over the 8 h period. Effects of continuous high level activation on mitochondrial and bouton areas were even more transient, disappearing 2 h after stimulation. These findings: (1) confirm previously reported effects of potentiating stimulation on synapse numbers and spine shape; (2) indicate that spine shape changes are not necessary for the maintenance of LTP; and (3) indicate that continuous high frequency activation which does not produce potentiation has different and non-persisting effects from potentiating stimulation.     

Inhibition of hexokinase leads to neuroprotection against excitotoxicity in organotypic hippocampal slice culture

During seizures, glucose concentrations are high in the hippocampus. Mitochondrial hexokinase (HK) catalyzes the first essential step of glucose metabolism and directly couples extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation. The neuroprotective effects of an HK inhibitor, 3-bromopyruvate (3-BrPA), on kainic acid (KA)-induced excitotoxic injury were investigated. Hippocampal slices were prepared from hippocampi of 6-8-day-old rats using a tissue chopper and placed on a membrane insert. After a treatment with KA (5 μM) for 15 hr, neuronal death was quantified by propidium iodide (PI), cresol violet, and TUNEL staining. KA-induced cell death was significantly prevented by 30 μM 3-BrPA treatment. According to Western blots, the expression level of phospho-Akt increased after 3-BrPA treatment. The induction of long-term potentiation (LTP) at 48 hr after 3-BrPA treatment tended to increase in the CA1 area compared with the KA-only group, but the difference was not significant. Blocking the PI3 kinase/Akt pathway using LY294002 reversed the neuroprotective effect of 3-BrPA. These results suggest that inhibition of HK may play a protective role against neuronal death in KA-induced excitotoxic injury.     

Multiple action of acetylcholine at a muscarinic receptor studied in the rat hippocampal slice

Responses of hippocampal pyramidal cells to topical application of acetylcholine (ACh) were measured in the in vitro hippocampal slice preparation. ACh but not cyclic GMP produced a short-latency hyperpolarization associated with a decrease in input resistance. This was followed by a long-latency but long duration depolarization associated in some cells with an increase in input resistance. This change in resistance followed the depolarization and outlasted it by 5--20 min, until complete recovery. During the depolarization there was a reduction in magnitude of EPSPs produced by activation of the Schaffer collateral excitatory afferents. The reversal potential for the hyperpolarization was about -95 mV, and it was blocked by 4-aminopyridine. The depolarization, but not the hyperpolarization was markedly attenuated in slices maintained in low (25 degrees C) temperature. The responses to ACh were blocked by atropine but not by D-tubocurarine. The hyperpolarization as well as the depolarization were present in slices treated with tetrodotoxin (TTX)., but were reduced in slices superfused with a low Ca2+-high Mg2+ medium, and in slices treated with Mn2+ and Co2+ ions. It is suggested that ACh causes a fast increase in gK+, followed by a long-lasting energy-dependent depolarization associated with action potential discharges, a decrease in conductance and a suppression of EPSPs.     

NMDA receptor-independent LTP in basal versus apical dendrites of CA1 pyramidal cells in rat hippocampal slice

The ability of hippocampal CA1 basal synapses to express N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (non-NMDA LTP) was studied and compared to the simultaneously induced apical dendritic non-NMDA LTP. Non-NMDA LTP in basal and apical dendrites was induced using stimulation pattern similar to the sharp wave-associated CA3 bursts. Basal dendritic non-NMDA LTP was input-specific and displayed similar development and magnitude to the apical dendritic non-NMDA LTP. Both apical and basal dendritic non-NMDA potentiations were inhibited by the voltage-dependent calcium channel (VDCC) inhibitor verapamil and the tyrosine kinase inhibitors genistein and levandustin A. However, the difference in the degree and time course of these inhibitions suggests involvement of distinct mechanisms in the two dendritic subfields. Hippocampus 1998;8:373–379. © 1998 Wiley-Liss, Inc.     

Anesthetic protection against anoxic damage in the rat hippocampal slice

Evoked population spikes were recorded from the dentate granule cell layer of hippocampal slices obtained from adult rats. These slices were subjected to short periods of anoxia in the presence of different anesthetics. The recovery of the population spike after anoxia was compared across treatments. Little or no recovery was found after 10 min of anoxia when no anesthetic (4 +/- 4%), 1.5% isoflurane (5 +/- 5%), or 15% isoflurane (0 +/- 0%) was present during the anoxic periods. However, the population spike did recover to 81 +/- 7% of its preanoxic amplitude within 1 h after the anoxia if thiopental 160 mg/liter was present in the perfusate during the anoxia. Fifteen percent isoflurane and 160 mg/liter thiopental were equipotent in reducing the amplitude of the evoked population spike before anoxia but only thiopental protected against the damage after 10 min of anoxia. Our results suggest that the blocking of the evoked population spike by thiopental is not the sole mechanism of its protection against anoxic damage. Isoflurane (1.5%) was able to provide a small degree of protection against shorter periods (7 min) of anoxia.