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Received 30 October 1997; accepted after revision 19 February 1998.
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ABSTRACT |
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INTRODUCTION |
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Enhancement of synaptic excitation and inhibition by the convulsant 4-aminopyridine (4AP) leads to the appearance of epileptiform activity (Rutecki, Lebeda & Johnston, 1987; Perreault & Avoli, 1992; Barkai, Friedman, Grossman & Gutnick, 1995). Synchronous GABA-mediated potentials and ictal discharges constitute the most typical pattern of 4AP-induced activity in the entorhinal cortex (Avoli et al. 1996). However, in some experiments interictal discharges can occur; these interictal events progress to ictal discharges, while synchronous GABA-mediated potentials are not detected during this pattern of activity (Avoli et al. 1996). Previous studies have revealed laminar differences in the functional organization of entorhinal cortex neurones, which depend on synaptic and intrinsic membrane properties (Jones & Heinemann, 1988; Alonso & Klink, 1993; Jones, 1994; Berretta & Jones, 1996). Both transmitter-mediated potentials and intrinsic voltage-dependent mechanisms contribute to epileptiform discharges (Schwartzkroin & Prince, 1980; Johnston & Brown, 1981; Gutnick, Connors & Prince, 1982; Dingledine, Hynes & King, 1986; Rutecki et al. 1987; Chagnac-Amitai & Connors, 1989; Perreault & Avoli, 1992; Traub, Miles & Jefferys, 1993). Hence, we employed field potential and intracellular recordings to determine the laminar contribution of entorhinal cortex neurones to 4AP-induced interictal and ictal discharges. In particular, we established the site of initiation of these epileptiform events by using knife cut dissection as well as the participation of NMDA- and non-NMDA-mediated potentials in layer II and IV-VI neurones. In addition we documented the occurrence of Ca2+-mediated spikes to the 4AP-induced epileptiform activity.
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METHODS |
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Preparation and maintenance of the slices
Adult, male Sprague-Dawley rats (200-300 g) were decapitated under halothane anaesthesia in accordance with the guidelines established by the Canadian Council on Animal Care. The procedures for preparing and maintaining combined hippocampus- entorhinal cortex slices have been previously described (Avoli et al. 1996; Nagao et al. 1996). In brief, horizontal brain slices (500 µm thick) were cut with a vibratome and were transferred to a recording chamber where they were maintained at a temperature of 33·5 ± 0·5°C in an interface between humidified gas (95 % O2-5 % CO2) and oxygenated artificial cerebrospinal fluid (ACSF, pH 7·4). The ACSF composition was (mM): 124 NaCl, 2 KCl, 2 MgSO4, 2 CaCl2, 1·25 KH2PO4, 26 NaHCO3 and 10 glucose. 4AP (50 µM, Sigma), 3,3-(2-carboxy-piperazine-4-yl)-propyl-1-phosphonate (CPP, 10 µM, Tocris Cookson), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM, Tocris Cookson) and NiCl2 (1 mM) were applied to the bath. The rate of perfusion (0·3-0·5 ml min-1) was kept constant in each experiment.
Recording procedures
Field potential recordings were made with glass pipettes that were filled with ACSF (resistance, 1-5 M
). Intracellular recording microelectrodes were filled with 3 M potassium acetate (resistance, 60-100 M) or 3 M potassium acetate and 50 mM 2-(tri-methyl-amino)-N-(2,6-dimethyl-phenyl)-acetamide (QX-314, a kind gift of Astra, Toronto, Canada) (resistance, 60-100 M). Field potential and intracellular electrodes were placed in layer II and/or in layers IV-VI (200-400 µm and 600-1000 µm from the pia, respectively) of the lateral portion of the entorhinal cortex (Köhler, 1988). The distance between field potential and intracellular electrodes was 100-200 µm.
Extracellular signals were fed to a Neuro Data amplifier, while intracellular signals were fed to an Axoclamp 2 amplifier with internal bridge circuit that allowed current to be passed through the intracellular microelectrode. The bridge was monitored carefully throughout the experiments and adjusted as necessary. Signals were displayed on a digital oscilloscope and on a Gould WindoGraf recorder; they were digitized by NeuroCorder and recorded on videocassette for later analysis.
Analysis and data base
The intracellular activity of thirty-seven neurones from thirty slices was recorded during bath application of 4AP. The resting membrane potential (RMP) measured after microelectrode withdrawal was
Table 1. Passive membrane characteristics of neurones layers II and IV-VI
4AP-induced epileptiform activity in the entorhinal cortex
Field potential recordings from the lateral portion of the entorhinal cortex revealed interictal discharges that occurred at a rate of 0·6 ± 0·7 s-1 (n = 30 slices) and long-lasting ictal discharges (duration, 76·7 ± 21·1 s; interval of occurrence, 8·1 ± 4·1 min; n = 22 slices) (Fig. 1A). This pattern of epileptiform activity occurred in 30 of 138 slices, while the majority of the slices generated spontaneous GABA-mediated synchronous potentials and ictal discharges (Avoli et al. 1996). GABA-mediated synchronous potentials were not recorded in these thirty slices which represent the data base of our study.
Simultaneous field potential recordings revealed different features of the epileptiform discharges recorded in layer II and layers IV-VI of the entorhinal cortex. Interictal discharges in layer II (350 µm sample in Fig. 1A) had a pronounced initial positive and a subsequent smaller negative component. At this recording site the ictal discharges were characterized by the rhythmic occurrence of population spikes superimposed on a rather brief negative DC shift. Interictal discharges in layer V (830 µm sample in Fig. 1A) were made of a negative component while the ictal discharges were associated with a pronounced negative DC shift as compared with layer II ictal discharges. Epileptiform discharges recorded in layers II and IV-VI were highly synchronized.
A, simultaneous recording of the interictal and ictal discharges from layer II (sample 350 µm) and layer V (sample 830 µm from the pia). Note the positive component of interictal discharges in layer II, the corresponding negative component in layer V and the larger DC shift during the ictal discharge in layer V. B, both interictal and ictal discharges disappear in layer II, but continue to occur in layer V after knife cut separation at a depth of 600 µm (i.e. between layers III and IV). C and D, plots of the changes in ictal discharge duration and interval in layers IV-VI after knife cut separation.
Site of origin of entorhinal epileptiform discharges
Simultaneous field potential recordings were performed before and after surgical separation of entorhinal layers II and IV-VI (n = 4 slices). In these experiments one electrode was placed at a depth 280-350 µm (layer II) and another at 830-1000 µm (layers V or VI) from the pia. As described above synchronized interictal and ictal discharges occurred at both recording sites under control conditions (Fig. 1A). However, epileptiform discharges disappeared in layer II (350 µm sample in Fig. 1B), but continued to occur in layer V (830 µm sample in Fig. 1B) after a knife cut was made at approximately 600 µm from the pia (i.e. between layers III and IV). The small amplitude potentials recorded in layer II under this experimental condition represented presumably volume conduction events. The duration of the ictal discharges recorded in the deep layers after surgical separation decreased from 75·0 ± 29·1 to 64·8 ± 22·2 s and the interval of occurrence from 8·5 ± 5·9 to 8·3 ± 3·3 min (n = 4 slices) (see Fig. 1C and D). Both differences were, however, not significant. Knife cut of the entorhinal cortex from the hippocampus in four additional experiments did not influence the occurrence of entorhinal interictal and ictal epileptiform discharges (not shown).
Intracellular features of the epileptiform potentials in layers IV-VI
The intracellular activity of seventeen cells was recorded at depths of 610-980 µm from the pia (layers IV-VI) in the presence of 4AP. Interictal depolarizations (amplitude, 29·4 ± 8·6 mV; duration, 386 ± 177·4 ms; n = 17) occurred synchronously with the negative field potential deflection and contained two components (Fig. 2A and Ba). The initial component was capped by a burst of action potentials (frequency up to 100 s-1) and corresponded to the initial, fast field potential deflection (arrows in Fig. 2Ba). The late component of the interictal depolarizations contained one to four 'humps' and coincided with the slow, negative field potential event (arrowheads in Figs 2Ba and 3A, -74 mV).
Ictal discharges recorded in these cells were characterized by large amplitude (31·5 ± 6·2 mV, n = 12) membrane depolarizations that were initiated by an interictal discharge with field potential and intracellular characteristics similar to those of the preceding interictal events (Fig. 2A). The initial component was capped by an action potential burst and corresponded to the initial, fast field potential event (arrows in Fig. 2Ba),while the late component progressed toward the membrane ictal depolarization (arrowheads in Fig. 2Ba). Fast depolarizing potentials (amplitude, 23-72 mV, measured from the level of the ictal depolarization) occurred at 5-12 s-1 during the ictal discharge. These rhythmic depolarizations corresponded to field potential population spikes and were capped by high frequency (up to 100 s-1) action potential bursts (Fig. 2Bb).
A, simultaneous field potential and intracellular recordings (upper and lower traces in this and subsequent figures) of the 4AP-induced epileptiform activity. Note the prolonged high amplitude depolarization underlying the ictal discharge. Ba, expanded trace of the ictal discharge onset and of the preceding interictal discharge. Ictal discharge is initiated by an interictal discharge which contains both an initial (arrows) and a subsequent prolonged (arrowheads) intracellular and field potential components similar to those of the preceding interictal discharge. Note that the late component of the interictal depolarization progresses into the ictal depolarization (arrowheads). Bb, expanded trace of the action potential bursts occurring synchronously with field potential population spikes during ictal discharge. RMP, -76 mV.
The interictal depolarizations recorded in layer IV-VI neurones were studied by changing the membrane potential with steady injection of positive or negative current through the intracellular microelectrode (n = 8, Fig. 3A). The amplitude of the initial component (measured up to the inflection of the first action potential in the burst, arrows in Fig. 3A) and of the late component (measured up to the first 'hump', arrowheads in Fig. 3A) changed in a different fashion during this procedure. During steady membrane depolarization the amplitude of the late component reached values similar to those of the initial component (Fig. 3A (-64 mV) and 3B). By contrast, during steady membrane hyperpolarization the late component did not change in amplitude, while the initial component increased linearly (Fig. 3A (-92 mV) and 3B). The duration of the action potential burst (measured at the level of the first action potential inflection) increased to 100 ms during injection of steady depolarizing current, and decreased to 27 ms during hyperpolarization (Fig. 3C). These modifications were associated with changes in the number of action potentials in the burst (cf. -64 and -92 mV samples in Fig. 3A). Interictal depolarizations were not followed by a hyperpolarizing potential even during steady membrane depolarization or at RMP (Fig. 3A).
A, at RMP (-74 mV), the interictal intracellular potential is characterized by an initial component with action potential burst (arrow), and a late component (arrowhead). During steady membrane depolarization (-64 mV) the late component is enhanced and action potential burst increases. By contrast, membrane hyperpolarization (-92 mV) depresses the late component and reduces the action potential burst. B, plot of the initial and late component amplitudes versus membrane potential. The amplitude of the initial component was measured up to the level of the first action potential in the burst (arrows), while the late component amplitude was measured up to the peak of the first 'hump' (arrowheads). C, plot of the action potential burst duration (measured at the level of the first action potential in the burst, arrows) versus membrane potential.
Intracellular features of the epileptiform potentials in layer II
The activity of ten neurones was recorded at depths of 220-380 µm (layer II) during 4AP-induced interictal and ictal discharges. In all cases the intracellular counterpart of the positive-negative sequence of interictal field potentials consisted of a depolarization (amplitude, 11·7 ± 5·8 mV; duration, 192·6 ± 47·9 ms; n = 10) that could trigger a single action potential (Fig. 4Ba). In 7 of 10 cells this depolarization was followed by a hyperpolarizing potential (amplitude, 4·8 ± 1·7 mV; duration, 536·4 ± 96·4 ms; n = 7; asterisk in Fig. 4Ba; see also Fig. 5B, -55 mV). Hyperpolarizations did not occur in neurones (n = 4) that were recorded with microelectrodes filled with QX-314, which is an intracellular blocker of GABAB-mediated inhibition (Nathan, Jensen & Lambert, 1990) and of voltage-gated Na+ channels (Gutnick et al. 1982).
A, simultaneous field potential and intracellular recordings of the epileptiform activity. Note the low amplitude, ictal depolarization. Ba, expanded traces of an interictal discharge. Note that the interictal depolarization is small in amplitude and short lasting; this intracellular event corresponds to a positive field potential (arrows), and is associated with single action potential discharge followed by a hyperpolarization (
The intracellular ictal discharges recorded in layer II neurones were characterized by long-lasting depolarization with an average amplitude of 11·6 ± 3·5 mV (n = 7) (Fig. 4A). In these neurones as well, the ictal discharge was initiated by an interictal event with extracellular and intracellular characteristics similar to those of the isolated interictal potentials (cf. Fig. 4Ba and b). The initial depolarizing potential of the ictal discharge corresponded to the positive component of the field potential (arrows in Fig. 4Bb) and progressed to a series of low amplitude membranedepolarizations. Fast depolarizing potentials (amplitude, 10-20 mV, measured from the level of the ictal depolarization) occurred rhythmically at 5-12 s-1 and were capped by single action potentials (Fig. 4Bc). These potentials occurred synchronously with field potential population spikes. Three neurones in layer II did not generate action potentials during the interictal depolarizations, while sporadic single action potentials occurred during low amplitude ictal depolarizations (Fig. 5A).
The amplitude of the interictal depolarizations recorded in layer II neurones decreased during steady membrane depolarization (cf. -55 and -72 mV samples in Fig. 5B). This procedure also made the late hyperpolarizing potential increase in amplitude. By contrast, steady membrane hyperpolarization increased the amplitude of the interictal depolarization, blocked the action potentials and reversed the late hyperpolarization at -85·5 ± 4·0 mV (n = 4) (Fig. 5B; -90 mV sample). The amplitude of the interictal depolarization recorded in these cells depended on membrane potential in a quasi-linear fashion (Fig. 5C).
A, interictal and ictal events recorded in this cell (260 µm from the pia) lack action potential discharge; only sporadic action potentials occur during the low-amplitude ictal depolarization. RMP, -68 mV. B, electrophysiological features of the interictal potentials in another layer II neurone (320 µm from the pia). At the RMP (-72 mV) the interictal intracellular potential contains a low-amplitude short-lasting depolarization with single action potential and subsequent hyperpolarization. Membrane depolarization (-55 mV) enhances the hyperpolarization, while steady membrane hyperpolarization (-90 mV) blocks the action potential and inverts the hyperpolarization. C, plot of the amplitudes of the interictal depolarizations versus membrane potential level.
Pharmacological features of epileptiform discharges
Bath application of the NMDA receptor antagonist CPP (10 µM) abolished the ictal activity (n = 4 slices; not shown), and modified the intracellular interictal depolarizations and the corresponding field potentials in layers IV-VI (cf. control and CPP samples in Fig. 6A). CPP reduced the duration of the interictal depolarizations from 394·7 ± 85·8 to 257·5 ± 86·2 ms (n.s., n = 4; Fig. 6C) and their amplitude from 28·8 ± 4·6 to 23·3 ± 5·4 mV (n.s., n = 4; Fig. 6C). The effects induced by CPP were characterized by depression of the late component of the interictal depolarization and disappearance of the corresponding late, field potential negative component. These changes were accompanied by enhancement of the initial positive component. The small changes in amplitude of the interictal depolarizations were, however, accompanied by significant reduction in action potential burst (Fig. 6A and C) and thus only single action potentials occurred during NMDA receptor blockade. CPP had no effect on the current-induced firing responses (Fig. 6B). CPP application did not influence the interictal depolarizations and the corresponding field potentials in layer II cells. The amplitude of these depolarizations changed from 10·3 ± 5·4 to 9·8 ± 6·2 mV and the duration from 195·6 ± 55·7 to 179·4 ± 61·4 ms (n = 3, n.s.). Further application of the non-NMDA receptor antagonist CNQX (10 µM, n = 3) abolished the interictal activity in the entorhinal cortex (Fig. 6D).
A, the NMDA receptor antagonist CPP (10 µM) decreases the interictal depolarization amplitude and duration, and abolishes the late negative field potential component. Note the block of action potential burst induced by CPP. B, CPP has no effect on action potential firing induced by intracellular injection of depolarizing current pulses. C, plots of the changes induced by CPP on the interictal depolarization duration, amplitude and action potential burst. D, further application of the non-NMDA receptor antagonist CNQX (10 µM) abolishes the interictal discharges. RMP, -75 mV.
Ca2+-mediated spikes and epileptiform discharges
The epileptiform activity generated by layer II and IV-VI neurones (4 and 6, respectively) was recorded with intracellular microelectrodes filled with potassium acetate and QX-314. Under these conditions all cells responded to depolarizing current pulses (100-200 ms, 0·2-1 nA) with single or double slow spikes (amplitude, 91·4 ± 9·2 mV; duration, 67·4 ± 8·4 ms; n = 10), that were elicited at membrane potentials more positive than -67 mV (Fig. 7A). Application of the low-threshold Ca2+ channel blocker Ni2+ (1 mM, n = 4) abolished these potentials (Fig. 7B) and made 4AP-induced synchronous activity disappear.
Interictal depolarizations recorded with potassium acetate- QX-314-filled microelectrodes were also capped by presumptive Ca2+-mediated spikes that corresponded to field potential population spikes (Fig. 7C). The Ca2+-mediated events associated with the epileptiform potentials were blocked by steady membrane depolarization to values more positive than -39·2 ± 3·5 mV (n = 6) with positive current injection (1-2 nA) (Fig. 7D). Under these conditions the inverted interictal potentials had an initial short-lasting component with a reversal potential of -43·3 ± 4·2 mV (n = 4) (arrow in Fig. 7D) and a subsequent component with a reversal potential of -24·3 ± 2·5 mV (n = 4) (arrowhead in Fig. 7D). Ca2+-mediated spikes (amplitude, 20-83 mV, measured from the ictal depolarization level) occurred rhythmically at 5-12 s-1 during the ictal discharge and corresponded to the field potential population spikes (Fig. 8A and B). Intracellular injection of long-lasting pulses of positive current (1-4 s, 0·5-1 nA) induced sequences of Ca2+-mediated spikes at 4-7 s-1 which did not correspond to any synchronous field potential event (Fig. 8C).
A, intracellular pulses of depolarizing current induce slow spikes with different amplitude and duration. RMP, -78 mV; cell located 880 µm from the pia. B, slow spikes are blocked by application of the Ca2+ channel blocker Ni2+ (1 mM). RMP, -70 mV; cell located 350 µm from the pia. C, slow, Ca2+-mediated spikes of different amplitude and duration occur during the interictal depolarization and are synchronized with field potential population spikes. D, Ca2+-mediated spikes associated with the interictal depolarization are blocked by steady membrane depolarization. Note the inversion of the initial (arrow) and subsequent (arrowhead) interictal depolarization components. In C and D same neurone as in A.
A, Ca2+-mediated spikes occur during interictal and ictal events and are synchronized with field potential population spikes. B, Ca2+-mediated spikes of different amplitude and duration occur during the ictal discharge. C, intracellular long-lasting pulses of the depolarizing current induce sequences of Ca2+ spikes with no synchronous field potential counterpart. Neurone in this figure had an RMP of -82 mV and was located 720 µm from the pia.
4AP-induced epileptiform activity in the entorhinal cortex
Our results indicate that the rat entorhinal cortex can generate interictal and ictal discharges during 4AP application. This pattern of activity deviates from what has been described under similar experimental conditions when GABA-mediated synchronous potentials and ictal discharges occurred in the entorhinal cortex (Avoli et al. 1996). Such an interictal-ictal pattern was detected in only one-fifth of the 4AP-treated entorhinal cortex slices. Bursting neurones play a substantial role in the initiation of interictal discharges (Traub & Wong, 1982; Miles & Wong, 1983; Connors, 1984; Chagnac-Amitai & Connors, 1989; Traub et al. 1993). Hence, the uncommon occurrence of interictal discharge in the entorhinal cortex may reflect the sparse presence of bursting neurones in this structure (Jones & Heinemann, 1988; Alonso & Klink, 1993). 4AP-dependent expression of GABA-mediated inhibition may also determine the patterns of synchronous activity (Rutecki et al. 1987; Perreault & Avoli, 1992). Accordingly, synchronous GABA-mediated potentials were not recorded in those slices that generated interictal activity. We have reported that µ-opioid receptor activation (which prevents release of GABA from inhibitory terminals; Capogna, Gähwiler & Thompson, 1993) abolishes GABA-mediated synchronous potentials and induces interictal discharges (Avoli et al. 1996).
However, the reversal potential of the interictal depolarizations induced by 4AP in the entorhinal neocortex had values more negative (i.e. between -43 and -24 mV) than those reported for interictal depolarizations induced by GABAA receptor antagonists (about 0 mV) in the neocortex (Gutnick et al. 1982) or in the hippocampus (Johnston & Brown, 1981). This finding may indicate that in the entorhinal cortex GABA-mediated potentials participate in interictal discharges as documented for 4AP-induced interictal depolarizations in hippocampal neurones (Rutecki et al. 1987).
Findings obtained with simultaneous field potential recordings from superficial and deep layers of the entorhinal cortex have shown that interictal and ictal discharges induced by 4AP are more robust in layers IV-VI. Moreover, both types of epileptiform activity continue to occur in the deep layers, but disappear in the superficial layers following surgical separation. Therefore, as proposed in previous studies (Jones & Heinemann, 1988; Jones & Lambert, 1990; Avoli et al. 1996) deep layer neurones in the lateral entorhinal cortex are the epileptiform activity generators.
Interictal potentials in layers IV-VI
Interictal depolarizations recorded in layer IV-VI cells include a late component that is depressed by steady membrane hyperpolarization, enhanced by membrane depolarization, and abolished by the NMDA receptor antagonist CPP. NMDA-mediated postsynaptic events are recorded in neurones located in the entorhinal cortex deep layers (Jones & Heinemann, 1988; Jones & Lambert, 1990; Berretta & Jones, 1996). Moreover, depression of the NMDA-mediated component abolishes the action potential burst associated with the interictal depolarization, an effect that occurs in spite of a small, non-significant change in interictal depolarization peak amplitude. Therefore, NMDA-mediated EPSPs contribute to the occurrence of action potential bursts, which in turn may play a crucial role in the synchronization and propagation of epileptiform activity across the entorhinal cortex. Reduction of epileptiform activity by NMDA receptor antagonists has been described in the hippocampus and neocortex (Herron, Williamson & Collingridge, 1985; Hwa & Avoli, 1991). Our results also demonstrate that the initial component of the interictal depolarization of layer IV-VI cells is non-NMDA mediated, since it is resistant to CPP, and it is abolished by CNQX.
Hyperpolarizing potentials do not follow the interictal depolarizations recorded in layer IV-VI cells either at RMP or during membrane depolarization. Since 4AP enhances postsynaptic inhibition (Rutecki et al. 1987; Perreault & Avoli, 1992; Barkai et al. 1995), the absence of post-burst hyperpolarization may indicate that in the entorhinal cortex deep layers, GABA-mediated inhibition plays a minimal role in terminating interictal discharges. Previous studies have shown that postsynaptic inhibition is weak in the entorhinal cortex deep layers (Jones, 1987; Jones & Heinemann, 1988).
Interictal potentials in layer II
NMDA-mediated potentials are recorded in layer II neurones under normal conditions (Jones, 1994), as well as during epileptiform discharges induced by GABAA receptor antagonists (Jones & Lambert, 1990) or bursting responses in chronically epileptogenic neurones (Bear, Fountain & Lothman, 1996). In our study we did not observe any NMDA-mediated component, which may be due to the potentiation of the GABA-mediated inhibition exerted by 4AP. In line with this view, hyperpolarizations followed the interictal depolarizations recorded in layer II cells, presumably exercising an inhibitory action on the late NMDA-mediated component. We are also inclined to consider these hyperpolarizations to be due to GABAB receptor activation, as indicated by their reversal potential (approximately -86 mV), duration (around 500 ms) and sensitivity to intracellular injection of QX-314 (Nathan et al. 1990). GABAB-mediated IPSPs are recorded in layer II cells in normal (Jones, 1994) and epileptogenic tissue (Bear et al. 1996) and can depress NMDA-mediated EPSPs (Jones, 1994). Non-NMDA-mediated interictal depolarizations in layer II neurones were twofold smaller than those recorded in layers IV-VI during NMDA receptor blockade. This characteristic may be due to differences in glutamate quantal release among layers (Berretta & Jones, 1996) or to the inhibitory action of the post-interictal hyperpolarization generated by layer II cells.
Ictal discharges
In layer IV-VI neurones the late NMDA-mediated component of the interictal depolarizations progressed to a long-lasting depolarization, thus giving origin to the ictal discharge. Findings obtained in this and previous studies (Jones & Heinemann, 1988; Avoli et al. 1996; Nagao et al. 1996) indicate that the occurrence of entorhinal ictal discharges involves activation of NMDA receptors. In the presence of 4AP, ictal depolarizations in layer IV-VI neurones had amplitude values (around 30 mV) similar to those of the NMDA-mediated interictal depolarizations.
Ictal and interictal depolarizations generated by layer II neurones had similar amplitudes (approximately 10 mV), thus reaching values that were threefold lower than analogous depolarizations recorded in layer IV-VI cells. Moreover, ictal discharges in layer II neurones were not accompanied by action potential bursts, while some cells remained silent. Neurones with similar properties (i.e. high threshold for action potential generation, non-NMDA-mediated EPSPs) have been described recently in the entorhinal cortex layer II under normal conditions (Empson, Gloveli, Schmitz & Heinemann, 1995). Since ictal discharges appear to 'attenuate' in layer II after propagating from the deep layers, it is likely that these neurones function as filters thus playing a role in controlling epileptiform activity output. Weakening of 4AP-induced epileptiform activity has been shown in superficial neocortical layers (Barkai et al. 1995).
Neurones in layers IV-VI generate rhythmic bursting activity during ictal discharge, while only a small percentage of these entorhinal cells have intrinsic burst responses under normal conditions (Jones & Heinemann, 1988). NMDA-mediated potentials in layers IV-VI may contribute to these bursts during ictal and interictal discharges. Moreover, since tonic firing is transformed into bursting by increasing [K+]o (Jensen, Azouz & Yaari, 1994; Andreasen & Lambert, 1995), different patterns of firing recorded from deep and superficial layer neurones during ictal discharges may also depend upon a different [K+]o homeostasis across the entorhinal layers. Indeed, [K+]o measurements during 4AP-induced ictal activity have shown twofold increases in the deep layers (up to 14 mM) as compared with those in layer II (up to 7 mM) (Avoli et al. 1996).
Ca2+-mediated spikes
Entorhinal neurones in layers II and IV-VI loaded with QX-314 generate slow spikes during intracellular injection of depolarizing current pulses as well as during interictal and ictal discharges. We consider these potentials to represent mostly low-threshold, Ca2+-mediated spikes since they are activated at relatively negative membrane potential values and are blocked by Ni2+ and steady membrane depolarization (Burlhis & Aghajanian, 1987; de la Peña & Geijo-Barrientos, 1996). The occurrence of voltage-dependent, Ca2+-mediated spikes during interictal depolarizations has been reported (Gutnick et al. 1982; Witte, Speckmann & Walden, 1987), while Ca2+-mediated dendritic bursts may contribute to 4AP-induced ictal discharges in the hippocampus (Traub, Borck, Colling & Jeffreys, 1996).
These Ca2+-mediated spikes occur synchronously with population spikes which correspond to rhythmic depolarizations and fast action potential discharges in layer II and IV-VI neurones recorded with potassium acetate-filled microelectrodes. Therefore, Ca2+-mediated events may underlie the rhythmic action potential discharge seen during the ictal activity. This process is more prominent in the deep entorhinal cortex layers where an NMDA-mediated mechanism contributes to epileptiform activity and may facilitate the occurrence of Ca2+-mediated currents (Dingledine et al. 1986).
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Acknowledgements
This study was supported by the MRC of Canada (grant MT-8109), the Savoy Foundation, the Hospital for Sick Children Foundation (grant XG-93056) and the Quebec Heart and Stroke Foundation. We thank Siobhán McCann for secretarial assistance.
Corresponding author
M. Avoli: 3801 University Street, Montreal, Quebec H3A 2B4, Canada.
Email: cyav{at}musica.mcgill.ca
Author's present address
V. Lopantsev: Department of Neurological Surgery, University of Washington Medical Center, 1959 NE Pacific Street, Box 356470, Seattle, WA 98195, USA.
This article has been cited by other articles:
-60 mV, the action potential amplitude (calculated from the baseline) was 90 mV and the apparent input resistance obtained from maximum voltage changes in response to hyperpolarizing current pulses (100-200 ms, -0·4 to -0·6 nA) was 27 M. Membrane characteristics are summarized in Table 1 for layer II and IV-VI cells. The amplitude and duration of the intracellular potentials were measured from RMP, unless otherwise specified. Quantitative results are expressed throughout this study as the mean ±
Layer II (n = 10) Layers IV-VI (n = 17) RMP (mV)1 -72·8 ± 6·4 -77·5 ± 3·7 Action potential amplitude (mV)2 98·1 ± 4·1 103·1 ± 7·1 Rm (M )2 47·6 ± 14·3 47·9 ± 12·5
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Figure 1. Field potential recordings of 4AP-induced epileptiform discharges in the lateral portion of the entorhinal cortex
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Figure 2. Interictal and ictal potentials in a layer V neurone (870 µm from the pia)
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Figure 3. Electrophysiological features of interictal discharges recorded in a layer V cell (810 µm from the pia)
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Figure 4. Interictal and ictal potentials in a layer II neurone (280 µm from the pia)
). Bb, expanded trace of the ictal discharge onset. Note that the ictal discharge is initiated by an interictal event with field potential and intracellular characteristics similar to those of the isolated interictal discharge. Bc, expanded trace of the single action potential discharges that occur synchronously with field potential population spikes during the ictal event. RMP, -73 mV. Action potentials are truncated in Ba-Bc.
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Figure 5. Epileptiform discharges in a layer II neurone which appears to be 'silent'
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Figure 6. Pharmacological properties of the interictal depolarizations generated by a layer IV-VI neurone (770 µm from the pia)
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Figure 7. Ca2+-mediated spikes recorded with potassium acetate-QX-314 filled microelectrodes in entorhinal cortex neurones
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Figure 8. Ca2+-mediated spikes recorded with potassium acetate-QX-314-filled microelectrodes during epileptiform discharges
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References
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