Different responses of CA1 and CA3 regions to hypoxia in rat hippocampal slice

1. To study the effects of brief periods of hypoxia on cellular functions in the rat hippocampal slice, extracellular and intracellular recordings were made from pyramidal neurons, and interstitial potassium activity ([K+]o) was measured in the pyramidal cell layers. Slices were perfused in an interface chamber at 36-37 degrees C with medium containing 8.5 mM [K+]o. Hypoxia was induced by switching the overflow gas from O2-CO2 to N2-CO2. 2. Brief periods of hypoxia (5-60 s) produced electrographic seizures with typical tonic and clonic components in 53% of 293 slices that generated spontaneous interictal bursts. Hypoxia-induced seizures were usually initiated in and restricted to the Ca1 region; only 2.5% of these slices generated seizures in CA3. In contrast to the CA1 region, the CA3 region could undergo spreading depression during hypoxia. The probability of seizure generation in CA1 was increased with increasing duration of hypoxia and was greatly reduced by lowering the bath temperature a few degrees. 3. [K+]o gradually increased in the CA1 and CA3 cell layers during the 20 s leading up to an hypoxia-induced seizure. [K+]o rose to approximately 9.8 mM (from a base line of 8.5 mM) in CA1 just before a seizure and to 11.4 mM during the seizure. After hypoxia, [K+]o reached a higher level in CA1 than in CA3, regardless of whether 1 microM tetrodotoxin was present to eliminate differences in cell firing in the two regions. CA1 pyramidal cells and glia gradually depolarized by several millivolts during and after hypoxia; no initial hyperpolarizing phase was detected. 4. Burst input from CA3 was necessary for hypoxia-induced seizures. The frequency and intensity of spontaneous burst-firing in CA3 remained steady in the period leading up to a CA1 seizure episode. In contrast, the intensity of synaptically driven bursts in CA1 grew markedly just before seizure onset. N-methyl-D-aspartate (NMDA) receptors participated in the crescendo of increasingly synchronous activity in CA1, because the competitive NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV, 30 microM), stereoselectively reduced seizure intensity. 5. Hypoxia-induced seizures were followed by a depressant phase, which was manifested most prominently by a prolonged (up to several minutes) reduction in the frequency and intensity of burst-firing in the CA3 region, hyperpolarization of CA1 neurons, and undershoot of [K+]o. In normal (3.5 mM) [K+]o, synaptically driven population spikes in CA1 were only reduced in amplitude by hypoxia; hypoxia did not induce seizures in 3.5 mM [K+]o.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epileptiform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition

Intracellular and extracellular recordings were made from pyramidal neurons in hippocampal slices in order to study spontaneous paroxysmal bursting induced by raising the extracellular potassium concentration from 3.5 to 8.5 mM. Extracellular recordings from all hippocampal subfields indicated that spontaneous bursts appeared to originate in region CA3c or CA3b as judged by burst onset. Burst intensity was also greatest in regions CA3b and CA3c and became progressively less toward region CA2. Intracellular recordings indicated that in 8.5 mM potassium, large spontaneous excitatory postsynaptic potentials (EPSPs), large burst afterhyperpolarizations, and rhythmic hyperpolarizing-depolarizing waves of membrane potential were invariably present in CA3c neurons. High potassium (8.5 mM) induced a positive shift (+9 mV) in the reversal potential of GABAergic inhibitory postsynaptic potentials (IPSPs) in CA3c neurons without changing input resistance or resting potential. This resulted in a drastic reduction in amplitude of the IPSP. Reduction of IPSP amplitude occurred before the onset of spontaneous bursting and was reversible upon return to normal potassium. A new technique to quantify the relative intensity of interictal-like burst discharges is described. Pentobarbital, diazepam, and GABA uptake inhibitors, which enhance GABA-mediated synaptic inhibition, reduced the intensity of potassium-induced bursts, whereas the GABA antagonist bicuculline increased burst intensity. Diphenylhydantoin and phenobarbital, anticonvulsants that have little effect on GABAergic inhibition, were without effect on spontaneous bursts. Burst frequency was reduced by bicuculline and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol but was unaffected by other drugs. Reduction of slice temperature from 35 to 19 degrees C dramatically reduced burst intensity but did not markedly affect burst frequency. We hypothesize that high potassium induces a rise in intracellular chloride concentration, possibly by activating an inward KCl pump or by a passive Donnan effect, which results in a decreased IPSP amplitude. With inhibition suppressed, the large spontaneous EPSPs that appear in high potassium cause individual CA3c neurons to fire. A combination of synaptic and electrical interactions among CA3c cells then synchronizes discharges into interictal spike bursts.     

Activity-evoked increases in extracellular potassium modulate presynaptic excitability in the CA1 region of the hippocampus

1. The effects of stimulus-evoked potassium release on the excitability of presynaptic axons were studied in the rat hippocampal slice preparation. Extracellular stimulation and recording in the stratum radiatum of CA1 yielded a characteristic field potential corresponding to the compound action potential of nonmyelinated afferents and subsequent postsynaptic activation of pyramidal cells. 2. Repetitive stimulation (1 s; 2-100 Hz) produced biphasic changes in the excitability of the afferents. Initial responses showed increased conduction velocity and variably increased amplitude; subsequent responses showed progressively decreasing conduction velocity and amplitude tending toward conduction block. Decreases in excitability were maximal at the end of stimulation and were more pronounced with higher stimulation frequencies. 3. When synaptic transmission was abolished with superfusate containing elevated [Mg2+] (6 mM) and decreased [Ca2+] (0.25 mM), kynurenic acid (1 mM), or adenosine (100 microM), the ability of the fibers to follow repetitive stimulation was enhanced, as indicated by a reduction in amplitude decrement of the presynaptic volley. The decrease in conduction velocity at the end of stimulation was less than half that obtained with intact postsynaptic activity. 4. Concomitant with changes in the excitability of CA1 afferents, the concentration of extracellular potassium ( [K+]o) increased up to 7 mM, as recorded in the stratum radiatum with potassium ion-sensitive microelectrodes. When postsynaptic activity was blocked, activity-evoked rises in [K+]o were reduced to less than 25% of their former value. This suggests that activity-evoked increases in [K+]o derive predominantly from postsynaptic elements. 5. Superfusion of solutions containing elevated [K+] produced biphasic changes in the excitability of CA1 afferents that were qualitatively similar to those produced by repetitive stimulation. Elevated [K+]o below 6 mM produced increased excitability, whereas [K+]o above 6 mM yielded decreased excitability. 6. These results demonstrate that in the CA1 region of the hippocampus, significant rises in [K+]o occur with activity and derive predominantly from postsynaptic elements. The conduction properties of CA1 afferents are sensitive to the level of [K+]o, whether altered artificially or by activity. These effects may constitute a mechanism of postsynaptic modulation of presynaptic conduction operating within a broad range of afferent firing frequencies in the hippocampus.     

Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice

Antagonism of the chloride-cotransport system in hippocampal slices has been shown to block spontaneous epileptiform (i.e., hypersynchronized) discharges without diminishing excitatory synaptic transmission. Here we test the hypotheses that chloride-cotransport blockade, with furosemide or low-chloride (low-[Cl(-)](o)) medium, desynchronizes the firing activity of neuronal populations and that this desynchronization is mediated through nonsynaptic mechanisms. Spontaneous epileptiform discharges were recorded from the CA1 and CA3 cell body layers of hippocampal slices. Treatment with low-[Cl(-)](o) medium led to cessation of spontaneous synchronized bursting in CA1 >/=5-10 min before its disappearance from CA3. During the time that CA3 continued to burst spontaneously but CA1 was silent, electrical stimulation of the Schaffer collaterals showed that hyperexcited CA1 synaptic responses were maintained. Paired intracellular recordings from CA1 pyramidal cells showed that during low-[Cl(-)](o) treatment, the timing of action potential discharges became desynchronized; desynchronization was identified with phase lags in firing times of action potentials between pairs of neurons as well as a with a broadening and diminution of the CA1 field amplitude. Continued exposure to low-[Cl(-)](o) medium increased the degree of the firing-time phase shifts between pairs of CA1 pyramidal cells until the epileptiform CA1 field potential was abolished completely. Intracellular recordings during 4-aminopyridine (4-AP) treatment showed that prolonged low-[Cl(-)](o) exposure did not diminish the frequency or amplitude of spontaneous postsynaptic potentials. CA3 antidromic responses to Schaffer collateral stimulation were not significantly affected by prolonged low-[Cl(-)](o) exposure. In contrast to CA1, paired intracellular recordings from CA3 pyramidal cells showed that chloride-cotransport blockade did not cause a significant desynchronization of action potential firing times in the CA3 subregion at the time that CA1 synchronous discharge was blocked but did reduce the number of action potentials associated with CA3 burst discharges. These data support our hypothesis that the anti-epileptic effects of chloride-cotransport antagonism in CA1 are mediated through the desynchronization of population activity. We hypothesize that interference with Na(+),K(+),2Cl(-) cotransport results in an increase in extracellular potassium ([K(+)](o)) that reduces the number of action potentials that are able to invade axonal arborizations and varicosities in all hippocampal subregions. This reduced efficacy of presynaptic action potential propagation ultimately leads to a reduction of synaptic drive and a desynchronization of the firing of CA1 pyramidal cells.     

Chronic epileptic foci in vitro in hippocampal slices from rats with the tetanus toxin epileptic syndrome

1. Minute doses of tetanus toxin were injected into the hippocampi of rats, under pentobarbitone anesthesia, to induce a chronic experimental epilepsy. The effects of this treatment were studied in vitro in hippocampal slices prepared 1-60 days after injection. 2. Epileptic activity was preserved in these slices in vitro, closely resembling that seen in vivo. Epileptiform afterdischarges were evoked by stimulation after survival times of greater than or equal to 3 days from injection. Spontaneous synchronous epileptic discharges were recorded from 7 days after injection. Both kinds of epileptiform activity were found with survival times up to 36 days but not beyond 44 days. This time course resembles the waxing and waning of the epileptic syndrome in vivo. 3. Two distinct types of spontaneous burst were seen. The first was a simple burst lasting 100-300 ms, reminiscent of the "interictal spike" of the clinical electroencephalogram. The second was much more prolonged, lasting several seconds. It consisted of a simple burst followed by a series of discrete afterbursts at 3-6/s and resembled the early stages of an epileptic seizure. Both types of burst were associated with slow field potentials that were positive at the cell-body layer. 4. Both the interictal and the seizure-like spontaneous epileptic discharges originated in the CA3b/c pyramidal cell region and propagated at 0.1-0.25 m/s along the cell layer toward the CA1 region. They occurred at very variable intervals, ranging from 20 s to 30 min. 5. Spontaneous epileptic bursts occurred in media containing 3 mM [K+]o to 5 mM in one-third of experiments during the period 1-4 wk after injection. Spontaneous bursts could be induced by increasing [K+]o to 5 mM in two-thirds of the remaining slices, which initially had produced evoked afterdischarges. 6. Intracellular recordings revealed that spontaneous field bursts were invariably associated with paroxysmal depolarization shifts (PDSs) and bursts of action potentials, suggesting that almost all the pyramidal cells in the region were recruited into the epileptic discharges. In some cells, smaller abnormal depolarizations were also seen; they were clearly larger than the spontaneous synaptic potentials but were not associated with field potentials. They may have been due to a more limited recruitment of pyramidal cells into partially synchronous bursts. 7. The tetanus toxin experimental epileptic syndrome differs from chronic models described previously in retaining in the hippocampal slice in vitro much of the spontaneous epileptic activity seen in vivo in the freely moving chronically epileptic rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. II. Role of extracellular potassium

The role of extracellular K+ (K+o) in nonsynaptic epileptogenesis induced in the CA1 area of rat hippocampal slices by lowering [Ca2]o was studied with K+-selective microelectrodes (KSMs). Extracellular field potentials and [K+]o were recorded simultaneously with 1-2 KSMs in the CA1 stratum pyramidale. In slices perfused with an oxygenated standard physiological solution (containing 2 mM Ca2+), base-line [K+]o was stable for several hours. The washout of Ca2+o was accompanied by a gradual tonic rise of [K+]o. Spontaneous and stimulus-evoked maximal seizurelike events (SLEs) appeared when [K+]o was approximately 0.5 mM above the initial 5 mM base line. These changes were reversible in normal medium. When K+o was pressure ejected in the CA1 stratum pyramidale of spontaneously active slices, a local rise in [K+]o of approximately 0.5 mM was necessary to trigger a SLE. A similar apparent [K+]o "threshold" was associated with SLEs evoked by electrical stimulation. Increasing [K+] in the perfusing solution by small increments (1 mM) markedly enhanced SLEs frequency and velocity of spread and decreased the period of absolute refractoriness that succeeded each paroxysm. Similar changes occurred during periods of transient hypoxia. Small [K+] decreases in the perfusate had the converse effects. Spontaneous SLEs were associated with phasic increases in [K+]o. In simultaneous [K+]o recordings from two layers, these transients were largest (up to 3.5 mM above base line) and rose more steeply at the stratum pyramidale. Toward the outer dendritic layers they became smaller, slower in time course, and delayed in onset. We conclude that the main source for these [K+]o transients are the hippocampal pyramidal cell bodies, which discharge intensely during a SLE, and that excess K+o is spatially dispersed around the discharge zone of the paroxysm. [K+]o continued to rise, though at a slower rate, throughout the course of a SLE. Following SLE termination, [K+]o decayed slowly to base line. The invasion of a CA1 region by a propagating SLE was preceded quite often by a slow rise in [K+]o. A sudden transition to a steeply rising [K+]o marked the explosive recruitment of this region into the discharge zone of the spreading paroxysm. The total (tonic and phasic) increase in [K+]o during SLEs did not surpass a maximal level of approximately 9 mM, which was the ceiling level of [K+]o in low [Ca2+]o. However, when spreading depression occurred, [K+]o rose up to 30-40 mM for several minutes. Spreading depression rarely appeared spontaneously despite the recurrence of SLEs, but could be provoked by repetitive electrical stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epileptiform activity induced by low chloride medium in the CA1 subfield of the hippocampal slice

1. Extracellular and intracellular recordings and measurements of the extracellular concentration of free K+ ([K+]o) were performed in the CA1 subfield of the rat hippocampal slice during perfusion with artificial cerebrospinal fluid (ACSF) in which NaCl had been replaced with equimolar Na-isethionate or Na-methylsulfate (hereafter called low Cl- ACSF). 2. CAl pyramidal cells perfused with low Cl- ACSF generated intracellular epileptiform potentials in response to orthodromic, single-shock stimuli delivered in stratum (S.) radiatum. Low-intensity stimuli evoked a short-lasting epileptiform burst (SB) of action potentials that lasted 40-150 ms and was followed by a prolonged hyperpolarization. When the stimulus strength was increased, a long-lasting epileptiform burst (LB) appeared; it had a duration of 4-15 s and consisted of an early discharge of action potentials similar to the SB, followed by a prolonged, large-amplitude depolarizing plateau. The refractory period of the LB was longer than 20 s. SB and LB were also seen after stimulation of the alveus. 3. Variations of the membrane potential with injection of steady. DC current modified the shape of SB and LB. When microelectrodes filled with the lidocaine derivative QX-314 were used, the amplitudes of both SB and LB increased in a linear fashion during changes of the baseline membrane potential in the hyperpolarizing direction. The membrane input resistance, as measured by injecting brief square pulses of hyperpolarizing current, decreased by 65-80% during the long-lasting depolarizing plateau of LB. 4. A synchronous field potential and a transient increase in [K+]o accompanied the epileptiform responses. The extracellular counterpart of the SB was a burst of three to six population spikes and a small increase in [K+]o (less than or equal to 2 mM from a resting value of approximately 2.5 mM). The LB was associated with a large-amplitude, biphasic, negative field potential and a large increase in [K+]o (up to 12.4 mM above the resting value). Changes in [K+]o during the LB were largest at the border between S. oriens and S. pyramidale. This was also the site where the field potentials measured 2-5 s after the stimulus attained their maximal amplitude. Conversely, field potentials associated with the early component of the LB or with the SB displayed a maximal amplitude in the S. radiatum. 5. Spontaneous SBs and LBs were at times recorded in the CA1 and in the CA3 subfield.(ABSTRACT TRUNCATED AT 400 WORDS)     

K-dependent inhibition in the dentate-CA3 network of guinea pig hippocampal slices.

1. The occurrence of potassium-dependent inhibitory postsynaptic potentials (K-IPSPs) in relation to burst discharges induced by 4-aminopyridine (4-AP; 30 microM) was studied in CA3, granule and hilar neurons in guinea pig hippocampal slices with the use of paired extra- and/or intracellular recording. 2. Slow small (2-5 mV) and large (up to 30 mV) K-IPSPs were observed in CA3, granule and in some hilar neurons during 4-AP applications in the presence of blockers for fast synaptic transmission, picrotoxin (50 microM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5-10 microM). Amplitudes of K-IPSPs were linearly related to voltage, and they reversed in sign close to -100 mV, as expected for synaptic potentials generated by an increase in K-conductance. 3. In CA3 neurons, 4-AP applied in the presence of picrotoxin elicited burst discharges and K-IPSPs. CNQX blocked the burst discharge activity and increased the amplitude of K-IPSPs. 4. In granule cells, 4-AP applied in the presence of picrotoxin elicited K-IPSPs and only inconsistently small excitatory postsynaptic potentials (EPSPs). The EPSPs were blocked by CNQX, but CNQX application did not affect the K-IPSPs. However, in granule cells it could be observed that blockade of Cl-inhibition by picrotoxin in the presence of CNQX increased the amplitude of K-IPSPs. 5. In hilar neurons, 4-AP applied in the presence of picrotoxin elicited mainly burst discharges. CNQX blocked the burst discharges only in a few cells. In most hilar neurons K-IPSPs were observed at the beginning of the 4-AP effect, but subsequently K-IPSPs were replaced by burst discharges. 6. To determine the type of cells that burst in picrotoxin and 4-AP, neurons were stained intracellularly with horseradish peroxidase. Neurons stained in the granule cell layer did not burst and were morphologically identified as granule cells. Neurons stained in the hilar region burst and were nonpyramidal, nongranule cells. Bursting cells stained in the CA3 area were all pyramidal cells. 7. The hilar neurons varied considerably in size and dendritic organization. They could be classified as aspiny and spiny cells, the latter including mossy cells. 8. We conclude that K-dependent inhibition may explain the long-lasting IPSPs observed in in vivo recordings from hippocampal cells. In a hippocampal lamella, burst discharge activity of hilar neurons including presumed excitatory mossy cells is associated with inhibition of granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low-magnesium epilepsy in rat hippocampal slices: inhibitory postsynaptic potentials in the CA1 subfield

Spontaneous, synchronous epileptiform discharges were recorded in both CA3 and CA1 subfields of rat hippocampal slices perfused with Mg2+-free medium. Surgical separation of the two areas abolished the spontaneous discharges only in the CA1 subfield. However, epileptiform responses in the isolated CA1 subfield could still be evoked by orthodromic stimulation. Intracellularly these stimulus-induced responses were characterized by a depolarization associated with a burst of action potentials. Stimulation of the alveus still evoked a hyperpolarizing potential, presumably a recurrent inhibitory postsynaptic potential (IPSP) in CA1 pyramidal cells. Both spontaneous and stimulus-induced epileptiform discharges were blocked by the selective antagonist of N-methyl-D-aspartate (NMDA) receptors DL-2-amino-phosphonovalerate (APV). APV also reduced the amplitude and duration of the IPSP induced by alveus stimulation. Thus, epileptiform discharges evoked by lowering Mg2+ in the CA1 subfield are associated with a preservation of inhibitory mechanisms. Furthermore the effects exerted by APV upon the IPSP implicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.     

Mechanisms of neuronal hyperexcitability caused by partial inhibition of Na+-K+-ATPases in the rat CA1 hippocampal region

Extra- and intracellular records were made from rat acute hippocampal slices to examine the effects of partial inhibition of Na(+)-K(+)-ATPases (Na(+)-K(+) pumps) on neuronal hyperexcitability. Bath application of the low-affinity cardiac glycoside, dihydroouabain (DHO), reversibly induced interictal-like epileptiform bursting activity in the CA1 region. Burst-firing was correlated with inhibition of the pumps, which was assayed by changes in [K(+)](o) uptake rates measured with K(+)-ion-sensitive microelectrodes. Large increases in resting [K(+)](o) did not occur. DHO induced a transient depolarization (5-6 mV) followed by a long-lasting hyperpolarization (approximately 6 mV) in CA1 pyramidal neurons, which was accompanied by a 30% decrease in resting input resistance. Block of an electrogenic pump current could explain the depolarization but not the hyperpolarization of the membrane. Increasing [K(+)](o) from 3 to 5.5 mM minimized these transient shifts in passive membrane properties without preventing DHO-induced hyperexcitability. DHO decreased synaptic transmission, but increased the coupling between excitatory postsynaptic potentials and spike firing (E-S coupling). Monosynaptic inhibitory postsynaptic potential (IPSP) amplitudes declined to approximately 25% of control at the peak of bursting activity; however, miniature TTX-resistant inhibitory postsynaptic current amplitudes were unaffected. DHO also reduced the initial slope of the intracellular excitatory postsynaptic potential (EPSP) to approximately 40% of control. The conductances of pharmacologically isolated IPSPs and EPSPs in high-Ca/high-Mg-containing saline were also reduced by DHO by approximately 50%. The extracellular fiber volley amplitude was reduced by 15-20%, suggesting that the decrease in neurotransmission was partly due to a reduction in presynaptic fiber excitability. DHO enhanced a late depolarizing potential that was superimposed on the EPSP and could obscure it. This potential was not blocked by antagonists of NMDA receptors, and blockade of NMDA, mGlu, or GABA(A) receptors did not affect burst firing. The late depolarizing component enabled the pyramidal cells to reach spike threshold without changing the actual voltage threshold for firing. We conclude that reduced GABAergic potentials and enhanced E-S coupling are the primary mechanisms underlying the hyperexcitability associated with impaired Na(+)-K(+) pump activity.     

Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons

The effects of ischemia were examined on CA3 pyramidal neurons recorded in hippocampal slices 2-4 mo after a global forebrain insult. With intracellular recordings, CA3 post-ischemic neurons had a more depolarized resting membrane potential but no change of the input resistance, spike threshold and amplitude, fast and slow afterhyperpolarization (AHP) or ADP, and firing properties in response to depolarizing pulses. With both field and whole-cell recordings, synaptic responses were similar in control and post-ischemic neurons. Although there were no spontaneous network-driven discharges, the post-ischemic synaptic network had a smaller threshold to generate evoked and spontaneous synchronized burst discharges. Thus lower concentrations of convulsive agents (kainate, high K(+)) triggered all-or-none network-driven synaptic events in post-ischemic neurons more readily than in control ones. Also, paired-pulse protocol generates, in post-ischemics but not controls, synchronized field burst discharges when interpulse intervals ranged from 60 to 100 ms. In conclusion, 2-4 mo after the insult, the post-ischemic CA3 pyramidal cells are permanently depolarized and have a reduced threshold to generate synchronized bursts. This may explain some neuropathological and behavioral consequences of ischemia as epileptic syndromes observed several months to several years after the ischemic insult.     

Long-term change in synaptic transmission in CA3 circuits followed by spontaneous rhythmic activity in rat hippocampal slices

The relevance of long-term potentiation (LTP) at excitatory synapses in CA3 circuits to generation of spontaneous epileptiform bursts in CA3 was investigated using rat hippocampal slices. CA3 pyramidal cells were antidromically stimulated through Schaffer collaterals. Evoked field potentials were extracellularly recorded from the stratum pyramidale and the stratum radiatum in CA3. Therefore, field potentials reflecting recurrent excitatory post-synaptic potentials (EPSPs) and inhibitory post-synaptic potentials (IPSPs) were positive at the stratum pyramidale and negative at the stratum radiatum. First, we tested how the amplitude of the evoked field potentials depends on a γ-aminobutyric acid (GABA_A) antagonist. Both of the positive and negative field potential peaks reduced in the medium containing penicillin (2 mM) or bicuculline (20 μM). This suggests that unmasked EPSPs due to suppression of IPSPs do not result in an increase in the evoked potentials. Second, CA3 pyramidal cells were antidromically stimulated by tetanic stimulation of Schaffer collaterals in order to induce LTP at synapses in CA3 circuits. Both of the positive and negative field potentials increased, suggesting that recurrent EPSPs were enhanced by tetanic stimulation. Induction of LTP at recurrent excitatory synapses was followed by spontaneous epileptiform bursts which persisted throughout experiments (1.5 h), while LTP of afferent synaptic potential evoked by hilar test stimulation was not induced. These results suggest that LTP at the afferent synapses is not necessary to spontaneous epileptiform bursts in CA3, but LTP at excitatory synapses between CA3 pyramidal cells contribute to spontaneous epileptiform bursts.     

Increasing extracellular potassium results in subthalamic neuron activity resembling that seen in a 6-hydroxydopamine lesion

Abnormal neuronal activity in the subthalamic nucleus (STN) plays a crucial role in the pathophysiology of Parkinson's disease (PD). Although altered extracellular potassium concentration ([K+]o) and sensitivity to [K+]o modulates neuronal activity, little is known about the potassium balance in the healthy and diseased STN. In vivo measurements of [K+]o using ion-selective electrodes demonstrated a twofold increase in the decay time constant of lesion-induced [K+]o transients in the STN of adult Wistar rats with a unilateral 6-hydroxydopamine (6-OHDA) median forebrain bundle lesion, employed as a model of PD, compared with nonlesioned rats. Various [K+]o concentrations (1.5-12.5 mM) were applied to in vitro slice preparations of three experimental groups of STN slices from nonlesioned control rats, ipsilateral hemispheres, and contralateral hemispheres of lesioned rats. The majority of STN neurons of nonlesioned rats and in slices contralateral to the lesion fired spontaneously, predominantly in a regular pattern, whereas those in slices ipsilateral to the lesion fired more irregularly or even in bursts. Experimentally increased [K+]o led to an increase in the number of spontaneously firing neurons and action potential firing rates in all groups. This was accompanied by a decrease in the amplitude of post spike afterhyperpolarization (AHP) and the amplitude and duration of the posttrain AHP. Lesion effects in ipsilateral neurons at physiological [K+]o resembled the effects of elevated [K+]o in nonlesioned rats. Our data suggest that changed potassium sensitivity due to conductivity alterations and delayed clearance may be critical for shaping STN activity in parkinsonian states.     

Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices.

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Na-K pump potassium regulation and IPSPs in seizures and spreading depression in immature rabbit hippocampal slices

1. Using the immature (8-12 days postnatal) rabbit hippocampal slice preparation, we investigated regional extracellular potassium concentration [( K+]o) changes that occur during spontaneous and evoked spreading depression (SD) episodes. We report here a difference between the CA1 and CA3 cell populations in the immature hippocampus with regard to 1) resting [K+]o, 2) magnitude of the [K+]o change during seizurelike events and SDs, and 3) susceptibility to SD episodes. Experiments were also performed to elucidate the roles that the Na-K pump and synaptic inhibition play in controlling SD onset, duration, and recovery. We demonstrated a major role for potassium regulation by the Na-K pump and a lesser modulatory role for inhibitory postsynaptic potentials (IPSPs) in preventing SD in the CA3 region. 2. Simultaneous intra- and extracellular recordings were made in the CA1 and CA3 regions of the immature rabbit hippocampus during spontaneous or evoked SD, while potassium ion-sensitive microelectrodes (K-ISMs) monitored changes in [K+]o. The CA1 region had 1) a higher frequency of spontaneous SD episodes than CA3, 2) a lower threshold to potassium-triggered SD, 3) a longer duration SD episode, and 4) smaller post-SD membrane potential and [K+]o undershoots (below the original resting membrane potential and resting [K+]o). 3. During the onset of a SD episode in the CA1 region, the local [K+]o rose either before or at the same time as the membrane potential depolarization. 4. In the CA3 region, spontaneous ictallike events consisting of tonic cell depolarization with repetitive activity followed by clonic afterdischarges were more likely to occur than SD episodes. During these ictallike episodes, [K+]o rose above the 10- to 12-mM ceiling level reported for adult CNS tissue during seizures. Increases in [K+]o evoked by repetitive stimulation were regulated at a lower level in CA3 (average [K+]o rise to 11.4 mM) than in CA1 (average [K+]o rise to 18.3 mM). 5. In CA3, bath application of 10 microM bicuculline or 3.4 mM penicillin did not change the frequency of spontaneously occurring SDs or the SD response threshold to local pressure ejection of 2 M KCl. However, blockade of IPSPs did lead to lower thresholds for SD or seizurelike episodes elicited by stimulation of the mossy fibers. 6. A single application of ouabain (10 microM) to CA3 by local pressure ejection caused a slow rise in local [K+]o measured with K-ISMs. The ouabain treatment also increased the frequency of spontaneous postsynaptic potential activity and decreased the amplitude and duration of CA3 pyramidal cell afterhyperpolarizations (AHPs).(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of excitation reveals inhibition of dentate spiny hilar neurons recorded in rat hippocampal slices.

1. Extracellular and intracellular recordings in rat hippocampal slices were used to compare the synaptic responses to perforant path stimulation of granule cells of the dentate gyrus, spiny "mossy" cells of the hilus, and area CA3c pyramidal cells of hippocampus. Specifically, we asked whether aspects of the local circuitry could explain the relative vulnerability of spiny hilar neurons to various insults to the hippocampus. 2. Spiny hilar cells demonstrated a surprising lack of inhibition after perforant path activation, despite robust paired-pulse inhibition and inhibitory postsynaptic potentials (IPSPs) in adjacent granule cells and area CA3c pyramidal cells in response to the same stimulus in the same slice. However, when the slice was perfused with excitatory amino acid antagonists [6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), or CNQX with 2-amino-5-phosphonovaleric acid (APV)], IPSPs could be observed in spiny hilar cells in response to perforant path stimulation. 3. The IPSPs evoked in spiny hilar cells in the presence of CNQX were similar in their reversal potentials and bicuculline sensitivity to IPSPs recorded in dentate granule cells or hippocampal pyramidal cells in the absence of CNQX. 4. These results demonstrate that, at least in slices, perforant path stimulation of spiny hilar cells is primarily excitatory and, when excitation is blocked, underlying inhibition can be revealed. This contrasts to the situation for dentate and hippocampal principal cells, which are ordinarily dominated by inhibition, and only when inhibition is compromised can the full extent of excitation be appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of extracellular space in hyperosmotic suppression of potassium-induced electrographic seizures

1. Focal electrographic seizures arose in the CA1 region of rat hippocampal slices bathed in elevated (8.5 mM) external potassium [( K+]o). High [K+]o also induced spontaneous interictal bursts that originated in area CA3 and propagated to CA1. To examine the contribution to electrographic seizure initiation of excitatory mechanisms that are influenced by extracellular volume, we studied the effect of hyperosmotic expansion of interstitial volume on seizure occurrence, interictal bursts, and excitatory synaptic transmission. The tissue electrical resistance was also measured leading up to and during seizures. 2. Media made 5-30 mosmol/kg hyperosmotic by addition of agents restricted to the extracellular space (mannitol, sucrose, raffinose, L-glucose, dextran) rapidly and reversibly abolished [K+]o-induced spontaneous CA1 seizures in 86% of slices tested. However, similar increases in osmolality effected by agents that access the intracellular compartment (D-glucose, glycerol) did not influence electrographic seizure occurrence. Hyperosmotic changes with plasma membrane impermeable compounds, but not permeable compounds, produced significant concentration-dependent decreases (1-10%) in the electrical resistance of CA1 stratum pyramidale. Because tissue resistance is proportional to extracellular volume, these results suggest that hyperosmotic suppression of electrographic seizures is associated with expansion of the extracellular space in hippocampal slices. 3. Measurement of electrical resistance of the CA1 stratum pyramidale during spreading depression and electrographic seizure revealed an increase in tissue resistance to 122% and 108% of control, respectively. Furthermore, a slight (approximately 2%) but significant increase in electrical resistance gradually occurred over the 20 s immediately preceding seizure generation. The observed increase in tissue resistance suggests extracellular space is decreased during these events. 4. Hyperosmolality did not alter CA3 interictal burst frequency. However, burst intensity, estimated from the total length of the burst waveform, was significantly reduced in both the CA3 (83% control) and CA1 region (67% control) when osmotic changes were imposed by plasma membrane impermeant compounds. Additionally, media made hypoosmotic by removal of 7.5 mM NaCl reversibly increased burst intensity. 5. High [K+]o potentiated excitatory synaptic transmission and excitatory postsynaptic potential (EPSP) spike coupling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Local synaptic circuits and epileptiform activity in slices of neocortex from children with intractable epilepsy.

1. Single and dual intracellular recordings were performed in neocortical slices obtained from tissue samples surgically removed from children (8 mo to 15 yr) for the treatment of intractable epilepsy. Electrical stimulation and glutamate microapplication were used to study local synaptic inputs to pyramidal cells. 2. In recordings with potassium-acetate electrodes, activation of presynaptic neocortical neurons with glutamate microdrops did not elicit a clear increase in postsynaptic potentials (PSPs) but did suppress current-evoked repetitive spike firing in recorded neurons. Bicuculline (10 microM) blocked this effect, suggesting it was caused by the activation of presynaptic gamma-aminobutyric acid (GABA) cells. In recordings with KCl electrodes, glutamate microdrops elicited an increase in the frequency and amplitude of depolarizing PSPs. Bicuculline (5-10 microM) blocked the glutamate-evoked PSPs, suggesting they were reversed GABAA-receptor-mediated inhibitory postsynaptic potentials (IPSPs). In one cell recorded with a KCl electrode (total n = 8), current-evoked spike trains elicited afterdischarges of reversed IPSPs, thus revealing a recurrent inhibitory circuit. Therefore local inhibitory synaptic circuits were robust and could be observed in tissue from patients as young as 11 mo. 3. In addition to short-latency (10-25 ms), monosynaptic excitatory postsynaptic potentials (EPSPs), electrical stimulation at low intensities sometimes elicited delayed EPSPs (20-60 ms). When GABAA-receptor-mediated synaptic inhibition was partially reduced in bicuculline (5-10 microM), electrical stimulation evoked large EPSPs at long and variable latencies (100-300 ms). Glutamate microapplication caused an increase in the frequency and amplitude of EPSPs; preliminary results suggest that glutamate microdrops were less likely to evoke EPSPs in tissue from younger patients (8-12 mo) than in slices from patients greater than 4 yr. Evidence for local excitatory synaptic circuits was thus found when synaptic inhibition was partially reduced. 4. After further reduction of inhibition in bicuculline (5-50 microM), electrical stimulation elicited epileptiform bursts. In pairs of simultaneously recorded neurons, bursts were generated synchronously from long-latency EPSPs (100-300 ms) in slices from patients as young as 8 mo. Reflected EPSPs at very long and variable latencies (500-1,100 ms) and repetitive epileptiform bursts could be evoked synchronously in pairs of cells. Glutamate activation of local presynaptic neurons elicited robust epileptiform events in recorded cells. This was seen in slices from patients as young as 16 mo. 5. These data provide physiological evidence for the presence of local inhibitory and excitatory synaptic circuits in human neocortex at least as early as 11 and 8 mo, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices

The effect of low extracellular Mg2+ concentration ([Mg2+]o) on neuronal activity was studied in rat hippocampal slices. After 20-40 min of perfusion with Mg2+-free medium, when [Mg2+]o declined to approximately 0.1-0.4 mM, spontaneous field potentials developed in the CA1 and CA3 regions, but not in the dentate gyrus. In the CA3 pyramidal cell layer, these potentials consisted of repetitive (0.3-0.5 Hz), 40- to 120-ms-long positive deflections (2-5 mV) with superimposed population spikes. In the stratum (str.) pyramidale of the CA1 region, positive-negative deflections (less than 3 mV) lasting for 30-80 ms were observed, which occurred with a frequency of 0.3-0.5 Hz. In some cases, longer lasting and rapidly recurring events were also observed. In CA3 pyramidal cells, the intracellular correlates of the field potential transients were 20- to 30-mV paroxysmal depolarization shifts (PDS) with superimposed bursts of action potentials, followed by large (greater than 10 mV), 500- to 1,200-ms-long afterhyperpolarizations (AHP). In contrast, pyramidal neurons of the CA1 area did not show PDSs; instead, sequences of excitatory postsynaptic potentials (EPSPs)/inhibitory postsynaptic potentials (IPSPs) accompanied the transient field potential changes. Occasionally, spontaneous EPSPs/IPSPs, occurring with high frequencies, could also be observed in CA1 without any field potential transients. In both hippocampal regions, the epileptiform activity evolved without significant alterations in the resting membrane potential (RMP) and input resistance (RN) of the neurons, although a 2- to 5-mV reduction in action potential threshold was noted. The spontaneous activity in Mg2+-free medium was readily suppressed by raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.6 to 3.6 mM. The perfusion of 10-30 microns DL-2-amino-5-phosphonovaleric acid (2-APV), an antagonist for the glutamate receptors of the N-methyl-D-aspartate (NMDA) type, also attenuated or reversibly blocked the spontaneous activity. Surgical isolation of area CA1 from CA3 ceased the occurrence of the transients in CA1 but not in CA3. The synaptic input/output curves were shifted to the left in the absence of [Mg2+]o. Threshold intensity for eliciting population spikes was 50-75% of that in normal medium. Paired-pulse facilitation was still present near threshold, but was reduced at higher stimulus intensities. Decreases in [Ca2+]o, produced by repetitive stimulation (20-Hz/5-10 s) of the Schaffer collateral/commissural pathway and monitored with ion-selective microelectrodes in the CA1 region, were enhanced in Mg2+-free medium.(ABSTRACT TRUNCATED AT 400 WORDS)