CA3-Released Entorhinal Seizures Disclose Dentate Gyrus Epileptogenicity and Unmask a Temporoammonic Pathway

Michaela Barbarosie,1 Jacques Louvel,2 Irène Kurcewicz,2 and Massimo Avoli1

 1Departments of Neurology and Neurosurgery, and Physiology, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada; and  2Centre Paul Broca and Institut National de la Santé et de la Recherche Médicale U109, 75014 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Barbarosie, Michaela, Jacques Louvel, Irène Kurcewicz, and Massimo Avoli. CA3-Released Entorhinal Seizures Disclose Dentate Gyrus Epileptogenicity and Unmask a Temporoammonic Pathway. J. Neurophysiol. 83: 1115-1124, 2000. We have investigated the propagation of epileptiform discharges induced by 4-aminopyridine (4-AP, 50 µM) in adult mouse hippocampus-entorhinal cortex slices, before and after Schaffer collateral cut. 4-AP application induced 1) ictal epileptiform activity that disappeared over time and 2) interictal epileptiform discharges, which continued throughout the experiment. Using simultaneous field potential and [K+]o recordings, we found that entorhinal and dentate ictal epileptiform discharges were accompanied by comparable elevations in [K+]o (up to 12 mM from a baseline value of 3.2 mM), whereas smaller rises in [K+]o (up to 6 mM) were associated with ictal activity in CA3. Cutting the Schaffer collaterals disclosed the occurrence of ictal discharges that were associated with larger rises in [K+]o as compared with the intact slice. Further lesion of the perforant path blocked ictal activity and the associated [K+]o increases in the dentate gyrus, indicating synaptic propagation to this area. Time delay measurements demonstrated that ictal epileptiform activity in the intact hippocampal-entorhinal cortex slice propagated via the trisynaptic path. However, after Schaffer collateral cut, ictal discharges continued to occur in CA1 and subiculum and spread to these areas directly from the entorhinal cortex. Thus our data indicate that the increased epileptogenicity of the dentate gyrus (a prominent feature of temporal lobe epilepsy as well), may depend on perforant path propagation of entorhinal ictal discharges, irrespective of mossy fiber reorganization. Moreover, hippocampal neuronal damage that is acutely mimicked in our model by Schaffer collateral cut, may contribute to "short-circuit" propagation of activity by pathways that are masked when the hippocampus is intact.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Entorhinal inputs to the dentate gyrus may be instrumental in the reverberation and amplification of epileptiform activity through the hippocampal-entorhinal loop (Paré et al. 1992). The dentate gyrus may sustain ictal activity during chronic epileptogenesis (Parent et al. 1997; Wuarin and Dudek 1996). Indeed, dentate seizure discharges in "naive" animals are shorter as compared with those recorded from epileptic rats (Stringer and Lothman 1989), lending support to the hypothesis that the dentate gyrus may amplify seizures in chronic, animal models of epileptogenesis.

The dentate gyrus may also gate the propagation of epileptiform discharge to the hippocampus (Collins et al. 1983; Jones and Lambert 1990; Walther et al. 1986). However, adequate stimulation of the contralateral CA3 or the ipsilateral angular bundle can overcome the dentate resistance to paroxysmal discharge generation, thus allowing robust ictal-like discharges to occur (Stringer and Lothman 1989; Stringer et al. 1989). This epileptiform activity most likely results from perforant path propagation of ictal events originating in the entorhinal cortex. Accordingly, the entorhinal cortex is susceptible to generate seizure activity in patients with temporal lobe epilepsy (Rutecki et al. 1989; Spencer and Spencer 1994) and in models of epileptiform discharge (e.g., Avoli et al. 1996a; Bragdon et al. 1992; Dreier and Heinemann 1991; Jones and Heinemann 1988; Nagao et al. 1996).

Propagation of entorhinal epileptiform discharges via the perforant path has been the object of several studies. However, direct propagation of epileptiform activity via the temporoammonic path remains elusive. Monosynaptic, entorhinal layer III neuronal inputs project directly to CA1 and subiculum (Steward and Scoville 1976; Witter et al. 1988; cf. Soltesz and Jones 1995;). Maccaferri and McBain (1995) have shown that the entorhinal cortex-CA1 pathway is under the control of oriens-alveus interneurons, which suppress the function of the entorhinal inputs to the CA1 region. Thus under normal conditions, entorhinal activity propagates to the hippocampus via the trisynaptic circuit. At the same time, Schaffer collateral inputs activate CA1 pyramidal neurons, which thus excite oriens-alveus GABAergic interneurons in this region and block the temporoammonic input.

In the present study, we have used a combined hippocampus-entorhinal cortex slice obtained from mouse brain tissue to investigate the propagation of ictal epileptiform discharges generated in the entorhinal cortex. First, we have characterized the role of the dentate gyrus in gating entorhinal ictal discharges toward the hippocampus proper. Second, we have determined whether cutting the Schaffer collaterals (and thus lifting control on the function of the temporoammonic input) discloses propagation of entorhinal epileptiform discharges via this pathway. We report here that in our model dentate gyrus epileptogenicity depends on the occurrence of entorhinal epileptiform discharges, not on synaptic reorganization of this subfield. Additionally, we show that entorhinal epileptiform discharges propagate directly to CA1 and subiculum only after Schaffer collateral cut, suggesting that the temporoammonic path input is masked by the trisynaptic propagation of epileptiform activity. Preliminary reports of this work have appeared in abstract form (Barbarosie et al. 1997, 1998).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adult, male, CD-1 or Balb-C mice (25-35 g) were decapitated under halothane anesthesia. Their brain was quickly removed and placed in cold (1-4°C) oxygenated artificial cerebrospinal fluid (ACSF). Horizontal slices were prepared following the the dissecting procedures described in detail by Dreier and Heinemann (1991) for rat combined hippocampus-entorhinal cortex slices, but were cut (550-600 µm thick) with a vibratome (see also Avoli et al. 1996a and Nagao et al. 1996 for previous rat studies performed in our laboratory). Slices that included the entorhinal cortex and the hippocampus proper (also comprising the subiculum and the dentate gyrus) were then transferred to a tissue chamber where they lay between oxygenated ACSF and humidified gas (95% O2-5% CO2) at 32-33°C. ACSF composition was (in mM) 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose. 4-Aminopyridine (4-AP; 50 µM) was directly applied to the perfusing medium. All chemicals were acquired from Sigma.

Field potential recordings were performed with ACSF-filled microelectrodes (tip diameter, 8µm; resistance, 2-10 MOmega ). Ion-selective microelectrodes were prepared according to the technique described by Heinemann et al. (1977). Details concerning the technique for measurements of ionic concentrations can be found in other publications from our laboratory (e.g., Avoli et al. 1996a,b). Unless otherwise indicated, the recording electrodes were positioned as follows: layers IV-V of the entorhinal cortex; the granule cell layer of the dentate gyrus; CA3 and CA1 pyramidal layer; and in continuation of the pyramidal layer in subiculum. A series of cutting experiments were performed using a microknife to establish the origin and the pathway used by epileptiform activity to propagate in the slice (Avoli et al.1996a; Barbarosie and Avoli 1997).

Field potential recordings as well as simultaneous field potential and [K+]o measurements were performed in >40 combined, mouse hippocampus-entorhinal cortex slices. The increases in [K+]o observed during the interictal and the ictal discharges induced by 4-AP were measured as maximal, absolute values. The time differences in ictal discharge onset were obtained from expanded traces of simultaneous field potential recordings, and measurements were made by taking into account the first sharp deflection of each trace at the beginning of the discharge (see Fig. 6, insets in b panels). Values in the text are expressed as means ± SD, and n represents the number of slices studied. Data were compared with the Student's t-test or the ANOVA test and were considered significantly different if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General features of 4-AP-induced epileptiform activity

Bath application of 4-AP to combined hippocampal-entorhinal slices induced, within ~1 h, the appearance of spontaneous, CA3-driven interictal-like potentials (duration: <1 s; arrows in Fig. 1B, see also arrowheads in the expanded recordings of Fig. 5B) and prolonged ictal-like discharges of entorhinal cortex origin (duration: 20-50 s; continuous line in Fig. 1B) (cf. Barbarosie and Avoli 1997). These potentials could be recorded simultaneously in all subfields (Fig. 1B). In the intact slice preparation, ictal discharges disappeared over time, whereas interictal discharges continued to be recorded during the experiment (cf. Barbarosie and Avoli 1997).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1. A: schematic illustration of the slice preparation used for our experiments. The microelectrode location is shown here for the recordings of part B. EC, entorhinal cortex; DG, dentate gyrus; and SUB, subiculum. B: simultaneous field potential and [K+]o recordings of 4-aminopyridine (4-AP)-induced epileptiform activity, after ~1 h incubation of an intact hippocampal-entorhinal slice, when ictal discharges are still present. Ictal and interictal discharges (highlighted in the CA3 trace by a continuous line and by arrows, respectively) are accompanied by rises in [K+]o in the entorhinal cortex, dentate gyrus and CA3 areas. Note that [K+]o associated with ictal discharges in entorhinal cortex and dentate gyrus are larger compared with rises in CA3.

Simultaneous field potential and [K+]o recordings revealed that rises in [K+]o, from a baseline value of 3.2 mM, were associated with both ictal and interictal discharges in the entorhinal cortex, dentate gyrus and in CA3 subfield (n = 22). In the intact slice preparation, the elevations in [K+]o seen during the ictal activity reached maximal values of 9.4 ± 2.1 mM (mean ± SD; n = 8) in the entorhinal cortex, 8.7 ± 1.9 mM (n = 7) in the dentate gyrus, and 5.1 ± 0.6 mM (n = 5) in CA3. In these same experiments, the interictal discharges were accompanied by increases in [K+]o that amounted to 4.0 ± 0.2 mM (n = 8) in the entorhinal cortex, 4.1 ± 0.2 mM (n = 8) in the dentate gyrus, and 3.5 ± 0.2 mM (n = 8) in CA3 (Fig. 1B).

Effect of Schaffer collateral cut on [K+]o elevations associated with 4-AP-induced epileptiform activity

After lesioning the Schaffer collaterals, hippocampal output activity was suppressed in the entorhinal cortex and dentate gyrus, and ictal events of entorhinal origin were either 1) disclosed when absent prior to Schaffer collateral cut, or 2) prolonged in duration if they did occur prior to the lesion (cf. Barbarosie and Avoli 1997). Figure 2 shows that the elevations in [K+]o associated with ictal discharges differed within the entorhinal cortex, the dentate gyrus, and the CA3 subfield according to the position of the recording electrodes. Therefore [K+]o measurements were performed in the deep layers of the entorhinal cortex, the granule cell layer of the dentate sector, and in the pyramidal cell layer of CA3, where the largest elevations in [K+]o were observed in coincidence with ictal activity. This type of laminar analysis also revealed that, with the exception of dentate interictal activity, field potential changed in polarity when recorded at different sites within a given area. This evidence, along with the elevations in [K+]o, indicates that with the exception of interictal discharges in the dentate area, the activity induced by 4-AP in different regions of the combined slice was not volume conducted (Fig. 2B).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2. Field potential and [K+]o profiles associated with 4-AP-induced epileptiform activity recorded after Schaffer collateral cut and thus after having disclosed ictal discharge occurrence. A: field potential and [K+]o recordings of ictal discharges obtained at different depths from the pia in the entorhinal cortex. B: field potential and [K+]o recordings associated with ictal discharges along the hilar-molecular axis of the dentate gyrus (a-c), in addition to the deep layers of the entorhinal cortex (d). C: field potential and [K+]o measurements performed along an axis normal to the CA3 pyramidal cell layer. Note that the highest [K+]o rises occur in the CA3 pyramidal and dentate granule cell layer and in the deeper layers of the entorhinal cortex. Note also that [K+]o rises in the dentate granule cell layer are comparable to those in the deep layers of the entorhinal cortex.

We investigated how the increases in [K+]o associated with the ictal activity were affected by Schaffer collateral cut (Fig. 3, A and B). We found that after this lesioning procedure, [K+]o associated with ictal activity reached peak levels of 13.7 ± 2.5 mM (n = 7) in the entorhinal cortex, 12.0 ± 2.2 mM (n = 7) in the dentate gyrus, and 5.1 ± 0.6 mM (n = 5) in CA3. The ictal-associated [K+]o elevations were of comparable magnitudes in the dentate and entorhinal sectors (P > 0.05). In addition, in both regions, [K+]o rise associated with the ictal discharges was higher than prior to severing this pathway (Fig. 3C; P < 0.05). In contrast, [K+]o rise in CA3 was not significantly affected by the Schaffer collateral cut, and these rises remained significantly lower than in the entorhinal cortex and the dentate gyrus (Fig. 3C; P < 0.05).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. Effect of cutting the Schaffer collaterals on the 4-AP-induced epileptiform discharges and the associated [K+]o rises in the entorhinal cortex, dentate and CA3 areas. A: field and [K+]o measurements of 4-AP-induced epileptiform activity in the intact hippocampal-entorhinal slice before (left panel) and after (right panel) ictal discharges stop occurring. B: after Schaffer collateral cut, ictal discharges are disclosed, and their associated rises in [K+]o are larger than before Schaffer collateral cut in the dentate gyrus and entorhinal cortex. Note the different calibrations of the [K+]o before and after the cut. C: summary of effects of Schaffer collateral cut on [K+]o rises associated with ictal activity in different limbic areas. Schaffer collateral cut results in both dentate and entorhinal sectors in significant (P < 0.05, asterisks) increases in [K+]o elevations associated with ictal discharges as compared with the intact slice preparation. Note also that the [K+]o increases associated with ictal activity are comparable in the dentate and entorhinal cortex both before and after Schaffer collateral cut, and that they are significantly lower in CA3. Note also that Schaffer collateral cut does not affect the changes in [K+]o associated with CA3 ictal discharge. Measurements were obtained from 7 slices for both dentate and entorhinal subfields and from 5 slices for CA3.

Our results show that 4-AP-induced epileptiform activity, in particular ictal discharges in dentate and entorhinal sectors are associated with prominent increases in [K+]o (cf. Figs. 1-3). Hence we investigated whether perforant path cut would affect the field potential and the [K+]o rises associated with ictal epileptiform discharges. Severing the perforant path resulted in blockade of both the dentate ictal discharge and the associated, local increase in [K+]o, whereas it did not influence the epileptiform discharge in the entorhinal cortex nor the corresponding [K+]o rise (Fig. 4; n = 3). Therefore in our preparation, dentate paroxysmal discharges were directly dependent on synaptic propagation of epileptiform activity from the entorhinal cortex. As reported in the rat combined hippocampus-entorhinal cortex slices (Avoli et al. 1996a; Nagao et al. 1996), perforant path cut also abolished the occurrence of ictal discharges in CA3 and CA1.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. Ictal discharges propagate to the dentate gyrus from the entorhinal cortex as demonstrated by neuronal pathway cuts and simultaneous field potential and [K+]o recordings from the dentate and entorhinal areas. A: field potential and [K+]o recordings in the dentate and entorhinal cortex after prolonged incubation with 4-AP (~2 h), when only interictal epileptiform events are recorded in the intact slice preparation. B: Schaffer collateral cut discloses ictal activity and associated [K+]o elevations that can be recorded in both dentate and entorhinal areas. C: lesioning the perforant path results in blockade of ictal discharge occurrence in the dentate gyrus as well as the associated rise in [K+]o. Recordings in A-C were obtained from the same experiment.

Involvement of the temporoammonic path in ictal discharge propagation

We next examined the pathway used by ictal discharges to propagate to the hippocampus, before and after having lesioned the Schaffer collaterals. To this end, we performed simultaneous recordings in the dentate gyrus, CA3, CA1, and entorhinal cortex and determined the temporal characteristics of the onset of ictal discharge reverberation from expanded recording traces. In five experiments we found that in the intact slice preparation, when ictal discharges are still present, propagation occurred via the trisynaptic circuit (Fig. 5). As shown in Table 1, the onset delays measured in the different areas of these slices corresponded to entorhinal-dentate-CA3-CA1 propagation (Table 1).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5. Propagation of ictal activity in the intact hippocampal-entorhinal slice, when ictal events are still present. A and B: simultaneous field potential recordings of ictal and interictal discharges performed in entorhinal cortex, CA3, and CA1 (A) and in entorhinal cortex, dentate gyrus, and CA1 (B). Expanded traces of the ictal discharge onset are also illustrated below. Note that reverberation of activity initiates in the entorhinal cortex and propagates to the hippocampus via the trisynaptic pathway. Arrowheads in B point at an interictal discharge of CA3 origin.


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Time delays for ictal discharge propagation in the hippocampal-entorhinal slice before and after Schaffer collateral cut

After Schaffer collateral cut, and thus after having disrupted the trisynaptic pathway, ictal discharges of entorhinal origin were still recorded in both CA1 area and subiculum (n = 8 and n = 7, respectively) where they were accompanied by rises in [K+]o (Fig. 6, panels in column a). By employing expanded recordings, and thus by measuring the discharge onset, we also discovered that seizure activity propagated to these regions directly, thus "short-circuiting" the trisynaptic path, at delays similar to those seen for the propagation to the dentate gyrus (Fig. 6, column b; Table 1).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. Field potential and [K+]o recordings performed in the entorhinal cortex, dentate gyrus, CA3, CA1, and subiculum show different modalities of ictal discharge propagation from the entorhinal cortex to the hippocampus after Schaffer collateral cut. Samples in column b are expanded traces of the onsets of the ictal discharges shown at low speed in column a. Vertical dotted lines indicate the discharge onset. In addition, this portion of the recording is further expanded in each inset where the discharge onset in each area is pointed by different symbols. A: when recording in entorhinal cortex, dentate gyrus, and in CA3, ictal discharge propagates to CA3 via the dentate gyrus; asterisk points at an interictal discharge of CA3 origin that is "volume conducted" to the dentate area. B: ictal discharge is recorded in CA1 even though Schaffer collaterals have been cut. Note that ictal activity propagates to CA1 and dentate gyrus with similar delays. C: ictal discharge is recorded in the subiculum after Schaffer collateral cut. Expanded trace shows similar propagation delays to both subiculum and dentate gyrus.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have used field potential and [K+]o recordings in the combined, mouse hippocampal-entorhinal slice preparation to understand the propagation of epileptiform activity within the limbic system. Our results can be summarized as follows. First, entorhinal seizures, which are released from CA3 control, propagate to the dentate gyrus where they induce robust ictal discharges comparable to those seen at the site of origin (i.e., the entorhinal cortex). Second, epileptiform discharges propagate via the trisynaptic circuit in the intact hippocampal-entorhinal slice, whereas direct propagation to CA1 and subiculum is uncovered by Schaffer collateral cut. We have previously shown that Schaffer collateral cut, which mimics neuronal cell loss observed in chronic animal models of epilepsy and temporal lobe epilepsy (TLE) patients might be sufficient to disclose recurrent seizures originating in the entorhinal cortex (Barbarosie and Avoli 1997).

Ictal discharge occurrence in the dentate gyrus

Considering hippocampal connections as strictly laminar in the transverse plane represents an oversimplification of the limbic circuit. For instance, the perforant path recruits the dentate gyrus via longitudinal connections that are topographically organized (Amaral and Witter 1989). In addition, electrophysiologists studying limbic epileptiform discharges in vivo have emphasized the importance of reciprocal connectivity among the hippocampus and entorhinal cortex that, in most in vitro slice preparations is lost (e.g., Paré et al. 1992). This may explain findings obtained from normal hippocampal-entorhinal slices, in which ictal discharges, generated in the entorhinal cortex, do not appear to propagate readily to the dentate gyrus via the perforant path (Behr et al. 1998; Jones and Lambert 1990; Mody et al. 1988). Our results show that entorhinal ictal discharges that are associated with rises in [K+]o can occur in otherwise "normal" slices, and do propagate to both dentate gyrus and CA3 area. Furthermore, the dentate ictal activity (as measured in terms of associated rise in [K+]o), correlates directly with the occurrence and strength of these events at the site of onset, i.e., the entorhinal cortex. By measuring [K+]o we also ruled out the possibility that field potential epileptiform discharges recorded in the hippocampal areas represented volume conduction phenomena. This is also supported by the inversion of polarity of the field potential discharges observed with laminar analysis and by the blockade of ictal discharges following lesion of the perforant path.

The spontaneous potentials induced by 4-AP were seen in CA3, entorhinal cortex, and dentate gyrus, thus indicating that these limbic areas were all recruited to give rise to epileptiform activity. Indeed, the robust ictal discharges and the associated elevations in [K+]o recorded in the dentate gyrus were reminiscent of the maximal dentate activation described by Stringer et al. (1989). Maximal dentate activation has been reproduced in vitro in low [Ca+2]o and high [K+]o medium (Pan and Stringer 1996, 1997; Patrylo et al. 1994; Schweitzer et al. 1992). In that model, dentate ictal activity depends primarily on intrinsic granule cell properties and ephaptic, nonsynaptic mechanisms because epileptiform dicharges are unaffected by excitatory amino acid receptor antagonists.

Herein, we have shown that under physiological concentrations of Ca2+ and K+, 4-AP can induce ictal discharges in the entorhinal cortex, and that these events propagate synaptically to the hippocampal areas. Accordingly, cutting the perforant path abolished both the occurrence of ictal discharges and the associated increases in [K+]o in the dentate gyrus, demonstrating synaptic propagation of the epileptiform activity. 4-AP-induced ictal events in the adult entorhinal cortex and hippocampus are dependent on N-methyl-D-aspartate (NMDA) receptor activation (Avoli et al. 1996a).

[K+]o during 4-AP-induced ictal epileptiform activity

Ictal discharges in the entorhinal cortex and dentate gyrus are associated with elevations in [K+]o that are significantly larger than in CA3. The [K+]o increases associated with the dentate ictal discharge (that correlate well with the [K+]o rises observed in the entorhinal cortex) can be explained through the high density of granule cells and the low extracellular space in this region. On the other hand, the low [K+]o associated with ictal discharge in CA3 versus dentate gyrus may be due to the reported preferential activation of interneurons by mossy fibers. Accordingly, the increased granule cell activity may suppress the excitability of the CA3 recurrent system (Acsady et al. 1998). Alternatively, presynaptic activation of metabotropic receptors may limit the bursting capacity of CA3 neurons and thus give rise to a smaller ictal discharge in CA3 (Scanziani et al. 1997). Moreover, bursting behavior of CA3 pyramidal neurons is tightly dependent on the pool size of available synaptic vesicles for release (Staley et al. 1998). These mechanisms may act in concert to protect excessive neuronal activity and damage to this region, which is critical in controlling the generation of ictal discharges within the entorhinal cortex (Barbarosie and Avoli 1997).

Laminar analysis of the ictal discharges recorded in CA3 indicates that in the combined hippocampus-entorhinal cortex slice, the largest [K+]o rises occur in the CA3 pyramidal cell layer. This is in agreement with a study made in juvenile, "isolated" rat hippocampal slices treated with 4-AP (Avoli et al. 1996b). In this preparation the [K+]o elevations associated with ictal discharges in CA3 attain maximal values in the pyramidal layer. However, they are preceded by an initial increase in [K+]o (which trigger them and coincides with a GABA-mediated synchronous potential) that was even greater than what was seen during the discharge and attained largest value in stratum radiatum. Our present findings indicate that, although ictal discharges in CA3 were associated with the largest [K+]o in the pyramidal layer, they were probably not initiated by an initial, GABA-mediated rise in [K+]o, as was the case in juvenile CA3. Rather, they simply resulted from propagation through the dentate gyrus of an ictal event originating in the entorhinal cortex.

Mossy fiber sprouting and chronic models of TLE

Mossy fiber reorganization and increased epileptogenicity of dentate granule cells is a hallmark of human TLE (Houser et al. 1990; Sutula et al. 1989) and chronic animal models of TLE (Behr et al. 1998; Cavazos et al. 1991; Represa et al. 1989; Sutula et al. 1988; Wuarin and Dudek 1996). Parent et al. (1997) have also reported that ectopic granule cell neurogenesis is associated with sprouting in this region and correlates with increased seizure activity. However, blocking mossy fiber sprouting does not interfere with the appearance of recurrent epileptic seizures (Longo and Mello 1997, 1998). Our results demonstrate that robust ictal discharges do occur in the dentate gyrus of "normal" slices (and hence in the absence of synaptic reorganization). Hence, although the dentate gyrus lacks the recurrent excitatory connections to support generation of seizure activity, our data show that it can be fully activated when entorhinal inputs impinge on it.

We have reported that CA3 neuronal damage, another hallmark of TLE (Ben-Ari 1985), mimicked by Schaffer collateral cut of an intact hippocampal-entorhinal slice, may be sufficient to disclose the generation of recurrent entorhinal seizures (Barbarosie and Avoli 1997). Therefore increased dentate gyrus epileptogenicity, evident in the chronic condition, may also result from the propagation of uncovered entorhinal cortex seizure activity.

Role of a temporoammonic pathway in epilepsy

It has been demonstrated that under normal conditions, depression of synaptic transmission between areas CA3 and CA1 discloses the operativity of a perforant path projection from the entorhinal cortex to the CA1 region (Maccaferri and McBain 1995). In our experiments, Schaffer collateral cut (which blocks CA3-driven excitatory inputs to both CA1 pyramids and interneurons) is sufficient to "short-circuit" the propagation of 4-AP-induced epileptiform activity via the entorhinal-CA1 path.

In chronic animal models of TLE, entorhinal layer III neurons are preferentially lost (Du et al. 1995), and inhibitory postsynaptic potentials appear impaired in both superficial and deep entorhinal layers in slices obtained from such animals (Bear et al. 1996; Fountain et al. 1998). It is attractive to postulate that CA1 pyramidal cell hyperexcitability observed in epileptic tissue (Williams et al. 1993) may, at least in part, be conferred via the direct excitation of CA1 pyramidal cells by disinhibited entorhinal neurons. Hence CA3 neuronal cell loss, and Schaffer collateral damage, also observed in models of TLE (Ben-Ari 1985), in addition to potential disinhibition of the entorhinal cortex, may contribute to disclose a direct entorhinal-CA1 pathway.


    ACKNOWLEDGMENTS

We thank T. Papadopoulos for secretarial assistance.

This study was supported by the Medical Research Council of Canada (Grant MT-8109), the Savoy Foundation, and the Hospital for Sick Children (Grant XG-93056). M. Barbarosie is the recipient of a Fonds de la Recherche en Santé du Québec studentship.


    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 21 May 1999; accepted in final form 25 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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