Janet L. Stringer

Recurrent spontaneous hippocampal seizures in the rat as a chronic sequela to limbic status epilepticus

A period of continuous hippocampal stimulation (CHS) establishes an acute condition of self-sustaining limbic status epilepticus (SSLSE) which is followed by chronic neuropathological changes reminiscent of hippocampal sclerosis encountered in epileptic patients. In the chronic (greater than or equal to 1 month) condition following CHS-induced SSLSE, extended electrographic monitoring in the hippocampus revealed spontaneous recurrent paroxysmal discharges. All 6 animals studied had persistent interictal spiking; 3 had multiple fully developed electrographic seizures. There was a marked diminution of paired pulse inhibition, demonstrated by a protocol known to reflect the potency of inhibition mediated by GABAA receptors. Hippocampal slices from animals that had previously experienced CHS-induced SSLSE demonstrated an increased excitability relative to slices from control animals as evidenced by epileptiform bursting in increased extracellular potassium ([K+]0) and decreased extracellular calcium ([Ca2+]0). These studies establish that CHS-induced SSLSE in rats provides an experimental model with recurrent spontaneous hippocampal seizures. Based on electrophysiological data we suggest that a decrease in GABA-mediated inhibition and/or altered sensitivity to extracellular ions may play roles in the development of such seizures.

Elimination of long-term potentiation in the hippocampus by phencyclidine and ketamine*

Long-term potentiation (LTP) of CA1 population spikes was elicited by cCA3 stimulation. Phencyclidine (PCP) and ketamine blocked the potentiation in a dose-dependent manner while diazepam and sodium thiopental had no effect. In midcollicular-transected rats where PCP has no depressant effect on the population spike, the drug still blocked LTP.

Reverberatory seizure discharges in hippocampal-parahippocampal circuits

Previously, a unique type of epileptiform discharge, recorded in the dentate gyrus, has been identified and termed maximal dentate activation. Maximal dentate activation is defined by the presence of bursts of large amplitude population spikes, associated with a secondary rise in the extracellular potassium and a negative shift of the dc potential. Prior work has linked maximal dentate activation to lengthening of afterdischarges when they are elicited in the hippocampus or outside of the hippocampus in the amygdala. The current study used two approaches to further examine the relationship of maximal dentate activation to seizures in limbic circuits in urethane-anesthetized rats. First, simultaneous recordings were employed to document that during maximal dentate activation, synchronous discharges occurred in the dentate gyrus, cornu Ammonis, subiculum, and entorhinal cortex. From anatomical work, these structures are known to be connected in a hippocampal-parahippocampal loop. The second approach used lesions of the entorhinal cortex to document the importance of this loop in the initiation and maintenance of maximal dentate activation. Both electrolytic and chemical (focal injections of tetrodotoxin) lesions of the entorhinal cortex blocked maximal dentate activation on the side of the lesion. However, maximal dentate activation was maintained on the opposite side, where the hippocampal-parahippocampal loop was intact. Altogether, these data support the hypothesis that maximal dentate activation is a marker for the presence of reverberatory, synchronized paroxysmal activity throughout the hippocampal-parahippocampal loop and that this loop behaves as a unit in epileptogenesis.

Repetitive seizures cause an increase in paired-pulse inhibition in the dentate gyrus

A paired-pulse stimulus protocol was used to measure angular bundle to dentate gyrus paired-pulse inhibition in rats before and after the occurrence of 36 or 72 seizures elicited from the contralateral CA3 region. The seizures showed progressive lengthening. There was a moderate increase in paired-pulse depression 1 h after 36 seizures and a further increase after 72 seizures. These data demonstrate that the same experimental protocol which produced a decrease in paired-pulse inhibition in the CA1 region caused the opposite effect in the dentate gyrus.

Maximal dentate activation: a tool to screen compounds for activity against limbic seizures

A model of partial complex (limbic) seizures in the anesthetized rat based on the phenomenon of maximal dentate activation was used to study the effect of various known antiepileptic drugs. Maximal dentate activation consists of a distinct triad of large amplitude population spikes, associated with a rise in [K+]0 to 10 mM and a negative shift of the DC potential. Repeated stimulation produces lengthening of the duration of maximal dentate activation (mimicking the lengthening of afterdischarges that occurs during kindling) and a decrease in the time to onset of maximal dentate activation. Diazepam (3 mg/kg), carbamazepine (50 mg/kg) and phenobarbital (60 mg/kg) caused a reversible reduction in the duration of maximal dentate activation. Carbamazepine (30 mg/kg) and phenobarbital (20 mg/kg) prevented the lengthening of maximal dentate activation that occurs with repeated elicitation while phenytoin (80 mg/kg), ethosuximide (300 mg/kg) and valproic acid (300 mg/kg) had no effect. The doses of these known antiepileptic drugs agree with the doses efficacious against limbic seizures in awake rats. This suggests that the ability to shorten the duration of maximal dentate activation provides a useful means to screen compounds for activity against partial complex seizures.

In vitro effects of extracellular calcium concentrations on hippocampal pyramidal cell responses

This study examined the effects of varying the extracellular Ca2+ concentration [( Ca2+]o) on membrane stabilization, response to paired stimuli, and long-term potentiation (LTP) in the CA1 pyramidal cells of the rat hippocampal slice. To approximate the in vivo state, 1.5 mM [Ca2+]o was used as the control condition. Raising [Ca2+]o caused a leftward shift of the input-output curve (stimulus intensity vs population spike amplitude) and reduced the tendency toward multiple spiking. Lowering [Ca2+]o shifted the input-output curve down and to the right. When [Ca2+]o was lowered to 0.75 mM an epileptiform pattern of extracellular field responses was produced. Paired-pulse phenomena were studied at an interstimulus interval of 20 ms using both input-output curves and action potential thresholds for single units. Only facilitation was seen using single unit thresholds. Using input-output curves, inhibition was seen in 2.5 and 3.5 mM [Ca2+]o at high stimulus intensities but not at all in 1.5 mM [Ca2+]. LTP was shown to be Ca2+-dependent, maximal at 1.5 mM [Ca2+]o and absent at 1.0 mM [Ca2+]o. LTP was present but less prominent in [Ca2+]o greater than 1.5 mM.

Maximal dentate activation is produced by amygdala stimulation in unanesthetized rats

In urethane-anesthetized rats, a process termed maximal dentate activation has been shown to be associated with the lengthening of afterdischarges that occurs with repeated hippocampal stimulation. Maximal dentate activation is a unique paroxysmal form of epileptiform discharges consisting of large amplitude population spikes in the dentate gyrus. The current experiments examined the relationship of maximal dentate activation to kindling of motor seizures in the awake animal. Both long duration (5 or 10 s) and short duration (1 s) stimulus trains in either the hippocampus or the amygdala were effective in eliciting maximal dentate activation. Repeated stimulation of the amygdala produced lengthening of afterdischarges and kindling of motor responses, but only after maximal dentate activation had appeared in response to the stimulus. Over the course of amygdala kindling, the duration of maximal dentate activation lengthened with increasing severity of behavioral seizures. This evidence supports the hypothesis that maximal dentate activation is a marker for the presence of seizures distributed throughout limbic circuits. In addition, the data suggest that maximal dentate activation may be an important process involved in the acquisition of kindled responses.

Effect of phencyclidines on hippocampal pyramidal cells

Phencyclidine (PCP) and several behaviorally active or inactive structural analogs were administered i.v. to urethane-anesthetized rats in order to determine their effects on CA1 pyramidal cell discharges elicited by contralateral CA3 (cCA3) stimulation. PCP and the behaviorally active m-amino derivative (m-NH2 PCP) depressed, in a dose-dependent manner, the amplitude of the population spike evoked in CA1 by a cCA3 stimulation (ED 50s: 0.9 mg/kg for PCP, 0.5 mg/kg for m-NH2 PCP). However, the behaviorally inactive derivatives m-nitro (m-NO2 PCP) and PCP methyliodide (PCP CH3I) were ineffective up to 10 mg/kg. PCP (0.1-0.3 mg/kg i.v.) also decreased the duration of inhibition of CA1 discharges in a paired-stimulus paradigm; this was in contrast to the effects of thiopental and diazepam. In midcollicular-transected, urethane-anesthetized rats, the inhibitory effect of PCP on cCA3-CA1 transmission was not observed but the drug was still as effective as in intact rats in the paired-stimulus paradigm. In animals subjected to 6-hydroxydopamine lesions of the hippocampal noradrenergic innervation (average 85%) decrease in NE content), the potency of PCP in inhibiting cCA3-CA1 transmission was the same as in a group of sham-operated controls. These results suggest the following conclusions: (i) PCP exerts at least 2 separate types of effects in CA1, both of which result from a central action of the drug; (ii) PCP decreases the monosynaptic excitation of CA1 pyramidal cells and this action requires the integrity of brainstem afferents; (iii) PCP may decrease recurrent inhibition or afterhyperpolarization in CA1 via a mechanism which is independent of these connections and, therefore, could result from a direct action of the drug at the level of the hippocampus; (iv) finally, no evidence was found to suggest that the noradrenergic innervation of the hippocampus is critically involved in the action of PCP on CA1 discharges.

Spreading depression and reverberatory seizures induce the upregulation of mRNA for glial fibrillary acidic protein

The present study evaluates the relative roles of seizure activity and spreading depression in upregulating glial fibrillary acidic protein (GFAP) mRNA expression. Stimulating electrodes were placed bilaterally in the angular bundle, and recording electrodes were placed bilaterally in the dentate gyrus of adult rats. Intense electrographic seizures were induced by delivering stimulus trains through one stimulating electrode. In some cases, spreading depression accompanied the seizures, while in other cases, the seizures occurred in the absence of spreading depression. Animals were killed 24 h following the last stimulus train, and the forebrains were prepared for quantitative in situ hybridization. Seizure activity and spreading depression led to significant increases in GFAP mRNA levels in the hippocampal formation. Seizure activity alone (without spreading depression) induced a 4-fold increase in GFAP mRNA levels in the hilus and molecular layer of the dentate gyrus and in stratum lacunosum-moleculare of the hippocampus. When seizure activity was accompanied by spreading depression, there was a 10-fold increase in GFAP mRNA levels in these same regions. Regional differences within the hippocampal formation in glial cell response were evident. While GFAP mRNA levels in stratum lacunosum-moleculare of the hippocampus were upregulated by seizure activity and spreading depression, levels in hippocampal stratum radiatum of the hippocampus remained unchanged. The results suggest that abnormal neuronal activity can influence glial cell gene expression and that spreading depression is a stronger signal than seizure activity in upregulating GFAP mRNA levels.

Epileptiform discharges induced by altering extracellular potassium and calcium in the rat hippocampal slice

During and after intense neuronal activity the concentration of extracellular potassium ([K+]o) increases while the concentration of calcium ([Ca2+]o) decreases. The present study examined the effect of increased [K+]o alone, and with a parallel decrease in [Ca2+]o, on overall excitability, long-term potentiation (LTP), and the appearance of epileptiform discharges. [K+]o and [Ca2+]o were varied over the range in which they fluctuate in vivo. Hippocampal slices were first equilibrated in a control artificial CSF containing 3.1 mM K+ and 1.5 mM Ca2+ and then reequilibrated in an identical solution except that the K+ was increased to 3.55, 4, 5, 6, or 8 mM with and without a decrease in Ca2+ to 1.0 mM. Raising [K+]o caused a leftward shift of input-output curves. Lowering [Ca2+]o to 1.0 mM had no effect on the ability of [K+]o to shift the input-output curve to the left. LTP was not changed by increasing [K+]o. Lowering [Ca2+]o to 1.0 mM blocked LTP and increasing the [K+]o did not overcome this blockade. When [K+]o alone was altered, the [K+]oS at which epileptiform bursts occurred 50% of the time were 5.6 and 7.6 mM for stimulus-locked and spontaneous bursting, respectively. The combination of decreased [Ca2+]o and increased [K+]o made slices considerably more prone to epileptiform activity. In 1.0 mM [Ca2+]o, the [K+]o at which 50% of the slices showed stimulus-locked bursting was decreased to 3.6 mM while that for spontaneous discharges was 5.4 mM. The sensitivity of hippocampal slices to [K+]o and [Ca2+]o, and the synergistic actions of alterations of these ions, indicates that even small changes in the aggregate extracellular ionic milieu may be important in epileptogenesis.