Characteristics of Plateau Activity During the Latent Period Prior to Epileptiform Discharges in Slices From Rat Piriform Cortex

Rezan Demir, Lewis B. Haberly, and Meyer B. Jackson

Departments of Physiology and Anatomy and Center for Neuroscience, University of Wisconsin Medical School, Madison, Wisconsin 53706


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Demir, Rezan, Lewis B. Haberly, and Meyer B. Jackson. Characteristics of Plateau Activity During the Latent Period Prior to Epileptiform Discharges in Slices From Rat Piriform Cortex. J. Neurophysiol. 83: 1088-1098, 2000. The deep piriform region has an unusually high seizure susceptibility. Voltage imaging previously located the sites of epileptiform discharge onset in slices of rat piriform cortex and revealed the spatiotemporal pattern of development of two types of electrical activity during the latent period prior to discharge onset. A ramplike depolarization (onset activity) appears at the site of discharge onset. Onset activity is preceded by a sustained low-amplitude depolarization (plateau activity) at another site, which shows little if any overlap with the site of onset. Because synaptic blockade at either of these two sites blocks discharges, it was proposed that both forms of latent period activity are necessary for the generation of epileptiform discharges and that the onset and plateau sites work together in the amplification of electrical activity. The capacity for amplification was examined here by studying subthreshold responses in slices of piriform cortex using two different in vitro models of epilepsy. Under some conditions electrically evoked responses showed a nonlinear dependence on stimulus current, suggesting amplification by strong polysynaptic excitatory responses. The sites of plateau and onset activity were mapped for different in vitro models of epilepsy and different sites of stimulation. These experiments showed that the site of plateau activity expanded into deep layers of neighboring neocortex in parallel with expansions of the onset site into neocortex. These results provide further evidence that interactions between the sites of onset and plateau activity play an important role in the initiation of epileptiform discharges. The site of plateau activity showed little variation with different stimulation sites in the piriform cortex, but when stimulation was applied in the endopiriform nucleus (in the sites of onset of plateau activity), plateau activity had a lower amplitude and became distributed over a much wider area. These results indicate that in the initiation of epileptiform discharges, the location of the circuit that generates plateau activity is not rigidly defined but can exhibit flexibility.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies both in vivo (Croucher et al. 1988; Piredda and Gale 1985, 1986; Racine et al. 1988; Stevens et al. 1988) and in vitro (Hoffman and Haberly 1991, 1996) have identified the piriform cortex (PC) as a brain region with an unusually high seizure susceptibility. Microelectrode recordings in brain slices have identified the endopiriform nucleus (En), situated beneath deep layer III of the PC, as a focus for the initiation of epileptiform discharges (Hoffman and Haberly 1991, 1993). Subsequent voltage imaging experiments showed that under some conditions layer VI of the neighboring neocortex can also contribute (Demir et al. 1998).

When slices of PC are stimulated electrically with currents just above the threshold for generation of epileptiform discharges, a pronounced latency of up to 100-150 ms elapses before a discharge appears (Demir et al. 1998, 1999; Hoffman and Haberly 1989, 1991). However, a slice is not completely quiescent during this "latent period." An accelerating buildup of electrical activity can be seen at the site of discharge onset (onset activity) (Demir et al. 1999; Hoffman and Haberly 1993). Similar activity in the hippocampus has provided support for models of discharge initiation by regenerative buildup through recurrent excitatory synapses (Traub and Miles 1991; Traub et al. 1989). Multisite voltage imaging has revealed an additional form of latent period activity with very different temporal qualities. A low-amplitude, sustained depolarization (plateau activity) appeared at the beginning of the latent period and continued until discharge onset (Demir et al. 1999). Plateau activity generally occurred at a site distinct from the site of discharge onset, at the boundary region of En and deep layer III of PC; onset activity generally occurred deeper within the En. The finding that CoCl2 and kynurenic acid blocked discharges when applied to the sites of discharge onset and plateau activity indicated that both types of latent period activity are required for the generation of interictal-like discharges (Demir et al. 1999).

In a recent study from this laboratory, the site of discharge onset was systematically mapped in PC slices under various conditions, including different rostrocaudal levels, different in vitro models of epilepsy, and different sites of stimulation in overlying PC (Demir et al. 1998). This study revealed variations in the site of onset, which imply that different structures could contribute to discharge generation under different conditions. Therefore we conducted a parallel study to map sites of plateau activity under the same range of conditions. Here we report variations in the site of plateau activity that occur in parallel with those seen in the site of onset. Under conditions in which the adjacent neocortex contributed to discharge onset, a parallel neocortical participation in plateau activity was also seen. The coordination between the sites of plateau and onset activity support the hypothesis that two distinct circuits feedback on one another to initiate epileptiform discharges. Electrical stimulation at the site of onset evoked low-amplitude plateau activity over a broader region, indicating that there is flexibility in this component of the discharge generating circuitry.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Piriform cortex slices

PC slices with a thickness of 350 µm were prepared from the brains of male Sprague Dawley rats (175-240 g) as described previously (Demir et al. 1998; Hoffman and Haberly 1991). Slices were cut with a Vibratome in a near coronal plane perpendicular to the brain surface. During slicing, subsequent storage, and recording, slices were maintained in artificial cerebrospinal fluid (ACSF) consisting of (in mM) 124 NaCl, 5 KCl, 26 NaHCO3, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose bubbled with 95% O2-5% CO2 (carbogen). Slices were defined as anterior, intermediate, and posterior PC according to stereotaxic level (Demir et al. 1998). Anterior PC slices taken from stereotaxic levels -0.70 to 1.60 mm contained lateral olfactory tract, claustrum, and agranular insular cortex (AI) and were further characterized by a thick layer Ia. Posterior slices were taken from stereotaxic levels -2.80 to -1.80 mm and were distinguished by the presence of anterior perirhinal cortex (PRha), and the absence of claustrum. Intermediate slices were taken from stereotaxic levels -1.80 to -0.70 mm and were characterized by an adjoining neocortex at the transition between the AI and PRha (see Figs. 4, 5, and 7 for labeled video pictures and sketches). Most of our previous work on plateau activity was conducted in intermediate PC slices (Demir et al. 1999), in which the claustrum was either small or absent.

After imaging experiments, all slices were fixed with 4% paraformaldehyde, sectioned at 60 µm, and Nissl stained, as described previously (Demir et al. 1998). Identification of sites of plateau activity were based on examination of these stained sections.

In vitro models of epilepsy

We used the same two in vitro models as in our previous studies to make slices generate interictal-like activity (Demir et al. 1998, 1999). The first model, termed induction, consisted of an N-methyl-D-aspartate (NMDA) receptor-dependent transformation process (Hoffman and Haberly 1989; Stasheff et al. 1989). Slices were subjected to a transient period of spontaneous bursting in low-Cl- ACSF (in which 93% of the Cl- was replaced by isethionate) for 45-90 min at 34°C, and then returned to normal ACSF for optical recordings. Epileptiform discharges could be evoked in these induced slices for up to 7 h after return to normal saline. The second model, termed disinhibition, involved blockade of GABAA receptors by bath application of bicuculline methiodide (5-10 µM; Sigma, St. Louis, MO).

Recordings were made at 32 ± 2°C. Epileptiform events were evoked by electrical stimulation of slices with a saline-filled glass pipette with a tip diameter of 20-50 µm. Current pulses (200 µs) were delivered with a stimulus isolator. The threshold for discharge generation was determined by careful variation of stimulus intensity.

Voltage imaging

The voltage-sensitive fluorescent dye RH414 (Molecular Probes, Eugene, OR) was used to image epileptiform discharges. Before imaging, slices were stained for 30-45 min in 200 µM RH414 in ACSF bubbled with carbogen. A 464-element, hexagonally arranged, photodiode fiber optic device (Chien and Pine 1991) served to measure fluorescence. The optical imaging setup used in this laboratory follows that of Wu and Cohen (1993) and has been described previously (Demir et al. 1998). It employs an upright epifluorescent microscope (Reichert-Jung Diastar), equipped with a 100-W tungsten-halogen light source, a 475- to 565-nm band-pass excitation filter, a 590-nm dichroic mirror, and a 610-nm longpass emission filter. Imaging data were collected with a Zeiss ×5 Fluar objective (NA = 0.25) that gave a distance between neighboring photodetector fields of 144 µm. The output of each photodetector was individually amplified to a final level of 0.2 V/pA of photocurrent, digitized, and read into a Pentium computer. Fluorescent signals were high-pass filtered with a 500-ms time constant and low-pass filtered with a corner frequency of 300 or 500 Hz. Trans-illuminated pictures of slices were taken with a charge-coupled device camera, and read into a PC with a frame-grabber (Data Translation, Marlboro, MA). Most video pictures were recorded with a lower magnification ×2.5 objective to facilitate visualization of anatomic landmarks.

Data acquisition and analysis

Optical signals were acquired and analyzed with the computer program Neuroplex (OptImaging, Fairfield, CT). Traces displayed individually represent averages of signals from two to seven neighboring photodiodes from a single trial. Fluorescence traces were overlaid on video images with computer programs written in IDL (Research Systems, Boulder, CO) (Demir et al. 1998; Jackson and Scharfman 1996). The contours for the site of discharge onset were drawn around the first photodetectors where the fluorescence change surpassed 50-70% of its maximum amplitude. This cutoff excludes plateau activity, which has an amplitude ~25% of the maximum amplitude of an interictal-like discharge. The contours for plateau activity were prepared manually by identifying detectors where a sustained depolarization was clearly visible above baseline noise. We have shown previously that plateau activity falls off sharply at a discreet boundary (see Fig. 7 of Demir et al. 1999), so that in spite of the subjectivity of this manual method there is ordinarily little error in demarcating the region of plateau activity. In the present study, some of the fluorescence traces showed overlap between local responses and plateau activity when experiments were performed with stimulation in En at or near the site of plateau activity (see RESULTS). In these cases there was more uncertainty in mapping activity. This may have led to overestimation of the size of the region showing plateau activity within ~300 µm of the stimulus electrode (see Fig. 6B).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Onset and plateau activity during the latent period

Previous work from this laboratory identified plateau and onset activity as two specific forms of abnormal electrical activity occurring during the latent period between electrical stimulation and discharge onset in PC slices (Demir et al. 1999). These are illustrated with fluorescence traces from induced (Fig. 1A) and disinhibited (Fig. 1B) slices. These traces all show the interictal-like epileptiform discharges typically seen with these two in vitro models of epilepsy. As pointed out previously (Demir et al. 1998), discharges in the disinhibited model are longer in duration as compared with the induced model. Other differences were observed in the sites of onset and plateau activity, as will be discussed in detail below.



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Fig. 1. Epileptiform discharges and different forms of latent period activity prior to discharge onset. A: results from an induced slice. B: results from a disinhibited slice. The video image on the left shows the piriform cortex (PC) slices (intermediate in both cases) from which the optical recordings were taken. Traces on the right, from the indicated sites, show onset activity (1), plateau activity (2), and rapidly decaying local responses in layer II of the overlying PC (3). All traces show epileptiform discharges, although in B3 the amplitude is very small. Brackets indicate onset and plateau activity in the relevant traces. Dashed vertical line indicates time of stimulus (94 µA in A and 74 µA in B, which was applied in layer Ib at sites indicated by the jagged line symbol). Dotted vertical lines indicate the time of discharge onset at the site of onset. Each trace represents an average of 6 neighboring detectors.

Onset activity was seen in both models [top traces (1) of Fig. 1, A and B]. Onset activity was a ramplike depolarization at the site of onset, beginning ~20-60 ms after stimulation and culminating in an epileptiform discharge. The middle traces of this figure (2) show plateau activity, with a roughly constant, low-amplitude depolarization beginning within ~10 ms of stimulation and continuing during the entire latent period. The bottom traces (3) show local cortical responses to stimulation followed by discharges. Mapping of the plateau and onset sites will be presented below, but in general, plateau activity was seen at a site distinct from the site of discharge onset. Outside the sites of discharge onset and plateau activity, little electrical activity was seen during the latent period. At these sites, an epileptiform discharge either erupted from a flat baseline or immediately followed the rapidly decaying local response seen near the site of stimulus. With shorter duration latent periods, the local response occasionally showed temporal overlap with the epileptiform discharge (compare traces A3 and B3 of Fig. 1). In general, stimulus currents slightly above threshold were used to make the latent period as long as possible and thus provide optimal resolution of latent period activity.

Properties of local responses

Responses from the site of plateau activity increased in amplitude in a graded manner in response to increasing stimulus currents (Demir et al. 1999). In contrast, epileptiform discharges were all-or-none. Here we analyzed stimulus-response relationships during the latent period both at the site of plateau activity and at other sites in the PC slice. Figure 2 shows optical signals from different sites in a disinhibited slice. These sites include the site of discharge onset in the dorsal-most part of En (Fig. 2Ba), the site of plateau activity at the border of En and deep layer III (Fig. 2Bb), a site in superficial layer III (Fig. 2Bc), and a site in layer Ib near the site of electrical stimulation (Fig. 2Bd). Responses evoked by three subthreshold and one suprathreshold stimuli are displayed. The amplitudes of subthreshold responses at these sites increased in a nonlinear fashion with increasing stimulus intensity (Fig. 2: columns 1 and 2). This behavior was observed not only at the site of plateau activity (Fig. 2Bb), but also at other sites in the slice wherever local responses could be seen (Fig. 2Bc; Fig. 3, B1-B5). The only exception to this was responses very near the stimulus site, where responses were predominantly monosynaptic excitatory synaptic potentials (and possibly axonal spikes) evoked directly by the stimulus current (Fig. 2Bd). Because the nonlinear increase in amplitude is steepest at stimulus intensities subthreshold for epileptiform discharge generation, it will be referred to here as "subthreshold nonlinear behavior."



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Fig. 2. Responses to subthreshold stimuli. A: a video image of a disinhibited intermediate slice with arrows pointing to the locations from which fluorescence traces in B were taken. B: traces show responses from the site of discharge onset (a), the site of plateau activity (b), superficial layer III of the overlying PC (c), and the site of stimulus in layer Ib of piriform cortex (d). Stimulus currents are indicated above. Note the disproportionate increase in responses when the stimulus intensity was increased from 55 to 65 µA (b and c). Traces represent averages of 2 neighboring detectors from the indicated sites. The site of stimulus is indicated by the jagged line symbol.



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Fig. 3. Linear and nonlinear stimulus-response plots. Peak amplitude of electrically evoked fluorescence change was plotted against stimulus current in control (A) and disinhibited (B) slices. Measurements were taken from a site in layer III. Both A and B show data from the same slice, before and after disinhibition with bicuculline. Stimulation was applied in layer Ib. The control plot could be fitted by a line, but the disinhibition plot was nonlinear (B). Linear or sigmoidal functions were drawn through the data to emphasize the different behaviors. Note the different scales for the 2 plots; bicuculline produced a large increase in amplitude for the same stimulus current. The threshold for the epileptiform discharge was 55 µA in this experiment. Traces on the right side represent responses used in B. Note the disproportionate increase in peak amplitude from B2 to B3.

Stimulus-response plots illustrate these different behaviors for control (Fig. 3A) and disinhibited (Fig. 3B) slices. The control plots were fitted by linear functions (5/5 slices), and the disinhibited plots were fitted by sigmoidal functions (5/5 slices). The linear stimulus-response behavior seen in control slices stands in striking contrast to the nonlinear behavior of disinhibited slices. In induced slices, stimulus-response plots from the site of plateau activity were linear (Demir et al. 1999). Thus in this particular respect, the induced model was closer to control than to the disinhibited model.

Mapping of plateau activity: stimulation in PC

To determine the precise location of plateau activity, contours were drawn around the region where it was observed (Fig. 4, A2-D2, blue shaded regions). In addition, contours showing the sites of onset were included (Fig. 4, A2-D2, red shaded regions) to emphasize the different site of plateau activity. The slices were Nissl stained after imaging experiments, and the photographs are shown above each site map (Fig. 4, A1-D1) to assist in relating the contours to anatomic regions. In these experiments, epileptiform activity was evoked by stimulus currents less 10% above the threshold for discharge generation. This provided the longest latency so that the activity preceding discharge onset could be most reliably assessed. Stronger stimulus currents increase the amplitude of plateau activity. The size of both the plateau and onset sites are also increased, but remain in the same basic location (Demir et al. 1999).



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Fig. 4. Mapping sites of onset and plateau activity. Slices were Nissl stained (A1-D1) after site mapping (A2-D2). Contours in A2-D2 indicate the sites of discharge onset (red) and plateau activity (blue) for induced anterior (A2), disinhibited anterior (B2), induced posterior (C2), and disinhibited posterior (D2) slices. In induced slices plateau activity extends into layers V and VI of the neighboring AI (A2) and PRha (C2) in anterior and posterior slices, respectively. In contrast, in disinhibited slices (B2 and D2) plateau activity did not extend into neocortex. Sites of stimulus are marked by the white jagged lines. Threshold stimulus currents were 70 µA (A2), 117 µA (B2), 600 µA (C2), and 120 µA (D2). CPu, caudate-putamen; ec, external capsule; Cl, claustrum; En, endopiriform nucleus; LOT, lateral olfactory tract; AI, agranular insular cortex; RF, rhinal fissure; PC, piriform cortex; PRha, anterior perirhinal cortex. Arrowheads mark the border between PC and AI in A1 and B1 and between PC and PRha in C1 and D1.

Previous work has shown that in induced slices, the site of discharge onset consists of the dorsal-most part of the En and the adjacent parts of layer VI of neocortex (AI in anterior slices and PRha in posterior slices) (Demir et al. 1998). In both induced anterior and posterior slices, the site of plateau activity included the boundary between En and PC deep layer III (Fig. 4, A2 and C2, blue shaded regions). In anterior slices plateau activity extended into layers V and VI of the adjoining AI (n = 8; Fig. 4A2), and in posterior slices plateau activity included small portions of layers V and VI of adjoining PRha (n = 18; Fig. 4C2). The contribution of deep layers of PRha to plateau activity in posterior slices was somewhat less than that of deep layers of AI in anterior slices (compare Figs. 4, A2 with C2). The extension of the site of plateau activity into AI or PRha in induced slices parallels the extension of the site of onset into the same neocortical structures.

In contrast to induced anterior slices, in disinhibited anterior slices the site of plateau activity was smaller and confined to a portion of En bordering deep layer III of PC (n = 6; Fig. 4B2). In disinhibited posterior slices (n = 5; Fig. 4D2) plateau activity was not confined to En, but was also seen in a substantial part of neighboring deep layer III of PC. As reported previously (Demir et al. 1998), the sites of onset in disinhibited anterior and posterior slices consisted of a site in En, without any involvement of deep layers of neighboring AI or PRha (red regions in Fig. 4, B2 and D2). The site of plateau activity showed a similar trend of not extending into adjoining parts of neocortex. Thus in disinhibited slices neither onset nor plateau activity appear in neighboring neocortex, and this was the case for both anterior and posterior slices. Comparison of the results from induced and disinhibited slices suggests that even as these sites vary, plateau activity and onset activity maintain a characteristic spatial relationship during the initiation of an epileptiform discharge.

In contrast to anterior and posterior slices, in intermediate slices the sites of plateau activity and onset activity were not influenced by choice of model. In induced (n = 20; Fig. 5) and disinhibited (n = 6) intermediate slices, plateau activity was confined to a portion of En and a part of adjacent PC layer III. In these slices, discharge onset was seen at essentially the same site for both models of epilepsy used in this study (Demir et al. 1998). Likewise, the site of plateau activity did not differ significantly between induced versus disinhibited intermediate slices. A summary of the site of plateau activity in different planes of slicing along the rostrocaudal axis is presented in Table 1.



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Fig. 5. Sites of plateau activity with different stimulation sites in PC. A: a photograph of a Nissl-stained intermediate PC slice (see Fig. 4 for abbreviations). An arrowhead marks the border of PC and AI/PRha. B1-B3: sites of onset (red) and plateau (blue) activity in an induced slice are indicated by the shaded contours. Epileptiform activity was evoked by stimulation in layer Ib (B2), superficial layer III (B2), and deep layer III (B3). Sites of stimulus are marked by the bold jagged lines. The threshold stimulus currents were 55 µA (B1), 75 µA (B2), and 40 µA (B3). Note that the site of plateau activity did not change significantly with different stimulus sites in the overlying PC.


                              
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Table 1. Sites of plateau activity

For a given slice, the site of plateau activity was relatively constant as long as the site of stimulation remained in PC. In experiments where two (n = 6) or three (n = 6) different stimulation sites within PC were compared, the site of plateau activity showed a variability of only one to three detector fields (150-450 µm; Fig. 5, B1-B3). In general, with stimulation in overlying PC, this small variability in the site of plateau activity was comparable to that described previously for the site of onset (Demir et al. 1998). Further, the variations in plateau site and onset site tended to occur in parallel, again supporting the idea that the sites maintain a characteristic spatial relationship.

Although most experiments showed plateau activity in the regions indicated in Table 1, the results of some experiments deviated from this pattern. In one of eight induced anterior slices, the site of plateau activity was located primarily in a portion of the En at the boundary with deep layer III, with little contribution from AI and deep layer III of PC. In this slice, the site of onset was localized in the central portion of En rather than the dorsal-most part and, unlike most induced anterior slices, did not include layer VI of AI. In 14 of 18 induced posterior slices, the site of plateau activity was as indicated in Table 1, but in two slices deep layer III of PC was excluded. In these slices the site of plateau activity was confined to a deep part of PRha and a portion of En just below the boundary with deep layer III of PC. In the two remaining slices the site of plateau activity included a portion of En and deep layer III of the overlying PC without any involvement of deep layers of PRha. Four of 20 induced intermediate slices failed to show plateau activity at the usual site consisting of adjoining parts of En and deep layer III of PC. In these slices, plateau activity was mainly concentrated in a part of En just below the border with deep layer III of PC.

In disinhibited slices, there was much less variability. Out of 17 disinhibited slices from different planes, only 1 (an intermediate slice) showed a small deviation from the typical pattern. In this slice, plateau activity was contained within the En near its boundary with deep layer III of the overlying PC.

Mapping of plateau activity: stimulation in En

When stimulation was applied in En, plateau activity was observed, but it was much lower in amplitude. Furthermore, the region where it was observed was larger, far more variable, and shifted relative to the location seen with stimulation in the overlying PC (Fig. 6). Because the threshold for generating a discharge was generally about 10-fold lower for stimulation at the sites of onset and plateau activity compared with stimulation in PC, the graded local responses were small. Plateau activity was correspondingly also small but was clearly visible in seven of nine slices (8 intermediate and 1 posterior). In these slices, plateau activity showed an expansion toward the site of onset. With either stimulation at the site of discharge onset (Fig. 6B) or the site of plateau activity (Fig. 6C), plateau activity extended from the original site seen with stimulation in PC (Fig. 6A) toward the site of discharge onset. Although the presence of local responses in the region created some uncertainty in mapping plateau activity (for example, Fig. 6B, trace 1), the extension of plateau activity in the ventral direction was at a considerable distance from the stimulus electrode so that mapping was unambiguous (Fig. 6B, trace 2). With these deep stimulation sites there was substantial overlap between the sites of onset and plateau activity. By contrast, stimulation in overlying PC produced plateau activity and onset at sites showing little or no overlap (Demir et al. 1999).



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Fig. 6. Plateau activity evoked by stimulation at the sites of discharge onset and plateau activity. Video images (A-C) taken during recording show contours for the sites of onset (red) and plateau activity (blue), with electrical stimulation in layer II of PC (glass electrode; A), in the site of onset (B), and in the site of plateau activity (C). Sites of onset and plateau activity were 1st determined for stimulation in layer II (A), and epileptiform events were then evoked by electrical stimulation at those sites (B and C). The site of onset remained roughly the same (red contours), but the site of plateau activity (blue contours) changed. Overlap between local responses and plateau activity (trace 1 of B) created some uncertainty in mapping plateau activity, so the blue contour close to the stimulus site is not as accurate as elsewhere in the figure (see METHODS). Threshold stimulus currents were 250 µA (A), 14 µA (B), and 16 µA (C) (note the lower threshold at the sites of onset and plateau activity). Although signals during the latent period were small due to the lower thresholds at these sites, onset and plateau activity could still be distinguished in the fluorescence traces numbered 1 and 2 in B and C. Traces were taken from the same sites in A-C, as indicated by the numbers 1 and 2 in the video images. Each trace is an average of 6 neighboring detectors from a single trial.

The site of onset was also examined for discharges evoked by stimulation in En and was found to be very similar to the site of onset seen with stimulation in overlying PC, for all conditions tested (Demir et al. 1998). Thus the location of onset activity was more consistent and well defined than the location of plateau activity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated subthreshold responses in the induced and disinhibition models of epilepsy. The stimulus-response plots for subthreshold stimulus currents were highly nonlinear in disinhibited slices, both at the site of plateau activity as well as most other sites. The amplitude of the fluorescence change increased steeply over a narrow range of stimulus currents below the threshold for epileptiform discharge generation (Fig. 3B). Epileptiform behavior is intrinsically nonlinear, in that a discharge is evoked when the stimulus current exceeds a characteristic threshold. The present observation suggests that there is an additional form of nonlinearity that can be associated with epileptiform behavior. This type of behavior was never observed in control slices (Fig. 3A), where stimulus-response relations were linear, but it was also not seen in subthreshold responses in induced slices (Demir et al. 1999, Fig. 4C). Thus subthreshold nonlinearity is not necessary for epileptiform behavior, and its presence in the disinhibited model may reflect a difference in the underlying mechanisms of discharge generation. Both models clearly increase neuronal excitability, but induction increases excitability without introducing nonlinear responses to stimuli below the discharge threshold.

Previous work has shown that the induction and disinhibition models also differ in discharge onset site, and in the duration of the interictal-like discharge (Demir et al. 1998). It is likely that in the induced model the changes brought on by spontaneous bursting in low-Cl- are more restricted spatially. Because GABAA receptors are ubiquitous, and are blocked throughout the slice by bath application of bicuculline, disinhibition will lead to a more global change in excitability, not only in En but also in overlying PC. In induced slices, linear behavior was seen even at the plateau site, but subthreshold responses at the site of onset deeper in En were too small to assess. Thus we cannot rule out the possibility that localized changes in excitability in the induced model lead to subthreshold nonlinearity at a highly restricted location.

One possible explanation for the subthreshold nonlinear behavior seen in disinhibited slices is positive feedback through recurrent excitatory synaptic pathways. Such fiber systems have been described in the PC, both between pyramidal cells and between pyramidal and deep multipolar cells (Behan and Haberly 1999; Luskin and Price 1983). Disinhibition would presumably unmask di- and/or polysynaptic potentials, which can be evoked in PC slices (Tseng and Haberly 1989). From this perspective, the more linear stimulus-response behavior seen in layer I (Fig. 2, bottom row of traces) could reflect differences in the strength of synaptic inputs to apical versus basal dendrites of pyramidal cells. Over short distances, excitatory inputs from association fibers in the PC project predominantly to pyramidal cell basal dendrites located in layer III (Haberly and Presto 1986; Rodriguez and Haberly 1989). Subthreshold nonlinear behavior was clearly seen in this region (Figs. 2B and 3B). Pyramidal cell axons typically traverse longer distances before ascending to layer I to innervate apical dendrites. These projections are less likely to be preserved in slices, so direct activation of association axons by stimulation in layer Ib would evoke mostly monosynaptic events, and these show a linear dependence on stimulus current.

In this work a detailed mapping study was conducted in slices of PC to locate the sites where plateau activity can be seen before the onset of epileptiform discharges. These sites are summarized in Table 1 and sketched on drawings of slices in Fig. 7 to make their spatial relationships clear. Although the site of plateau activity varied with the different conditions tested, it always included a portion of En at the boundary with deep layer III of the overlying PC. Additional sites where plateau activity was observed depended on the rostrocaudal level of the slice, as well as the in vitro model used to generate epileptiform behavior. A portion of deep layer III at the boundary with En and deep layers of neocortex also showed plateau activity under some conditions. These regions thus possess properties that allow them to produce a weak and roughly constant depolarization. It is not known whether this depolarization depends on intrinsic regenerating potentials or reciprocal excitatory synaptic interactions, but the neurons in these regions must have the cellular properties or synaptic circuitry capable of sustaining plateau activity for the entire latent period, which can last >100 ms.



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Fig. 7. Sites of onset and plateau activity. Solid blue contours indicate the site of plateau activity on sketches of PC slices from different stereotaxic levels for the 2 models used in this study. Results from 4 typical experiments are shown for each condition. The dashed red ellipses show the site of onset reported by Demir et al. (1998). Many of the imaging experiments used in that previous study were also used in the present study. Note that although the contours demarcating sites of onset and plateau activity overlap when results from multiple experiments are combined, in individual experiments there was little if any overlap (Figs. 4 and 5). All of the data summarized here are for stimulation in PC. Sites were more variable and overlapped more when stimulation was applied in En (Fig. 6). Dashed line indicates the boundary between PC and neocortex. For label definitions, see Fig. 4.

Participation of neocortical structures adjoining PC had been reported previously in the onset of epileptiform discharges (Demir et al. 1998). In induced anterior and posterior PC slices the sites of onset not only consisted of the dorsal portion of En, but also a portion of deep layer VI of adjoining neocortex (either AI or PRha, for anterior and posterior slices, respectively). Here we found that as the site of onset expanded to include deep layers of the neighboring cortex, there was a parallel expansion of plateau activity toward deep layers of neocortex (in Fig. 7, compare anterior-induced with anterior-disinhibited, and posterior-induced with posterior-disinhibited). It is noteworthy that participation of adjacent neocortex in both onset and plateau activity is seen only in the induced model of epilepsy, and not in the disinhibited model. The finding of different degrees of neocortical participation in discharge onset led us to hypothesize previously that there are regional variations in synaptic circuitry (Demir et al. 1998). Thus if GABAA receptors play a greater role in limiting synchronous firing in En, this could account for preferential onset in En in the disinhibited model. The present finding of a similar exclusion of plateau activity from adjoining neocortex in the disinhibited model argues for a similar role for GABAA receptors at the boundary region between En and deep layer III of PC (the principle site of plateau activity). The properties of the adjoining neocortex that allow it to contribute to discharge onset in the induced model may be related to those properties that allow it to contribute to plateau activity.

The sites of both onset and plateau activity varied, and it is significant that the variation of these two sites occurred in parallel. This is consistent with the idea that functional interactions between the two circuits generate these forms of electrical activity. The parallel spread into neocortex preserves a characteristic spatial relation, and this spatial relation may be necessary for the communication between these two circuits that allows them to amplify electrical activity and initiate an epileptiform discharge. This supports the hypothesis that the sites of onset and plateau activity work together to generate an epileptiform discharge, presumably through reciprocal excitatory synaptic pathways (Demir et al. 1999).

The variation in the site of plateau activity was far more pronounced when the stimulus electrode was moved to the site of discharge onset in En. The threshold was very low in these experiments, so that plateau activity had a much lower amplitude and became distributed over much of the En. This suggests that the properties that are necessary for plateau activity are distributed widely through the En and neighboring areas of PC and neocortex. By contrast, the site of onset was generally smaller and remained at essentially the same location (Fig. 6). What may make the site of onset unique is its strong reciprocal excitatory synaptic connections with a large surrounding area. Plateau activity can then appear at variable locations within this larger region, according to where the stimulus is applied. But the discharge onset will occur at the one location receiving strong excitatory input from all these areas.


    FOOTNOTES

Address for reprint requests: M. Jackson, Dept. of Physiology, SMI 127, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706.

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 30 July 1999; accepted in final form 18 October 1999.


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ABSTRACT
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DISCUSSION
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