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Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State University, Fort Collins, Colorado 80523
Submitted 9 February 2004; accepted in final form 8 April 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Schwartzkroin and Prince 1977; Wong and Prince 1979; Wong and Traub 1983), because this region has excitatory interconnections between pyramidal cells (Miles and Wong 1983, 1986, 1987; Miles et al. 1984; Wong and Traub 1983). Network simulations in computer models of CA3 pyramidal cells that are interconnected by recurrent excitatory synapses can reproduce many of the properties of the all-or-none bursting characteristics of CA3 pyramidal cells (Traub and Wong 1982, 1983a, b; Traub et al. 1987a, b), which supports the hypothesis that these epileptiform bursts are driven and synchronized by a network of neurons with excitatory interconnections. Moreover, in immature rats, a stage when it is thought that there are more local excitatory synaptic connections in the brain, the disinhibited hippocampus has a higher susceptibility to generate epileptiform bursts. The propensity for and the duration of the bursts in the CA3 area are thought to decline as neuronal connections are pruned during maturation (Gomez-Di Cesare et al. 1997; Swann and Brady 1984; Swann et al. 1986, 1993, further suggesting that the local excitatory circuits participate in the generation of epileptiform activity.
Considerable evidence has shown that, during epileptogenesis, local excitatory circuits are formed through axonal sprouting in the dentate gyrus, a region that normally has few local excitatory neuronal connections in either human tissue (Babb et al. 1991; Ben-Ari et al. 1981; Franck et al. 1995; Sutula et al. 1989) or in animal models (Buckmaster and Dudek 1997a, b; Molnar and Nadler 1999; Patrylo and Dudek 1998; Sutula et al. 1998; Tauck and Nadler 1985; Wuarin and Dudek 1996, 2001). These newly formed local circuits would be responsible for recurrent excitation and contribute to synchronization, and thus may be an important contributor to seizures. More recently, both anatomical and physiological evidence has suggested that similar synaptic reorganization via axonal sprouting also occurs in the CA1 area during epileptogenesis and appears to contribute to seizure activity (Esclapez et al. 1999; Lehmann et al. 2000; Meier and Dudek 1996; Perez et al. 1996; Smith and Dudek 2001, 2002). To further address this hypothesis, we analyzed the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), and used the approach of flash photolysis of caged glutamate to detect local excitatory circuits (Callaway and Katz 1993; Dalva and Katz 1994; Wieboldt et al. 1994) in hippocampal CA1 from control rats versus rats with kainate-induced epilepsy. We first confirmed the results of Esclapez et al. (1999) that the frequency of sEPSCs was higher in rats with kainate-induced epilepsy. We also found that the CA1 area of epileptic animals had significantly more local circuits than controls. These data support the hypothesis that, during epileptogenesis, axonal sprouting and formation of new excitatory local circuits, as seen in the dentate gyrus, also appears to occur in the CA1 area of the hippocampus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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All procedures with animals used in this study were approved by and in compliance with the guidelines of the Colorado State University Animal Care and Use Committee. The kainate treatment protocol has been described previously (Hellier et al. 1998; Smith and Dudek 2001; Wuarin and Dudek 2001). Briefly, male Sprague-Dawley rats (Harlan) weighing 
175 g were injected hourly with kainate (5 mg/kg, ip). Motor seizures normally occurred after one to three injections. Animals had recurring class IV/V seizures (Ben-Ari 1985; Racine 1972) for 
3 h. Control rats received saline injections in parallel with kainate-treated rats. After kainate treatment, rats were monitored for 1–2 h/day, 3–5 days/wk (i.e., 6 h/wk) to determine whether they developed spontaneous motor seizures, and the frequency and severity of recurrent seizures were analyzed. Due to the progressive nature of epileptogenesis in this model and the increase in the frequency of spontaneous seizures, the seizure rate presented in this paper was based on data from the last month before the animals were killed for the slice experiment.
Slice preparation
Three to 9 mo (5.91 ± 0.55 mo) after kainate or saline treatment, when kainate-treated rats had developed spontaneous seizures, rats were anesthetized with halothane and decapitated with a guillotine. Rats were coded and obtained from the vivarium by another person so that the experimenter was blind to the type of the animal (epileptic or control). The brains were quickly dissected out and placed in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 1.3 CaCl2, 1.3 MgSO4, and 11 glucose. Transverse 300-µm-thick hippocampal slices were cut parallel to the base of the brain with a vibroslicer (Campden Instrument, Lafayette, IN), mostly from the temporal part of the hippocampi. Slices were trimmed, and the CA1 region was isolated from CA3/CA2 by a knife cut (Fig. 1A) and incubated in a storage chamber preheated to 32–34°C for 2 h to recover.
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Slices were submerged and mounted in a recording chamber perfused with oxygenated ACSF at room temperature. The GABAA-receptor antagonist, bicuculline methiodide (Sigma, 30 µM), was routinely added to suppress inhibitory synaptic transmission. Whole cell patch-clamp recordings were performed with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Patch pipettes were pulled from borosilicate glass capillaries (OD, 1.65 mm; ID, 1.2 mm; Garner Glass, Claremont, CA) with a P-87 Flaming-Brown puller (Sutter Instruments, Novato, CA) and filled with intracellular solution containing (in mM) 130 K-gluconate, 1 NaCl, 5 EGTA, 10 HEPES, 1 MgCl2, 1 CaCl2, 2 ATP, and 5 biocytin. The pH was adjusted to 7.2 with 5 M KOH. Patch pipettes had resistances of 2–5 M
. The series resistances of whole cell configuration and the cell input resistance were estimated from the amplitude of the initial capacitive transient and steady-state current in response to a 5-mV, 30-ms hyperpolarizing pulse. Series resistance was uncompensated and monitored during each experiment. Only data without an apparent series resistance change during the experiment were included in this study. All signals were low-pass filtered at 2 kHz, sampled at 10 kHz, and recorded with pClamp 8.0 software (Clampex) through a Digidata-1320A digitizer (Axon Instruments).
Flash photolysis of caged glutamate
The UV flash used for photolysis of caged glutamate was generated from a xenon lamp (Chadwick-Helmuth, El Monte, CA), transmitted through an epifluorescence attachment to an upside-down Optiphot microscope (Nikon), and was focused by a high-numerical aperture, oil-immersion objective (x40, Nikon). The location of the photostimulation was guided by a HeNe laser (Oriel instruments), mounted on the epifluoresence attachment. The intensity of each flash was 100–200 W-s (50–100 mJ), controlled by a Strobex power supply model 238 (Chadwick-Helmuth). The duration of each flash stimulation was 0.5 ms, given once every 20 s, determined by a A-65 timer (Winston Electronics). Slices were viewed with a monochrome charge-coupled device (CCD) camera (Cohu, San Diego, CA) through a video monitor. Photostimulations were applied at sites 150–200 µm apart along the CA1 cell body layer. Caged glutamate was purchased from Molecular Probes (Eugene, OR).
The rationale of focal flash photolysis of caged glutamate is to use glutamate as a neuronal excitant to activate a relatively small population of presynaptic neurons, independent of fibers-of-passage. This is shown schematically in Fig. 1B. Briefly, a strong flash stimulation uncages glutamate and thus excites the neurons at a particular site to generate action potentials. If none of the excited neurons have a connection with the recorded neuron, no EPSCs are evoked in the recorded neuron (Fig. 1B1). If one (or more) of the excited neurons has excitatory connections to the recorded neuron, EPSCs will be consistently generated and recorded (Fig. 1B2). Based on our previous studies, glutamate evokes action potentials when applied to the somatodendritic regions of neurons in CA1 and CA3 (Christian and Dudek 1988a, b) and in the dentate gyrus (Christian and Dudek 1988a; Wuarin and Dudek 1996), but does not cause action potentials when applied to axons of passage. Figure 2 shows that flash stimulations directed at the recorded neuron uncaged glutamate in the illuminated area, depolarized the somatodendritic regions of the neuron, and caused the recorded neuron to fire action potentials. Under our experimental conditions, the CA1 pyramidal cells from both control and kainate-treated rats responded to somatic flash stimulations with a period of repetitive action potentials; the period of repetitive firing was variable in duration and in the number of action potentials (usually lasting for hundreds of milliseconds; Fig. 2, A1–A3 and B1–B3). Sometimes only a few action potentials were evoked, followed by a prolonged depolarization, which presumably led to action potentials in the axons (Fig. 2, A4 and B4).
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; Su et al. 2002). In our study, glutamate photoactivation of CA1 pyramidal cells from kainate-treated rats—similar to pyramidal cells from control rats—evoked repetitive firing without obvious burst firing. Nonetheless, intrinsic burst-firing neurons may be more numerous in the CA1 area of kainate-treated rats, but this would have minimal effects on the outcome under our experimental conditions, because glutamate photoactivation normally induced at least a few action potentials in both control neurons and neurons from kainate-treated rats. Thus in both the control and experimental groups, the presynaptic neurons would be expected to fire enough action potentials to activate local excitatory circuits. Our results, similar to those of Wuarin and Dudek (2001), indicated that recorded neurons had two clearly distinct types of responses to focal flash photolysis of caged glutamate at surrounding sites: 1) evoked repetitive EPSCs to virtually every flash or 2) no repetitive EPSCs to surrounding stimulation sites (see RESULTS). Even if differences did occur between the two groups, they were relatively minor, and our analysis classified the results into positive versus negative responses and did not include a comparison of possible differences in the duration or robustness of the evoked EPSCs. If intrinsic burst-firing was induced or enhanced in the CA1 pyramidal cells of kainate-treated rats, the responses in those preparations with increased local excitatory circuits would likely be augmented. Data analysis and statistics
The pClamp 8.0 (Clampfit, Axon Instruments) and MiniAnalysis 5.0 programs (Synaptosoft, Leonia, NJ) were used for qualitative and quantitative data analysis. The sEPSCs were detected using MiniAnalysis and were visually checked to minimize errors. A 3-min sample of whole cell recording per cell was used for measuring sEPSC frequency and amplitude. To minimize potential sampling errors, a fixed number of sEPSCs from each neuron (i.e., the 1st 50 sEPSCs) was pooled for constructing histograms for amplitude and interval distributions and for calculating cumulative probability. Values of all sEPSC intervals and amplitudes were logarithmically transformed in the histograms. The Kolmogorov-Smirnov (KS) two-sample, two-tailed test was used to compare the cumulative probability of sEPSC intervals and amplitudes between control and epileptic groups. The mean sEPSC interval and amplitude were averaged across neurons (i.e., n = cells, not events). The 
2 test was employed to compare the ratios between groups. The Student's t-test were used for comparisons between two groups. Data are expressed as means ± SE, and 
= 0.05 in all tests.
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RESULTS |
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(P > 0.05) and 172 ± 22 versus 162 ± 19 M (P > 0.05), respectively, and these values were not significantly different. The main data are summarized in Table 1.
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At the beginning of each experiment, sEPSCs were recorded for 3–5 min in voltage-clamp mode at resting membrane potential. In this study, sEPSCs were observed in 62.2% of the neurons (n = 37, 0.2–3.5 Hz) from epileptic animals (Fig. 3B), and were also present in 42% of the neurons (n = 48, 0.1–1.1 Hz) from control rats (Fig. 3A). The histogram of inter-EPSC intervals (logarithmically transformed value) from both control and epileptic groups fit a Gaussian distribution (Fig. 4A). The peak of the inter-EPSC distribution of the epileptic group (2.78) was clearly shifted to the left, compared with that in the control group (3.21; Fig. 4A). Consistently, the cumulative probability plots for inter-EPSC interval in both groups were also significantly different (Fig. 4A, inset; P < 0.001, KS test). The mean sEPSC frequency averaged from cells of the epileptic group was 1.17 ± 0.17 Hz (n = 23), which was significantly higher than that in the control group (0.45 ± 0.06 Hz, n = 20; P < 0.001, Student's t-test). In contrast, the distributions of sEPSC amplitude of the control and epileptic groups were similar. The histograms of both groups fit a Gaussian distribution (Fig. 4B), and the peaks of the two histograms were identical (i.e., 1.25; Fig. 4B). Note that a small fraction of the sEPSCs in the epileptic group had a relatively large amplitude 2 (i.e., 100 pA), and this amplitude range was not observed in the control group (Fig. 4B). This also reflected a deviation in the tail of the amplitude cumulative distribution (Fig. 4B, inset), but the difference was not significant (P = 0.07, KS test). Similarly, the mean amplitude averaged across neurons of the control and epileptic groups was not significantly different (20 ± 1.17 pA, n = 20 vs. 25.2 ± 4.1 pA, n = 23; P > 0.05, Student's t-test). Thus the CA1 pyramidal cells from kainate-treated rats exhibited an increased sEPSC frequency, but not amplitude, relative to the controls. These data were consistent with the results of an earlier study using rats that had been systemically treated with pilocarpine and rats that had been injected intraventricularly with kainate (Esclapez et al. 1999).
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Our data that the frequency of sEPSCs in the isolated minislices was increased in epileptic rats supports the hypothesis that the excitatory synaptic drive to CA1 pyramidal neurons was increased, possibly due to formation of new synaptic connections. To further address this question, flash photolysis of caged glutamate was used to compare the local excitatory connectivity in CA1. Flashes were aimed at sites along stratum pyramidale, on both sides of a recorded neuron (Fig. 5A), over a distance of 200–600 µm from the recorded neuron. Using this approach, 4 of 48 CA1 pyramidal neurons (8%) from control rats were observed to receive excitatory connections from other CA1 pyramidal cells (data not shown). In contrast, a significantly higher connection rate (12 of 37, 32%, P < 0.001, 2 test) was found in CA1 pyramidal cells from rats with kainate-induced epilepsy. Normally four to seven spots per neuron were flashed along s. pyramidale. Of the 12 CA1 pyramidal neurons from the epileptic group that responded to flash stimulations, 7 neurons responded to one flash site, 4 neurons to two sites, and 1 neuron to at most five stimulation sites. Altogether, 63 sites were flashed, and 20 of them (32%) evoked responses in the 12 neurons. An example of the responses of a CA1 pyramidal cell to flash stimulations is shown in Fig. 5. Flash stimulations aimed at different sites of s. pyramidale (Fig. 5A) evoked either no response (Fig. 5B1) or a burst and/or a train of EPSCs that lasted up to several hundred milliseconds (Fig. 5, B2 and B3). These durations of repetitive EPSCs were consistent with the duration of the action potential bursts in the presynaptic neurons (Fig. 2). The responses were reproducible during repeated flash stimulations at the same sites (Fig. 5, B2 and B3), and they did not occur when the shutter was closed (data not shown). These data provide comparatively direct physiological evidence that more local excitatory connections are formed in the CA1 area during epileptogenesis.
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Neurons that receive excitatory inputs from local neurons would be expected to have more sEPSCs. We analyzed the sEPSC frequency in neurons with versus without evidence for local excitatory inputs from the flash photolysis experiments. The neurons that had repetitive EPSCs to photostimulation tended to have a higher sEPSC frequency than the neurons without EPSCs to photostimulation, but the difference was not significant (1.35 ± 0.30 vs. 0.81 ± 0.15 Hz, P > 0.05). However, normally two to three neurons were recorded per rat, and another analysis was performed where the kainate-treated rats (vs. neurons) were grouped into those with EPSCs to local stimulation and those without EPSCs. If any neuron from the same rat responded to photostimulation with EPSCs, this rat was considered as a "positive response" rat, and the sEPSC frequency of all neurons from this rat were averaged as the sEPSC frequency for this particular rat. In this analysis, those rats with evoked EPSCs to the photostimulation protocol had a higher mean sEPSC frequency than those in the latter group (1.39 ± 0.20 vs. 0.40 ± 0.11 Hz, P < 0.05). Thus the animals with neurons that responded to photostimulation had a significantly higher frequency of sEPSCs, supporting the hypothesis that this group had increased recurrent excitation.
Afterdischarges appeared in CA1 pyramidal cells from rats with kainate-induced epilepsy, but not in neurons from control rats
During the experiments to examine the direct responses of CA1 pyramidal cells to somatic flash stimulations, four of the CA1 pyramidal neurons from the epileptic rats responded to the somatic flash stimulations with afterdischarges (Fig. 6) composed of depolarizing shifts and repetitive action potentials. The afterdischarges were not observed in the controls. This is consistent with a previous study (Meier and Dudek 1996), which showed that in bicuculline the isolated CA1 area of rats with kainate-induced epilepsy responded to stimulation of stratum radiatum with evoked afterdischarges (and in some cases, spontaneously generated repetitive bursts); these events were not seen in slices from control rats.
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To explore whether the epileptic rats with more severe seizures were more likely to have formed new local excitatory connections in the CA1 area, we compared the seizure rates of kainate-treated rats for the last month before the in vitro electrophysiological experiments with their synaptic responsiveness to focal flash photolysis of caged glutamate. Of the 15 kainate-treated rats used in this study, 8 were found to receive local synaptic inputs from within CA1. The mean overall seizure rate of these eight rats was not significantly higher than that of the remaining seven rats that were not found to receive local excitatory inputs in the flash photolysis experiments (0.6 ± 0.1 vs. 0.4 ± 0.1 seizure/h, P > 0.05). However, the rate of occurrence of severe seizures (i.e., class 5; Ben-Ari 1985; Racine 1972) in the eight rats with detectable local inputs was significantly greater than that of the other seven rats without detectable local inputs (0.28 ± 0.05 vs. 0.12 ± 0.03 seizure/h, P < 0.05). This result suggests that rats with a detected neuronal interaction had experienced more severe seizures.
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DISCUSSION |
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; Ben-Ari et al. 1981; Franck et al. 1995; Sutula et al. 1989) and animal models (Buckmaster and Dudek 1997a,b; Molnar and Nadler 1999; Patrylo and Dudek 1998; Sutula et al. 1998; Tauck and Nadler 1985; Wuarin and Dudek 1996, 2001). A central question in epilepsy research is how widespread is this phenomenon? The CA1 area of the hippocampus in normal adult animals is known to have relatively few excitatory connections (Christian and Dudek 1988b; Deuchars and Thomson 1996; Knowles and Schwartkroin 1981). In epileptic animals, however, highly synchronized, synaptic-driven network bursts are readily evoked or generated spontaneously (Esclapez et al. 1999; Franck and Schwartzkroin 1985; Meier and Dudek 1996; Perez et al. 1996; Smith and Dudek 2001; 2002), suggesting an enhanced synaptic activity and probably also a reduced inhibition (Best et al. 1993; Dinocourt et al. 2003; Morin et al. 1998) in this area during epileptogenesis. Reconstruction of biocytin-labeled CA1 pyramidal neurons from epileptic rats has revealed that these cells have significantly more extensive axonal arborizations in stratum oriens (Esclapez et al. 1999; Perez et al. 1996; Smith and Dudek 2001), and these axon collaterals even invade s. pyramidale and stratum radiatum of the CA1 region (Esclapez et al. 1999). Furthermore, the mean axon length and branching points of the CA1 pyramidal neurons from epileptic animals are significantly greater than that in controls (Perez et al. 1996). These data provided morphological evidence of axonal sprouting in CA1 during epileptogenesis. In this study, we have provided physiological evidence that rats with kainate-induced epilepsy exhibited an increased sEPSCs frequency and an increased number of local excitatory connections assessed with focal flash photolysis of caged glutamate; these results were sometimes associated with afterdischarges in the CA1 area of the hippocampus. Increased frequency of sEPSCs in CA1 pyramidal cells of rats with kainate-induced epilepsy
The frequency of sEPSCs is primarily controlled by presynaptic factors (i.e., activity of presynaptic neurons, the number of axon terminals, and/or the transmitter-release probability; Katz 1962), whereas the amplitude of sEPSCs is determined largely by postsynaptic mechanisms (e.g., available postsynaptic receptors; Jonas et al. 1993; Perkel and Nicoll 1993; Tang et al. 1994). While miniature EPSCs (mEPSCs) result from spontaneous transmitter release by both local and projection axons, the sEPSCs recorded in our experiments would be expected to result from mEPSCs and action potential-dependent transmitter release of intact local axons (i.e., in CA1), since the projected axons from CA3/CA2 and more remote areas were cut during slice preparation (see METHODS). Therefore an increased frequency of sEPSCs in CA1 pyramidal neurons from epileptic rats is most likely from newly formed local excitatory circuits in CA1 during epileptogenesis. However, we do not rule out the possibility of an enhanced transmitter release probability in glutamatergic synapses in the CA1 area during epileptogenesis. These results are consistent with others reported earlier (Esclapez et al. 1999), which show an increased sEPSC frequency in CA1 pyramidal cells from rats that had been treated systematically with pilocarpine and rats that had been injected intraventricularly with kainate.
Glutamate microstimulation to study recurrent excitation in CA1 of kainate-treated rats
The enhanced sEPSC frequency in minislices from epileptic rats suggests an increase in local synaptic activity in CA1 that possibly involved formation of new connections between CA1 pyramidal cells during epileptogenesis. Previous studies using electrical stimulation (Esclapez et al. 1999; Franck and Schwartzkroin 1985; Meier and Dudek 1996; Perez et al. 1996; Smith and Dudek 2001) showed that the CA1 area generated epileptiform bursts in slices from epileptic rats. Glutamate microdrop application in the CA1 pyramidal cell layer evoked EPSCs in CA1 pyramidal cells from epileptic rats (Smith and Dudek 2002), which also supports the hypothesis that new local circuits formed during epileptogenesis. However, electrical stimulation would inevitably activate axons of passage, and glutamate drops have poor spatial resolution; therefore, a better approach to study the recurrent excitation issue is the use of flash photolysis of caged glutamate, where the amount of glutamate and location of the stimulation can be precisely controlled. Theoretically, the most direct analysis of neuronal connections requires dual recordings, which also has disadvantages for this purpose.
Focal photoactivation of caged glutamate versus dual recording for quantitative analysis of recurrent excitation
The flash photolysis data in this study further support the sEPSC data, showing that the local excitatory connections in CA1 had increased in rats with kainate-induced epilepsy. Theoretically, the best way to address this issue requires dual intracellular recordings to compare monosynaptic excitatory connections in control and epileptic rats. However, the extremely low detection rate for neuronal connectivity in the CA1 area (0–1%, Deuchars and Thomson 1996; Knowles and Schwartkroin 1981) demands many hundreds of pairs of recordings to be able to detect a potential difference between groups. For instance, if the connection rate between CA1 pyramidal cells in control animals is 1% (Deuchars and Thomson 1996), and if we hypothesize that the connection rate in CA1 from epileptic rats is 2%, a minimal 800 pairs per group is required to be able to detect the difference (P = 0.045). If the differences between the two groups is smaller, an even higher number of recordings is required, which makes it quite difficult (if not impractical) to assess the differences between the control and epileptic rats. Instead, we used the focal flash photolysis of caged glutamate for this purpose. Because the uncaged glutamate during each flash stimulation excites a small group of neurons under our experimental conditions (Wuarin and Dudek 2001), each flash stimulation would be equivalent to recordings of tens of pairs of neurons. Furthermore, the detected interactions in bicuculline would be a mixture of monosynaptic, multisynaptic, or/and polysynaptic EPSCs, thereby greatly enhancing the detection rate. In fact, using this approach, we detected local interconnections in 8% of the neurons from control rats and in 32% of the neurons from epileptic rats. The CA1 connection rate in epileptic rats (32%) was lower than that in dentate granule cells with robust mossy fiber sprouting (66%) studied by the same technique (Wuarin and Dudek 2001), but was significantly higher than the controls (P < 0.001), suggesting the formation of new connections in CA1. If enough connections are formed, the CA1 area is expected to be capable of generating network bursts, as in the CA3 area (Wong and Prince 1979; Wong and Traub 1983). Therefore the afterdischarges observed in CA1 minislices in this study are consistent with our sEPSC and flash-photolysis data and further support the hypothesis that more excitatory connections are formed and an interconnected synaptic network is present in the CA1 area of rats with kainate-induced epilepsy.
Conclusions and potential relevance to epilepsy
We observed significantly increased local excitatory connections in the CA1 area associated with an enhanced sEPSC frequency, thus providing new and more direct electrophysiological evidence supporting the hypothesis that new local excitatory circuits have formed via axonal sprouting during epileptogenesis in CA1. A recent study has reported that a similar synaptic reorganization may occur in neocortex (Li and Prince 2002), and another study has proposed that more widespread cross-regional synaptic reorganization may occur during epileptogenesis (Lehmann et al. 2001). Clearly, although an increasing body of evidence supports the hypothesis that formation of new local excitatory circuits may be important for epileptogenesis, other mechanisms such as alterations of GABA-mediated inhibition, intrinsic membrane properties, N-methyl-D-aspartate receptors, and nonsynaptic factors may also play an important role.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. E. Dudek, Dept. of Biomedical Sciences, Anatomy and Neurobiology Section, 1617 Campus Delivery, Colorado State Univ., Fort Collins, CO 80523 (E-mail: ed.dudek{at}colostate.edu).
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