Hippocampus-Entorhinal Cortex Loop and Seizure Generation in the Young Rodent Limbic System

Maria Elisa Calcagnotto, Michaela Barbarosie, and Massimo Avoli

Montreal Neurological Institute, Department of Neurology and Neurosurgery and Department of Physiology, McGill University, Montreal, Quebec H3A 2B4, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Calcagnotto, Maria Elisa, Michaela Barbarosie, and Massimo Avoli. Hippocampus-Entorhinal Cortex Loop and Seizure Generation in the Young Rodent Limbic System. J. Neurophysiol. 83: 3183-3187, 2000. Application of the convulsant 4-aminopyridine (4AP, 50 µM) to adult mouse combined hippocampus-entorhinal cortex (EC) slices induces interictal and ictal discharges originating from CA3 and EC respectively. In this model of limbic seizures, ictal discharges disappear over time and are reestablished after Schaffer collateral cut, a procedure that blocks interictal propagation from CA3 to EC. Here we tested whether this form of network plasticity is operant in hippocampus-EC slices obtained from young (10-25 day-old) mice. In these experiments 4AP elicited interictal (duration = 100-250 ms; interval = 0.7 ± 0.2 s, mean ± SD, n = 20) and ictal (duration = 267 ± 37 s; interval = 390 ± 37 s, n = 20) discharges in both CA3 and EC. However, in young mouse slices the ictal events occurred throughout the experiment, whereas Schaffer collateral cut abolished CA3-driven interictal discharges in EC without influencing ictal activity (n = 10). Perforant path lesion prevented the spread of EC-driven ictal events to CA3, where interictal and short ictal discharges (duration = 32 ± 11 s; interval = 92 ± 9.7 s, n = 8) continued to occur. Hence, two independent forms of ictal activity were seen in CA3 and in EC after separation of these structures. In intact hippocampus-EC slices, ictal discharges were reduced by an N-methyl-D-aspartate receptor antagonist (n = 10). Under these conditions, Schaffer collateral cut abolished ictal activity in EC, not in CA3 (n = 6). Thus the young mouse hippocampus-EC loop has different properties as compared with adult tissue. These differences, which include the inability of hippocampal outputs to control ictal discharge generation in EC and the ability of the loop to sustain ictal activity, may contribute to the low-seizure threshold seen in young individuals.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Application of 4-aminopyridine (4AP) or Mg2+-free medium to adult mouse hippocampus-entorhinal cortex (EC) slices elicits ictal epileptiform activity that originates in the EC and propagates to the hippocampus, along with interictal discharges initiating in CA3 (Barbarosie and Avoli 1997; for studies in other species cf. Avoli et al. 1996a; Bragdon et al. 1992; Dreier and Heinemann 1991; Nagao et al. 1996; Wilson et al. 1988). Hippocampal output activity in the adult mouse limbic system exerts an unexpected control on the EC propensity to generate ictal discharges. Accordingly, 1) interictal discharges occur throughout the experiment, but ictal activity disappears within 1-2 h; and 2) Schaffer collateral cut abolishes interictal activity in EC and makes ictal discharge reappear in this structure.

Application of 4AP to "isolated" hippocampal slices obtained from young (10-25 day-old) rats induces ictal and interictal discharge initiating in CA3 and GABA-mediated synchronous potentials (Avoli et al. 1996b). In contrast, only interictal discharges are seen in isolated hippocampal slices obtained from adult (>30-day-old) animals (Perreault and Avoli 1991), thus indicating age-dependent differences in hippocampal excitability that may reflect the low-seizure threshold seen in early childhood (Aicardi 1986). It is unknown whether brain maturation influences seizure generation in the EC, and even more important, whether the hippocampal control exerted on EC excitability is operant early in life. Here we used the 4AP model in combined hippocampus-EC slices obtained from young mice to answer these two questions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BALB/c mice (10-25 day-old) were decapitated under halothane anesthesia. Their brains were removed and placed in cold oxygenated artificial cerebrospinal fluid (ACSF). Horizontal slices (600-µm thick) were cut with vibratome and transferred to a tissue chamber where they were placed between oxygenated ACSF and humidified gas (95% O2-5% CO2) at 32-34°C. The ACSF composition was (in mM): NaCl 124, KCl 2, KH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 26, and glucose 10. 4AP (50 µM): 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP, 10 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) were bath applied. Chemicals were acquired from Sigma and Tocris Cookson.

Slices included the EC and the hippocampus proper. Simultaneous field potential recordings were made with ACSF-filled glass pipettes that were usually positioned in layers IV-V of the medial EC and in CA3 stratum radiatum. Signals were fed to DC amplifiers and displayed on a Gould-pen chart recorder. Cuts were made with a microknife to establish the origin and propagation pathway(s) of the 4AP-induced synchronous activity (Avoli et al. 1996a; Barbarosie and Avoli 1997). Measurements in the text are expressed as mean ± SD, and n represents the number of slices studied. Data were compared with the Student's t-test or the analysis of variance (ANOVA) test and were considered significantly different if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Several types of spontaneous synchronous activities occurred in CA3 and in EC after 1.5 h of 4AP application (Fig. 1). Interictal discharges (arrows in Fig. 1Aa) had intervals of 0.7 ± 0.2 s (n = 20) and originated in CA3 from where they propagated to the EC as indicated by their time onset (Fig. 1Ab). Ictal discharges also occurred simultaneously in CA3 and EC with similar durations (262 ± 37.1 s, n = 20) and intervals of occurrence (390 ± 66.3 s, n = 20) (Fig. 1Aa, continuous line). However, in contrast to what was reported in the adult mouse (Barbarosie and Avoli 1997), ictal activity did not disappear over time. Rather, it continued to occur at a steady rate throughout the experiment (Fig. 1B).



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Fig. 1. Spontaneous activities induced by 4-aminopyridine (4AP) in young mouse hippocampus-entorhinal cortex (EC) slices. A: simultaneous recordings in CA3 and EC demonstrate the occurrence of interical (arrows in a) and ictal discharges (continuous line in a). Ab: 2 interical discharges shown at high speed; note that the discharge initiates in CA3 and propagates to the EC. Ac: ictal discharge onset is characterized by a negative-going event that is clearly recorded in EC. B: rate of occurrence of ictal discharges in 6 slices over time. Note that a steady rate is seen during the first 6 h of 4AP application. C: isolated negative potentials (*) and a spreading depression-like episode that is recorded in the EC only.

Isolated field potentials with intervals of occurrence ranging 15-120 s could also be recognized (Fig. 1C, asterisks). These events were most often of negative polarity and could initiate ictal discharges (Fig. 1Ac, arrowhead). 4AP-induced, isolated field potentials are blocked by either GABAA receptor antagonists or a µ-opioid receptor agonist (unpublished observations in the mouse, but see Avoli et al. 1996a,b for the rat). Hence, they will be referred to as GABA-mediated synchronous potentials. In 15/20 experiments we also observed spreading depression-like episodes (cf. Avoli et al. 1996b) that, however, remained confined to the EC (Fig. 1C).

We also determined the site of origin and the modalities of propagation of 4AP-induced discharges by performing selective cuts of neuronal pathways. By doing so we established the reciprocal influences exerted by the activities occurring in CA3 and EC. Schaffer collateral cut abolished CA3-driven interictal discharges in EC without altering the ictal activity that continued to occur with similar characteristics in both CA3 and EC (n = 10, Fig. 2A). Cutting the perforant path (PP) (which presumably abolishes the propagation of EC activity to the hippocampus proper) did not modify ictal discharges in the EC (Fig. 3A, control), but decreased the duration of CA3 ictal events (32 ± 11.6 s as compared with 260 ± 37.9 s before cut, P < 0.0001) and increased their occurrence (interval = 92 ± 9.7 s versus 380 ± 52.5 s before cut P < 0.0001, n = 8).



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Fig. 2. A: effects of Schaffer collateral cut on 4AP-induced interictal and ictal discharges. Interictal events are not recorded in the EC after the cut, but ictal discharges are unchanged in both areas. B: application of the N-methyl-D-aspartate- (NMDA) receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP) (10 µM) reduces ictal discharge duration in an intact hippocampus-EC slice. Schaffer collateral cut during CPP application abolishes ictal discharges in the EC, thus indicating that NMDA receptor independent ictal activity originates in CA3. C: duration and interval of occurrence of ictal discharges recorded in 6 intact slices in control (i.e., 4AP in the bath) and after addition of CPP. Note that CPP decreases the duration of the ictal events and reduces their interval of occurrence. *, statistically significant difference.



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Fig. 3. 4AP-induced epileptiform activity in isolated hippocampus-EC slices. A: under control conditions 2 forms of ictal discharges are recorded in CA3 and EC; note that ictal discharges in CA3 are shorter than those recorded in EC. CPP abolishes the ictal discharges in EC, but ictal activity continues to occur in CA3. B: duration and interval of occurrence of the ictal discharges recorded in CA3 and EC in 6 isolated slices in the presence of 4AP (control) and after addition of CPP. *, statistically significant difference.

Complete disconnection between the hippocampus proper and the EC resulted in the independent occurrence of ictal discharges in these two structures (n = 10) (Fig. 3A, control). Ictal events in the isolated EC had duration and intervals similar to those seen in the intact slice (duration = 262 ± 37.1 s and interval = 390 ± 66.3 s in the intact slice; duration = 240 ± 48.9 s and interval = 340 ± 96.9 s in the isolated EC; n = 5; P > 0.5). In contrast, CA3 ictal events in the isolated slice were shorter in duration (32 ± 11.6 s versus 262 ± 37.1 s before the cut, P < 0.0001) and occurred at shorter intervals (92 ± 9.7 s versus 390 ± 66.3 s; n = 5; P < 0.001) as compared with the intact preparation (Fig. 3A). The electrographic characteristics of CA3 ictal discharges in the isolated mouse hippocampus were similar to those of isolated rat hippocampal slices (Avoli et al. 1996b).

Next, we analyzed the pharmacological profile of the epileptiform activities induced by 4AP by using excitatory amino acid receptor antagonists. Ictal discharges originating in CA3 were isolated with the N-methyl-D-aspartate (NMDA) receptor antagonist CPP. In intact hippocampus-EC slices CPP reduced ictal discharge duration and interval of occurrence (n = 10; Fig. 2, B and C). In these experiments (n = 6) Schaffer collateral cuts abolished ictal discharges in EC, but did not block CA3-driven ictal events that were however shorter than in control (duration = 23 ± 4 s versus 32 ± 11.6 s, P > 0.1; interval = 46 ± 5.8 s versus 92 ± 9.7 s; P < 0.01). CPP also abolished spreading depression-like episodes. Finally we studied CPP effects after complete separation of the hippocampus and EC. This pharmacological procedure blocked EC ictal activity without influencing ictal discharges in CA3 (n = 10; Fig. 3). Application of the non-NMDA receptor antagonist CNQX abolished all epileptiform activities in both CA3 and EC, whereas the synchronous GABA-mediated potentials continued to occur (n = 2, not shown but see Avoli et al. 1996a for the rat studies).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reciprocally connected, hippocampus-EC slices obtained from young mice respond to 4AP by generating the following: 1) interictal discharges that initiate in CA3 and propagate to the EC; and 2) ictal discharges due to an NMDA receptor-dependent, synchronizing mechanism in the EC. These data are similar to those seen in adult hippocampus-EC slices during the initial 1-2 h of 4AP application (Barbarosie and Avoli 1997). However, two differences emerge by comparing these previous findings and those reported here. First, ictal discharges in the young mouse occur throughout the experiment (up to 6 h). Second, two independent sites of ictal discharge generation can be identified either by isolating the two limbic structures or by antagonizing the NMDA receptor.

Ictal discharges in the intact, young mouse hippocampus-EC slice originate in the EC because they are unaffected by disruption of input activity rising from CA3. This finding was evident both with Schaffer collateral cut (which abolishes interictal activity propagation from CA3 to CA1) and after complete separation of the EC from the hippocampus proper. By contrast, ictal discharges in CA3 were altered by cutting the perforant pathway, a procedure that disclosed an independent site of ictal discharge generation similar to what was reported in young isolated hippocampal slices (Avoli et al. 1996b). The prominent role played by the EC in ictal discharge generation is in line with evidence obtained in several models of limbic seizures (Avoli et al. 1996b; Dreier and Heinemann 1991; Nagao et al. 1996; Pare et al. 1992). Moreover, a dysfunction of the EC has been reported in temporal lobe epileptic patients (Rutecki et al. 1989; Spencer and Spencer 1994), where surgical removal of this structure may be essential for achieving seizure control (Goldring et al. 1992).

In combined hippocampus-EC slices obtained from adult rodents, NMDA receptor antagonist abolishes ictal activity (Avoli et al. 1996b; Nagao et al. 1996). By contrast, in intact young mouse hippocampus-EC slices this pharmacological procedure only reduced the duration of the ictal discharges. Indeed, ictal discharges continued to occur in CA3 and EC although with reduced duration. This NMDA receptor-independent, ictal activity originated from CA3 and propagated to the EC as shown by cutting the Schaffer collaterals, which effectively abolished ictal events in EC. Ictal discharges that are insensitive to NMDA receptor antagonists are induced by 4AP in the young rat, isolated hippocampal slice (Avoli et al. 1996a). Interestingly, disconnection of the hippocampus from the EC in the presence of NMDA receptor antagonists decreased the duration of CA3 ictal discharges, thus suggesting that in the young rodent brain, even when NMDA receptors are not functional, the hippocampus-EC loop can sustain and perhaps amplify ictal activity.

An additional difference between the epileptiform activity recorded in the young and in the adult hippocampus-EC slice is the occurrence of ictal activity that continues to occur throughout the experiment. We can exclude that the inability of interictal activity to control EC ictal activity was due to a lack of functional inputs from the hippocampus proper to the EC because these interictal discharges propagated from CA3 to EC. Rather the resistance of ictal discharges may reflect the ability of a young CA3 network to induce non-MNDA receptor-mediated ictal events. These discharges are disclosed in the young mouse combined slice through isolation from EC inputs or during blockade of NMDA receptors by CPP.

Hyperexcitability and increased seizure propensity characterize the early stages of CNS development (Schwartzkroin 1993). Observations in young individuals also suggest that the immature CNS has a high susceptibility to generate seizures (Aicardi 1986). Our results demonstrate the following: 1) early in life the hippocampus-EC loop is unable to control ictal activity; 2) both hippocampus (CA3) and EC can produce ictal activities mediated by non-NMDA and NMDA receptors respectively; and 3) EC generates, spreading depression-like episodes that are not seen in adult slices. Hence, our study provides some novel explanations for why the young brain is more susceptible to generating seizures.


    ACKNOWLEDGMENTS

We are grateful to T. Papadopoulos for secretarial assistance.

This work was supported by grants from the Medical Research Council of Canada (MT-8109), the Savoy Foundation, the Hospital for Sick Children Foundation (XG-93056), and the Quebec Heart and Stroke Foundation.

Present address of M. Barbarosie: Dept. of Neuroscience, Howard Hughes Medical Institute, Brown University, Providence, RI 02912.


    FOOTNOTES

Address for reprint requests: M. Avoli, 3801 University, Montreal, Quebec H3A 2B4, Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 December 1999; accepted in final form 7 February 2000.


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0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society




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