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1 INSERM U739, CHU Pitié-Salpêtrière, UPMC, 105 boulevard de l'Hôpital, Paris 75013, France
2 INSERM U497, Ecole Normale Superieure, 46 rue d'Ulm, Paris 75005, France
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Abstract |
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800 µm, KA injection induced a strong increase in extracellular firing which ceased after 2–4 min. Pyramidal cells in this zone fired and depolarized to a potential at which action potentials were no longer evoked. No further activity was detected near the injection site for 3–5 h. In longitudinal slices of the CA3 region, firing induced by KA injection spread at a velocity close to 1 x 10–4 mm ms–1. The velocity increased to 1 x 10–1 mm ms–1 when synaptic inhibition was blocked, suggesting that inhibitory processes normally restrict the spread of firing. At distances of 1.5–2.5 mm, KA injection induced a short-term increase in firing which was maintained, and often increased and rhythmic at gamma frequencies at 2–5 h after injection. We also examined slices prepared from animals injected with KA, at a delay of 2–5 h corresponding to the expression of status epilepticus. Near the injection site, Gallyas silver staining revealed cellular degeneration, and no activity was recorded. Interictal-like activity was generated by ipsilateral slices distant from KA injection. Contralateral slices also generated an interictal-like activity, but no cell death was detected. Hippocampal oscillations generated at distant sites may be associated with status epilepticus.
(Received 27 July 2005;
accepted after revision 18 October 2005;
first published online 20 October 2005)
Corresponding author R. Miles: INSERM U739, CHU Pitié-Salpêtrière, 105 boulevard de l'Hôpital, 75013 Paris, France. Email: rmiles{at}chups.jussieu.fr
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Introduction |
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In this study we examined events intervening between the application of KA and the emergence of status epilepticus. We made focal intrahippocampal applications of KA in order to distinguish between its local and distant effects (Magloczky & Freund, 1993; Riban et al. 2002). KA was injected in acutely prepared slices to investigate immediate events. Slices prepared from animals injected in the same way were also studied to examine the emergence of status epilepticus. Intracellular, multi-unit and field responses were recorded close to and distant from the site of KA injection, to discriminate between the contributions of these different sites to status epilepticus. We found that KA injection caused a strong but transient increase in multi-unit firing near the injection site, and that no further activity was detected at this site. The firing initiated by KA spread slowly through slices. At distant sites, a maintained and often rhythmic activity persisted for several hours after injection. We conclude that the behavioural manifestations of status epilepticus may depend in part on persistent population activities generated at hippocampal sites distant from the injection.
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Methods |
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Intrahippocampal kainate acid injection
Adult male mice were anaesthetized with 4% chloral hydrate (120 ml kg–1; Sigma France) and 4% urethane (1000 ml kg–1; Sigma, France), and placed in a stereotaxic frame. Injections were made through a stainless steel cannula of outside tip diameter 0.28 mm, connected to a 0.5 µl microsyringe (Hamilton, Fisher Labosi, France). A volume of 50 nl kainic acid (Sigma, France) dissolved at 20 mM in 0.9% NaCl was injected into the right dorsal hippocampus (Bouilleret et al. 1999). The electrode was maintained in place for 5 min to limit reflux along the injection track. Control animals were prepared identically and injected with the same volume of NaCl. Injections were made at stereotaxic coordinates of Bregma: anterioposterior (AP) =–1.8 mm, mediolateral (ML) =–1.8 mm, dorsoventral (DV) =–1.8 mm. This corresponds to a site in the dorsal hippocampus in the apical dendritic zones of the CA1 region near the hippocampal fissure. Animals typically recovered from anaesthesia after 2–5 h. On recovery they displayed behavioural signs of status epilepticus, including stereotyped turning movements and immobile states.
In experiments to examine the effects of kainic acid in tissue from injected animals, slices were prepared at 1.5–2.5 h after injection, before the animals recovered from anaesthetic. Recordings began at 3–4 h after kainic acid injection. In these experiments the injection solution contained both 20 mM kainic acid and the fluorescent tracer microruby (10 mg ml–1, 3000 Da, Invitrogen, Cergy Pontoise), so that the site of injection could be identified.
In some experiments we examined the extent of diffusion of 50 µl Rhodamine dextran (10 kDa, lysine fixable, Invitrogen, Cergy Pontoise, France) injected in vivo using the same system. Animals were anaesthetized, injected, and after 3 h perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed, cryoprotected overnight in 30% sucrose in PB. Serial sections of thickness 60 µm were cut at –25°C and permeabilized in a PB solution containing 0.1% Triton and 0.1% gelatin. Neuronal Nissl substance was stained with A488-Neurotrace (Invitrogen). Fluorescence images were acquired with a Leica confocal microscope TCSP2. Neurotrace was detected using the 488 nm line of an argon laser for excitation, and Rhodamine dextran excited by the 543 nm line of a green neon laser. Typically, 40 confocal sections (1024 x 1024 pixels) were scanned per tissue section of thickness 60 µm. Examination of 20 representative tissue sections from 80 sections cut around the injection site was sufficient to include all tissue that contained Rhodamine dextran. 3D rendering (Fig. 7D) was done on stacks of 800 images using the programme Amira (TGS, Mercury Computer Systems, Merignac, France).
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Slices were prepared from mice after KA injection, or from uninjected animals. In both cases an intracardiac perfusion was made under chloral hydrate/urethane anaesthesia, with a cold (2–5°C) artificial cerebrospinal fluid (ACSF) containing (mM): 26 NaHCO3, 1 KCl, 9 MgCl2, 1 CaCl2, 248 sucrose and 10 D-glucose. After decapitation, the brain was removed. Hippocampal–cortical slices (Walther et al. 1986) or longitudinal CA3 slices (Miles et al. 1988) of thickness 400 µm were cut with a vibratome (Vibratome, St Louis, MO, USA) and placed in an interface recording chamber. They were perfused at 3–4 ml min–1 with an oxygenated (95% O2/5% CO2) ACSF containing (mM): 124 NaCl, 4.5 KCl, 1 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 10 glucose (pH 7.4, 295–310 mosmol l–1) at 35°C, while their upper surface was exposed to a humidified 95% O2/5% CO2 atmosphere.
Procedures for KA injection into slices were similar to those used in injecting animals. The same volume and concentration, 50 nl of 20 mM KA in ACSF, or 50 nl of a saline solution of 0.9% NaCl was injected from an identical cannula system with a Hamilton syringe. The cannula was advanced until it just entered the slice before injection. Injections were made at similar apical dendritic sites of the CA1 region in hippocampal–cortical slices, and at apical dendritic sites in longitudinal slices of the CA3 region.
Recording methods and data analysis
Recordings were made during a period of 1–6 h after KA or saline injection. Intracellular recordings from hippocampal and cortical neurones were made using glass microelectrodes filled with 2 M potassium acetate, and bevelled to a final resistance of 50–100 M
. Membrane potentials were measured using an Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA). Multi-unit activity and field potentials were recorded with extracellular electrodes made from tungsten wire of 50 µm diameter (Phymep, Paris, France). Up to four electrodes were mounted on holders controlled by separate manipulators. Differences in potential between each tungsten electrode and a reference Ag–AgCl electrode were measured using a 4-channel amplifier (AM Systems, model 1700, Carlsborg, WA, USA). Extracellular signals were amplified 1000 x and filtered with pass band between 1 Hz and 10 KHz. Signals were digitized at 10–20 kHz, with a voltage resolution of 0.6 µV for extracellular signals and 25 µV for intracellular signals, using a 12-bit, 16-channel analog-to-digital converter (Digidata 1200 A, Axon Instruments), and visualized on a PC using the programme Axoscope (Axon Instruments).
Action potential frequency was measured from multi-unit records using routines written in the Labview environment (National Instruments Austin, TX, USA). Spikes were detected using an ‘up-only’ algorithm, and a user-defined threshold (Cohen & Miles, 2000). The programmes are available at http://glab.bcm.tmc.edu. Power spectra were constructed from extracellular records, filtered to pass frequencies lower than 100 Hz, using the Clampfit programme (Clampfit 9, Axon Instruments). A Fourier transform was applied to records of duration 2–5 min, using a rectangular window, 50% overlap and a sample number of 32768 which corresponds to a spectral resolution of 0.3 Hz.
Morphology
Cell death was studied using the Gallyas silver impregnation technique (Gallyas et al. 1980). Animals were killed at 3 h after the injection of kainate or physiological saline. They were perfused intracardially with cold ACSF, followed by 100 ml of fixative containing 4% paraformaldehyde and 15% saturated picric acid dissolved in 0.1 M phosphate buffer (PB). The brains were then removed, 100 µm-thick sections were cut with a Vibratome and washed in PB. The Gallyas staining procedure consists of a pretreatment with alkaline hydroxylamine, washing in acetic acid, impregnation in silver nitrate in the presence of ferric ions, washing in citric acid, development, and a second wash in acetic acid. It reveals a black silver precipitate which accumulates in the cytoplasm of degenerating cells, while healthy cells are coloured orange. After impregnation, sections were washed in PB, mounted on gelatine-coated slides and covered with DePeX (Laboratoire d.b.h., France).
Distinct populations of interneurones were examined by immunostaining against parvalbumin (PV), calbindin (CB), calretinin (CR) and somatostatin (SOM). Animals were fixed as before with paraformaldehyde and picric acid. The hippocampus and nearby cortex was dissected and sections of thickness 60 µm were cut with a vibratome. Endogenous peroxidase activity was blocked by 1% H2O2, and non-specific staining suppressed by 5% milk powder and 2% bovine serum albumin containing 0.1% Triton-X. Monoclonal mouse antibodies against PV (dilution 1 : 3000, Sigma), CB and CR (1 : 3000 each, Swant, Bellinzona, Switzerland), and rat polyclonal antibody against SOM (1 : 200, Chemicon International, Temecula, USA) were applied with 0.1% Triton-X 100 for 24 h at 4°C. Immunostained elements were visualized using biotinylated antimouse and antirat immunoglobulin G (1 : 250, Vector Laboratories, Burlingame, CA, USA) as the secondary antiserum followed by avidin–biotinylated horseradish peroxidase complex (ABC; 1 : 250, Vector). The immunoperoxidase reaction was developed by 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma), as a chromogen. Sections were osmicated (0.25% OsO4 in PB, 30 min), dehydrated in ethanol, and mounted in Durcupan (ACM, Fluka).
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Results |
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Kainic acid (KA) injection into hippocampal slices caused an increase in neuronal activity. Subsequent effects differed at sites close to and distant from the injection. While the separation is rather artificial, we will refer to local actions as those induced within 800 µm of the injection site and those at sites beyond 1200 µm as distant.
KA was injected at apical dendritic sites of the CA1 region in hippocampal–cortical slices (n= 23). Records from sites near the injection in the CA1 and CA3 regions as well as in the dentate gyrus and the subiculum (Fig. 1A E1 and E2) revealed an increase in firing with latency 150–2000 ms. Action potential frequency in multi-unit records increased from values of 68.3 ± 82.7 Hz. (Fig. 1B) to 449.1 ± 203.5 Hz. The increase in firing was accompanied by field potential oscillations at frequencies of 30–100 Hz (Fig. 1C, E1 and E2). Sometimes two waves of increased activity were recorded by electrodes situated in CA3 (15 of 23 slices) and in the subiculum (7 of 23 slices). In all records from sites close to KA injection, spontaneous multi-unit activity ceased completely at 2.5 ± 3.0 min (n= 46 CA3 and subicular records), and did not recover (Fig. 1D, E1 and E2).
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Immediate actions of kainic acid injection in longitudinal slices of the CA3 region
We wished to examine the spread of firing induced by KA injection, and to compare local and distant effects in a similar structure. Experiments were therefore repeated in longitudinal slices of length 3–4 mm prepared from the CA3 region (Fig. 2).
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Records from multiple sites, revealed a unidirectional spread of activity induced by KA (Fig. 2A). The mean propagation speed, measured to the onset of the increase in firing, was 9 x 10–5± 6 x 10–5 mm ms–1 (Fig. 2E). This velocity is slower than that of conduction in non-myelinated hippocampal axons (0.2 mm ms–1; Raastad & Shepherd, 2003), and might indicate that activity spreads by the diffusion of kainate (Nicholson, 2001). At the site of injection, activity ceased in most slices (22 of 26) at 8.34 ± 6.77 min after KA injection. In contrast, activity was maintained at sites more distant than 1 mm. In 15 records from distant sites in 26 slices, the frequency of firing remained increased after 15 min.
KA-induced firing spread slowly throughout longitudinal slices. We compared this spread to that of epileptiform discharges induced by disinhibition (Miles et al. 1988). In the presence of bicuculline (20 µM; n= 5), a spontaneous interictal-like activity propagated throughout the CA3 region (Fig. 3A). Its velocity, measured from differences in latencies of events recorded with three extracellular electrodes, was 0.12 ± 0.08 mm ms–1 (n= 5). We then injected KA into disinhibited slices. It induced a local increase of activity, which spread at a speed of 0.11 ± 0.07 mm ms–1 (mean ±S.D., n= 5) much more rapidly than in the absence of bicuculline (Fig. 3A). These data suggest that synaptic inhibition normally prevents the rapid spread of KA-induced firing. In the presence of synaptic inhibition, the spread seems to be mediated by mechanisms other than axonal or polysynaptic transmission.
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500–800 µm. Injection of KA on one side of the cut induced an increase in firing, while activity was not changed on the non-injected side of the cut (n= 4). Cellular effects of kainic acid injection
Extracellular data showed that firing increased dramatically and then stopped close to KA injection sites, while activity at a distance increased and was then maintained. We next examined the cellular correlates of these two responses in records from close and distant sites. As shown in Fig. 4, pyramidal cells close to the injection site were strongly depolarized by KA injection (n= 4). The depolarization, sometimes preceded by a hyperpolarization, induced firing of duration 5–90 s at frequencies up to 200 Hz (Fig. 4A), correlated with the increase of activity in local extracellular records. After firing, all cells continued to depolarize to potentials of –40 to –10 mV, and stopped firing. The depolarization block of Na+-dependent action potentials was maintained. Pyramidal cell input resistance was reduced from 35 ± 9 M (mean ±S.D., n= 4) before KA application, to 3 ± 1 M after injection (Fig. 4B). Hyperpolarizing current injection could not repolarize cells to potentials at which action potentials were generated.
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We observed different behaviours at sites close to and distant from KA injection during the time period of 30 min to 5 h after KA injection (Fig. 6). Close to the injection site, no extracellular unit activity was detected during this period (Fig. 6A, E1), and intracellular records could not be obtained in either hippocampal–cortical slices (n= 9) or longitudinal CA3 slices (n= 22).
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Gamma-frequency oscillations emerged with a delay of 20–30 min after KA injection. Figure 6E shows power–frequency histograms for one recording constructed at different times before, during and after the excitation induced by KA injection. The increase in the coherence of oscillations was quantified by measuring the mean power in the gamma frequency range 30–100 Hz. For 10 slices, the mean power increased from a control value of 0.07 ± 0.04 µV2 to a peak of 2.8 ± 3.89 µV2 during the KA-induced increase in firing. At 10–20 min after KA injection, the power was reduced to 0.13 ± 0.12 µV2. A considerable increase in the power of oscillations then occurred, to 5.08 ± 6.15 µV2 at 50–70 min and 4.01 ± 6.34 µV2 at 90–120 min after injection (Fig. 6E).
Long-term actions of kainic acid injection in ex vivo slices
KA injection in slices led to a maintained suppression of activity at the injection site, and an increased, often synchronous, activity at distant sites. We compared these effects with the activity generated by slices from animals injected with KA (Fig. 7). In this way, we could not only compare the effects of in vitro and in vivo KA injections, but also examine the effects of KA injection in the contralateral, non-injected hippocampus. KA was injected together with the fluorescent marker microruby so that the injection site could be identified. Up to six hippocampal–cortical slices were cut and kept in order that records could be made from ipsilateral sites close to and distant from the injection. Slices were also prepared from the hemisphere contralateral to the injection, and stored in order. Figure 7D shows a 3-D reconstruction of the spread of the tracer Rhodamine dextran (molecular mass 10 kDa) injected with kainate. At 3 h, the molecule was detected at distances of 500 µm from the injection site in the dorsal–ventral plane of the hippocampus and in the plane of the CA1 dendritic axis, with a rather larger spread of 800 µm especially towards the subiculum in the plane orthogonal to the dendritic axis and along the hippocampal fissure.
Little or no multi-unit activity was detected near the injection site in ipsilateral slices from six animals during the period 2–5 h after KA injection in vivo (Fig. 7A). In contrast, slices prepared from distant sites generated a robust extracellular activity (Fig. 7B). This consisted of unit discharges together with interictal-like field potentials of duration 40–120 ms, which recurred at a frequency of 0.5–3 Hz. This activity was initiated in the CA3 or CA1 regions and propagated to the dentate gyrus and the subiculum (Fig. 7B, n= 10 ipsilateral slices).
An interictal-like activity was also recorded at 2–5 h after KA injection in hippocampal–cortical slices from the contralateral hemisphere (Fig. 7C, n= 15). Figure 7E shows that population oscillations were generated only by distant ipsilateral slices, and most frequently by contralateral slices distant from the exact counterpart to the injection site. We also examined longitudinal slices prepared from the ipsi- and contralateral CA3 region of injected animals (n= 6). Activity was robust, and interictal-like activity was generated by three of six slices (not shown). Thus, population oscillations are generated by both ipsilateral and contralateral hippocampus at a delay corresponding to the expression of status epilepticus in vivo. They are generated at a distance, rather than at the injection site, and while injected slices generate gamma oscillations, slices from injected animals generate interictal-like activity.
Effects of kainic acid injection on cell survival: Gallyas stain
We next used the Gallyas stain (Gallyas et al. 1980) to ask how neuronal cell death at distinct ipsilateral and contralateral sites was associated with electrical activity. Staining was done on tissue obtained from animals at 3 h after KA injection.
Near the injection site, many cells were black with silver accumulation (Fig. 8A) indicating cellular damage, in the CA1 and CA3 regions, but not the dentate gyrus. Cell death was reduced with distance. At 1.5–2 mm from the injection site, there was some loss of CA1 pyramidal cells, CA3 cells were less affected, and no cell death was evident in the dentate gyrus (Fig. 8B). There was no loss of pyramidal cells at any sections obtained from the hippocampus contralateral to KA injection (Fig. 8C; Table 1).
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A more detailed view of inhibitory cell survival (Fig. 9) was obtained by immunohistochemical studies on the distribution of cells expressing the markers parvalbumin (PV), calbindin (CB), somatostatin (SOM) and calretinin (CR). We compared results from animals killed at 3 h. after KA injection and after injection of saline solution.
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Discussion |
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Depolarization block and death of cells near the injection site?
We injected small volumes of high concentrations of KA both in vivo and in vitro in an attempt to mimic procedures used to produce status epilepticus in the intact animal (Bouilleret et al. 1999). Near sites of KA injection in vitro, multi-unit records revealed a strong but transient firing that lasted for 2 or 3 min. No further activity was recorded for the duration of the experiment (Figs 1 and 2). This is consistent with data showing that high concentrations of KA induce neuronal necrosis (Fujikawa et al. 2000), although slower apoptotic mechanisms may also be initiated (Pollard et al. 1994). Pyramidal cells depolarized to membrane potentials where firing was no longer observed (Fig. 4). A similar fast transition to a state of depolarization block occurs in the dorsal motor vagal nucleus following ischaemia (Muller & Ballanyi, 2003), and in the hippocampus after anoxia (Dzhala et al. 2001) or KA application (Robinson & Deadwyler, 1981). Such depolarizations induce a maintained calcium entry (Choi, 1987) and initiate cell swelling (Nadler et al. 1978; Oliva et al. 2002) which are precursors of cell death. The cellular degeneration near the injection site observed with the Gallyas stain (Fig. 8) confirmed that KA injection induces a local cell death.
Spread of kainate-induced excitation
The activity induced by KA injection propagated through longitudinal slices of the CA3 region at a velocity close to 1 x 10–4 mm ms–1 (Fig. 2). This is much slower than the spread of synchronous discharges initiated by disinhibition, 0.12 mm ms–1 (Miles et al. 1988). Disinhibition-induced synchrony in the CA3 region spreads by a polysynaptic process in which cell firing synaptically excites distant cells, which then excite still more distant cells. In contrast, intracellular records revealed that a travelling wavefront of inhibitory synaptic events preceded the KA-induced excitation in pyramidal cells (Figs 4 and 5). Since KA-induced firing spread more quickly in the presence of bicuculline (Fig. 3A), we conclude that synaptic inhibition normally prevents the propagation of KA-induced firing by polysynaptic pathways.
If the activity initiated by KA does not spread synaptically, how does it spread? KA might simply diffuse throughout the slice inducing firing as it spreads. Using reasonable values for the diffusion coefficient of KA (4 x 10–6 cm2 s–1), tortuosity (1.65) and extracellular volume fraction (0.15), the diffusion equation (Nicholson, 2001) suggests that at a distance of 2 mm, the peak concentration of KA should be reached after a delay of 30–50 min. Lower concentrations that may increase cell firing will arrive more quickly. However, neuronal firing induced by KA will also initiate local processes that facilitate propagation, including an increase in extracellular K+ and the liberation of glutamate. These processes should recruit cells to the excited ensemble, and accelerate the spread of firing. The spread of neuronal excitation induced by KA injection might then resemble a spreading depression (Lauritzen et al. 1988). Indeed, spreading depression in hippocampus propagates at speeds close to 10–4 mm ms–1 (Snow et al. 1983; Herreras et al. 1994; Peters et al. 2003), similar to those we measured.
A delayed emergence of 40–60 Hz synchrony at distant sites in vitro
KA induced high-frequency firing and oscillating field potentials in the gamma frequency range both in response to injection and with a delay at distant sites in CA3 slices. During these oscillations (Fig. 6B and C), interneurone firing was phase-locked to the local field potential, while pyramidal cells fired infrequently and received a rhythmic inhibition (Whittington et al. 1995; Fisahn et al. 1998).
The power of gamma frequency oscillations increased significantly, with a delay of several tens of minutes after KA injection (Fig. 6E). Maybe the power of oscillations is enhanced by the reconfiguration of neuronal circuits dependent on the loss of pyramidal cells and some types of interneurones, although we note the peri-somatic parvalbumin-positive cells were largely spared (Table 2). If KA induces a wave of activity that propagates by mechanisms similar to a spreading depression, but faster than diffusion, then a second wave of KA diffusion might underlie the delayed emergence of gamma oscillations. Alternatively, gamma frequency activity may itself initiate self-reinforcing synaptic (Whittington et al. 1997; Lauri et al. 2003), cellular (Melyan et al. 2002), or even axonal (Semyanov & Kullmann, 2001) plasticity. The present data do not let us discriminate between these different sites for changes that might underlie the delayed enhancement of gamma oscillations (Ben-Ari & Gho, 1988; Khalilov et al. 1999; Khalilov et al. 2003).
Differences between delayed activity in injected slices and slices from injected animals
At sites near in vitro KA injections, or in slices close to in vivo injection sites, we observed no neuronal activity after 2–5 h. The two approaches both revealed an enhanced, often rhythmic, activity at distant sites. However the nature of the activity was not the same. Gamma-frequency oscillations were observed at 2–5 h at distant sites in injected slices, whereas slices prepared from injected animals generated an interictal-like activity (Fig. 7). Previous work reveals a similar difference which remains unexplained. In slices for instance, bath application of similar concentrations of KA has been shown to induce either interictal-like (Westbrook & Lothman, 1983; Fisher & Alger, 1984) or gamma-frequency activity (Vreugdendhil et al. 2003; Fisahn et al. 2004).
The reason for this difference is not clear. Similar concentrations and volumes of KA were injected in the same way from the same catheter system in both slices and animals. Possibly gamma oscillations may be transformed with time into an interictal-like activity. Gamma oscillations were generated in injected slices at latencies between 20 min and 3 h 30 min after injection. Both ipsilateral and contralateral slices from injected animals generated interictal-like activity at latencies of 2–5 h after injection. One difference might be the circuits involved. Gamma activity was generated at distant CA3 sites after KA injection in longitudinal CA3 slices, but not at distant entorhinal sites after injections into combined hippocampal–cortical slices. Even so, interictal rather than gamma oscillations were observed in longitudinal CA3 slices prepared from injected animals. Another difference might be the injection site. In longitudinal slices, injections were made at apical dendritic sites of the CA3, region, while KA was injected in vivo in CA1 dendritic regions. Possibly, the distinct neuronal elements activated by injection at these different sites influence the nature of the delayed population activity. Finally, injection in the intact animal probably activates distant structures that may initiate distinct modulating influences absent in a slice. In order to affect hippocampal activity, such influences should operate in long-range recurrent fashion.
Relations between status epilepticus and kainate-induced activity in vitro
Is the delayed rhythmicity observed in vitro related to the behavioural manifestations of status epilepticus? While our data show that the injected site cannot contribute, delayed activity generated at distant sites in the injected and also in the contralateral hippocampus is probably involved. Clearly, activities of non-hippocampal structures are likely to be involved.
Both the injected and the contraleral hippocampus independently generated delayed interictal activity (Fig. 7B and C). A comparison may illuminate the nature of the effective stimulus needed to initiate this activity. It seems unlikely that injected KA penetrated to the contralateral hippocampus. Furthermore we could not detect a cell loss in the non-injected hippocampus (Fig. 8). Thus, population oscillations in the contralateral hippocampus probably depend on changes induced by activity in the injected hippocampus. Such changes have been described in a preparation comprising both hippocampi from young animals, and ascribed to activity-dependent changes in the reversal potential for GABAergic signalling (Khalilov et al. 2003). However it is difficult to interpret our data within the framework of a mirror focus. First, we showed that the duration of high-frequency firing at the injection site was less than 3 min. Secondly, the probability that contralateral slices that strictly mirrored the injection site generated delayed oscillations was less than that for slices contralateral to sites distant from KA injection (Fig. 7E). Thirdly, our data suggest that the spread of KA-induced activity by axonal propagation and synaptic transmission is suppressed by synaptic inhibition (Fig. 3A). We note that in young animals GABAergic signalling should be depolarizing, thus favouring an axonal and synaptic spread. One way to reconcile these constraints is to suggest that contralateral interictal activity is not induced by the immediate transient firing near the injection site, but rather results from activity-dependent mechanisms initiated by the delayed population oscillations generated at ipsilateral sites distant to KA injection. This hypothesis could be tested by comparing the time course with which interictal activities develop at distant ipsilateral sites and in the contralateral hippocampus in vivo.
Similar epileptic activity with different morphology
Both distant ipsilateral hippocampus and contralateral hippocampus generate a delayed interictal-like activity (Fig. 8). Patterns of cell survival at the time that the activity was generated were considerably different. There was a moderate cell loss of both CA1 and CA3 pyramidal cells, and also interneurones in all regions at distant sites ipsilateral to the injection site. In contrast, no loss of principal cells or interneurones was detected in contralateral hippocampus (Fig. 9). This suggests that morphologically different neuronal networks can generate similar population activities, and also that cell death is not a necessary factor for the generation of epileptiform activity in the hippocampus.
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