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Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, Virginia
Submitted 31 January 2007; accepted in final form 28 April 2007
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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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; Clark et al. 2000; Galaburda and Eidelberg 1982; Taylor et al. 2001) and epilepsy (Crino 2004; Kim et al. 2006; Segawa et al. 1979) that has a varied etiology, including genetic and acquired causes (Barkovich and Lindan 1994; Barkovich et al. 1995; Cantagrel et al. 2007; Chang et al. 2006; Iannetti et al. 1998; Mitchell et al. 2003; Piao et al. 2005; Richman et al. 1974). Despite identification of genes contributing to some forms of polymicrogyria, including GPR56, PAX6, and AH11 (Gleeson et al. 2004; Guerrini and Marini 2006; Mitchell et al. 2003; Piao et al. 2005), the connection between the structural abnormality and neurological dysfunction is ill-understood. These genes play a role in neuronal migration and differentiation (Fukumitsu et al. 2006; Little et al. 2004; Shashidhar et al. 2005; Tzoulaki et al. 2005). Changes in migration endpoint and differentiation can elicit subsequent abnormalities in connectivity (Chevassus-Au-Louis et al. 1998; Garbossa and Vercelli 2003; Jones et al. 1981; Prince et al. 1999). The mechanisms that produce the epileptiform activity are likely to occur subsequent to the formation of the initial structural abnormality. In fact there is often a "waiting period" before the onset of seizures in patients with epilepsy having a developmental cause (Bartolomei et al. 1999; Kobayashi et al. 2001; Park et al. 2006; Widdess-Walsh et al. 2005).
One goal of research with animal malformation models is to identify the mechanisms that initiate a propensity for hyperexcitability. The freeze-lesion model of microgyria mimics all aspects of the histopathology of human four-layered microgyria (Dvorak and Feit 1977; Dvorak et al. 1978; Jacobs et al. 1996). This type of microgyria involves the focal loss of deep layers IV–VIa, resulting in folded superficial layers above a glial scar and layer VIb. Interictal-like epileptiform activity can readily be evoked in vitro in slices containing the microgyral malformation (Jacobs et al. 1999a). Although overt spontaneous seizures have not been observed in freeze-lesioned animals, seizure susceptibility is increased in these animals (Scantlebury et al. 2004). Lack of seizures is not a limitation because our goal is to study the onset of epileptiform activity. In fact, seizures and hyperexcitability may induce additional alterations in the same biological properties that initiated the excitability changes (Jankowsky and Patterson 2001; Motte et al. 1998; Parent et al. 1999, 2006; Pazman et al. 1997; Sutula 2002; Sutula et al. 1992; Teskey et al. 2006; Vezzani and Hoyer 1999). Similar to the clinical waiting period, field potential epileptiform activity is not observed in this rat model until postnatal day (P) 12 (Jacobs et al. 1999a), despite the completion of microgyral structure by P7–P8 (Dvorak et al. 1978; Rosen et al. 1992). One approach to distinguishing mechanisms that generate the hyperexcitability from processes altered by it is to examine the model before onset of the hyperexcitability.
We believe that a major factor contributing to hyperexcitability in the microgyral rat is hyperinnervation of the epileptogenic area surrounding the malformation by excitatory afferents (Jacobs et al. 1999b). We first hypothesized that excitatory afferents might be redirected to this paramicrogyral region (PMG) because these afferents have lost many of their targets within the malformation. For instance, some thalamocortical afferents have lost their layer IV targets (both layer IV neurons and the dendrites of deeper-lying pyramidal neurons). Neurons with the appropriate molecular cues are present within layer IV of the surrounding PMG. The microgyral area from a single lesion typically extends approximately 1 mm, whereas single thalamocortical afferents have projection diameters of approximately 0.5–1.5 mm (Arnold et al. 2001). Thus layer IV neurons of the PMG would be found within the projection zone of most thalamocortical afferents. This may aid in redirecting the collaterals away from the malformed region and toward the PMG. Supporting this idea is our finding that within the PMG, miniature excitatory postsynaptic current (mEPSC) frequency was significantly increased for layer V pyramidal neurons (Jacobs and Prince 2005), suggesting that they are hyperinnervated by excitatory afferents. In addition, anatomical studies have shown that few thalamocortical afferents invade the malformed region yet a dense projection occurs in the adjacent PMG (Jacobs et al. 1999b; Rosen et al. 2000). Hyperinnervation of the PMG by excitatory afferents would be expected to be proepileptogenic. Thus if these changes begin before the onset of epileptiform activity, they may be an important contributing factor to the hyperexcitability associated with this malformation. Here we have used whole cell patch-clamp recordings to examine spontaneous (s) and evoked (e) EPSCs just before onset (P7–P11) of epileptiform activity in the rat freeze-lesion model of microgyria.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Pups were anesthetized with hypothermia, the skull was exposed, and a rectangular (2 x 5 mm) freezing probe maintained at approximately –5°C was placed on the skull over the somatosensory cortex for 5 s. The scalp then was sutured and the pup warmed up and returned to the dam. Rats aged P7–P11 were anesthetized with pentobarbital (55 mg/kg) and decapitated. Brains were removed and placed in a cold sucrose slicing solution containing (in mM) 2.5 KCl, 1.25 NaH2PO4·H2O, 10.0 MgSO4, 0.5 CaCl2·2H2O, 234 sucrose, 11 glucose, and 26 NaHCO3. Slices, 300 µm thick, were cut on vibratome in sucrose slicing solution and incubated in normal Ringer solution [artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 5 KCl, 1.25 NaH2PO4·H2O, 2 CaCl2·2H2O, 10 glucose, and 26 NaHCO3] at 34°C for 25 min and thereafter at room temperature.
Whole cell patch-clamp recordings were made under visual control in a submerged chamber at 32°C using a Multiclamp 700A amplifier (Axon Instruments). Recordings were made from layer V pyramidal neurons (0.3–2.5 mm from the sulcus or in homotopic control cortex) using glass micropipettes (2–5 M
, Garner Glass, Claremont, CA) filled with intracellular recording patch solution containing (in mM): 117 gluconic acid, 117 CsOH, 11 CsCl, 10 Hepes, 11 EGTA, 1 MgCl2·6H2O, 1 CaCl2·2H2O, and 0.5% biocytin.
EPSCs were recorded at a holding potential of –60 mV. Only recordings with an access resistance <21 m that varied <20% were accepted for analysis. Data were digitized on-line (20 kHz) using Clampex software (Axon Instruments). After the recording of spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs), 1 µM tetrodotoxin (TTX, 1:1,000, Sigma) was applied to the bath solution and recordings of miniature (m) EPSCs and IPSCs were begun after 5 min of wash-in. Currents were detected and measured using MiniAnalysis Software (by Synaptosoft). Only recordings with >50 events were accepted for analysis.
Stimulus parameters were similar to those that evoked epileptiform activities in previously described reports (Jacobs et al. 1999a). Pulses were applied within layer V, 
450 µm from recorded cells with a glass pipette filled with 1 M NaCl. A 20-µs-duration pulse was applied and the current intensity needed for evoking a detectable PSC was considered threshold. A series of stimuli was then applied by increasing the pulse duration to 40, 80, 160, and 320 µs (2, 4, 8, and 16 x threshold).
After recordings were completed, slices were immediately fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Slices were maintained in this fixative or placed in 0.1 M phosphate buffer (pH 7.4) after 24 h until sectioning or reaction for biocytin. In some cases slices were cryoprotected by immersion in 30% sucrose (in 0.1 M phosphate buffer) until they sank and then were resectioned on a freezing microtome at 40 µm. Slices were either stained for Nissl with cresyl violet or reacted for biocytin, using standard procedures (Vector ABC kit and DAB) to verify pyramidal cell morphology and the laminar position of the recorded cell. Measurements are reported as means ± SE. Student's t-test (Microsoft Excel) and ANOVAs (SigmaStat or SPSS) were used to test for significance.
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RESULTS |
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Spontaneous excitatory postsynaptic currents
The frequency of sEPSCs varied appreciably between cells, from 0.20 to 8.57 Hz in 65 control cells and from 0.15 to 16.43 Hz in 55 PMG cells. Mean sEPSC frequency was significantly greater in the PMG cell group relative to the control cells (Fig. 1, A and C, 3.0 ± 0.5 vs. 1.6 ± 0.2 Hz, respectively, t-test, P < 0.005). Peak amplitude, rise time, and decay time of sEPSCs were not significantly different between PMG and control cells (see Table 1). The sEPSCs in both control and PMG cells typically lasted approximately 1 ms (see decay time in Table 1). Thus events occurring over intervals shorter than this are likely to sum, creating a larger postsynaptic response. To examine this, we analyzed the incidence of "bursts" of sEPSCs (two or more events occurring with an interevent interval of <10 ms). PMG cells had significantly more bursts per minute than control cells (Fig. 1D, 7.3 ± 2.4 vs. 2.2 ± 0.5 bursts/min, respectively, t-test, P < 0.05). In addition, a significantly larger proportion of PMG cells contained bursts compared with control cells (89 vs. 70%, respectively, z-test, P < 0.05).
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EPSC frequency is independent of location within PMG cortex
We selected layer V pyramidal neurons within the PMG region previously shown to be epileptogenic at later ages (after P12) (Jacobs et al. 1999a). To determine whether there was a bias within this region, we examined the distribution of EPSC frequency versus either location from the sulcus or depth from the pia (Fig. 2). There was no correlation between frequency and distance from the sulcus for either sEPSCs or mEPSCs (Fig. 2A, Pearson product moment, R values of 0.00 and –0.07 for sEPSCs and mEPSCs, respectively, both NS).
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Evoked excitatory postsynaptic currents
Stimulation within layer V nearby the recorded cell produced short-latency EPSCs whose peak and area increased with increasing stimulus intensity. In some cases the EPSC came to a single peak, followed by a smoothly decaying current (see example in Fig. 3 A2). In other cells multipeaked short-latency EPSCs were observed (example in Fig. 3A1). Significantly, more PMG than control cells had multipeaked eEPSCs (60% of 15 control and 96% of 26 PMG cells, z-test, P < 0.05). The peak amplitude and area of the eEPSC were significantly larger in PMG compared with control cells (Fig. 3, B–D, two-way repeated-measures ANOVAs, P < 0.01). The interaction between stimulus intensity and group (PMG vs. control) was significant for the eEPSC area (P < 0.01), but not for peak amplitude (P = 0.06).
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; Jacobs et al. 1999a). This type of late activity was observed in only two of 15 control cells, one of which is shown in Fig. 3A2. In contrast, late activity was observed in 69% of 26 PMG cells (significantly more than controls, z-test, P < 0.005). In some cells the late activity was clearly evoked because it occurred with a similar latency on multiple stimulus presentations. Effect of age on excitatory postsynaptic currents
We initially hypothesized that during the normal period of synaptogenesis, numbers of excitatory inputs to PMG cells would be increased over control levels. To determine whether the number of functional synapses was changing during the ages examined (P7–P11), we separated the results by survival age. Surprisingly, for control cells, neither sEPSCs nor mEPSCs increased in frequency with age during this period (Fig. 4 A, one-way ANOVAs, NS). The sEPSC frequency showed a relatively stable mean from 1.5 ± 0.4 at P7 to 2.1 ± 0.6 Hz at P11. In contrast for PMG cells, the frequency of sEPSCs significantly increased with age from 0.9 ± 0.1 at P7 to 4.8 ± 1.1 Hz at P11 (Fig. 4A, one-way ANOVA, P < 0.05).
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For mEPSCs, a one-way ANOVA showed a significant increase in frequency with age for PMG cells but not for control cells (Fig. 4A). Control and PMG (experimental condition) mEPSC frequencies across survival age were compared with a two-way ANOVA that showed a significant difference for age, experimental condition, and interaction between these two (P values of <0.001, <0.005, and <0.02, respectively). The mEPSC frequency difference between control and PMG cells was largest at P10 and P11 (Fig. 4A). Two-way ANOVAs applied for mEPSC amplitude, area, and rise time showed no differences for either age, experimental condition, or interaction.
The number of EPSC bursts per minute significantly increased with age for PMG but not control sEPSCs (Fig. 4B, P < 0.005). A two-way ANOVA on sEPSC bursts per minute showed significant differences for age, experimental condition, and interaction (P < 0.05 for all). For mEPSCs we examined the CV of interevent interval versus age. Controls showed no significant difference in this measure over the P7–P11 age range (one-way ANOVA, NS). In contrast, in PMG cells, there was a significant decrease in CV with increasing age (one-way ANOVA, P < 0.05). These results show that variability in mEPSC interevent interval for PMG cells is highest, and most different from control at (P7–P9) ages when the mean frequency is not significantly different between control and PMG cells. In addition, variability in this measure is near control levels at P9–P10 ages, when the mEPSC frequency is significantly higher. These results further suggest a division in neuronal mechanisms for P7–P9 ages compared with P10–P11.
To examine the distribution of individual mEPSC events, cumulative probability plots for interevent interval were constructed (Fig. 5). For this analysis, the first 200 events were selected for the four cells closest to the mean, yielding 800 events for each group. The distribution was similar for control and PMG cells at ages P7–P9, but showed a significant divergence at ages P10 and P11, with a greater number of small interevent intervals for PMG cells (Kolmogorov–Smirnov tests, NS for P7–P9, P < 0.001 for P10 and P11). For PMG cells at P11, 61% of the events had an interevent interval of 1 s, whereas for control cells at P11, this was true for only 19% of events (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). The fact that there appears to be an increased excitatory synaptic input before the inception of epileptiform activity suggests that this mechanism may contribute to the onset.
If presynaptic release probability for afferents to these layer V neurons is unchanged in PMG cortex, then the increase in mEPSC frequency should be the result of hyperinnervation of layer V pyramidal neurons by excitatory afferents. Interestingly, the increase in mEPSC frequency in the PMG occurs at a time when functional level of synaptic input is stable in controls (Fig. 4A, Control mEPSCs). This is somewhat surprising because the density of dendritic spines and complexity of basal dendrites both continue to increase between P7 and P14 (Eayrs and Goodhead 1959; Wise et al. 1979). Additionally, the increase in mEPSC frequency seems to occur suddenly at P10. A number of studies have previously shown a maintenance of an immature state in microgyral animals, including retention of radial glial neurons (Rosen et al. 1994), normally eliminated pathways (Innocenti and Berbel 1991), and a slower action potential for microgyral neurons (Luhmann et al. 1998). This does not appear to be the case for mEPSC frequency because at P7, for instance, it is even lower in PMG than in controls. The increased variability (as measured by CV) in interevent interval at young ages suggests that some alterations in presynaptic mechanisms are under way, but not yet stabilized at P7–P9. At a time when the mEPSC frequency is significantly larger for PMG cells, the variability in mEPSC interevent interval has become stable (see Fig. 4).
Sprouting of connections commonly occurs in the CNS of both developing and adult animals when target sites become available, through loss of normal input (Carmichael 2003; Salin et al. 1995; Serfaty et al. 2005; Steward 1992; Stroemer et al. 1995; Uryu et al. 2001; van Praag et al. 1996; Villablanca and Hovda 2000). The missing neurons from the malformed region would have normally sent some axonal collaterals horizontally to the adjacent cortex (Burkhalter 1989; Jones 1984). This may account for some vacated target space. An additional source of new target space may come from the increase in basal dendritic length that occurs in the PMG (Di Rocco et al. 2002). It is also possible that synaptic density is increased on individual layer V neurons. This has not yet been studied.
Pathways that have lost targets within the eliminated middle and deep layers of the malformed region include thalamocortical, intracortical, and callosal afferents. We have presumed that it is more likely that these afferents will synapse on cells in an abnormal position (medial–lateral or anterior–posterior) of the correct layer rather than on cells of the incorrect layer but initially specified location. This presumption comes in part from coculturing studies that show that thalamic axons find neocortical layer IV independent of the position of the two pieces of tissue (Bolz et al. 1990, 1992; Yamamoto et al. 1989, 1992). Such studies have also shown that regional specificity is not maintained (Molnar and Blakemore 1991). This is in contrast to what is likely to occur when no targets of the correct layer are present. Using a teratogen to kill dividing neuroblasts expected to become layer IV neurons, a microcephalic cortex can be created (Ciaroni et al. 1989; Garbossa and Vercelli 2003; Spatz and Laqueur 1968). In this case, where no layer IV is present, thalamocortical axons will synapse on surviving layer III neurons (Jones et al. 1981). In addition, large cortical lesions that also eliminate the subplate often cause degeneration of thalamocortical neurons (Kolb and Cioe 2003; Loopuijt et al. 1995). We expect that with our transcranial lesions, the subplate likely survives (Dvorak and Feit 1977; Dvorak et al. 1978); however, there is still likely some thalamic degeneration. Hermann et al. (1997) showed that neonatal transcranial freeze lesions similar to ours, but over occipital cortex, caused a significant decrease in volume and cell number within the lateral geniculate nucleus (Herman et al. 1997). This group has also observed gender differences in cell size distribution within the medial geniculate nucleus, with only freeze-lesioned males being altered from controls (Herman et al. 1997). These thalamic changes are unlikely to be related to epileptogenicity because both males and females consistently show epileptiform activity (Jacobs et al. 1999a).
Interestingly, there appears to be a gradual change in sEPSC frequency over the age range examined, but a sudden change in these measures at P10 for mEPSCs. An increase in sEPSC frequency without a simultaneous increase in mEPSC frequency could be explained by the increased firing of cortical pyramidal neurons. Intrinsic properties of pyramidal neurons within the PMG have not yet been fully investigated, although changes have been observed for neurons inside the four-layered region (Luhmann et al. 1998). Changes observed there would not be likely to increase intrinsic excitability because a decrease in action potential amplitude and decrease in the firing frequency per current (F/I slope) were observed (Luhmann et al. 1998). A change in intrinsic properties of cortical neurons has been demonstrated in other malformation models. In the telencephalic internal structural heterotopia (TISH) model, neurons within the heterotopia rest at more depolarized levels (Trotter et al. 2006). In the methyl azoxymethanol acetate (MAM) model of microcephaly and focal cortical dysplasia, neurons of the heterotopic region lack the Kv4.2 potassium channel, making them more likely to burst (Castro et al. 2001). In addition, deafferentation in adult cortex can increase the percentage of neurons identified as intrinsically bursting (Topolnik et al. 2003). It is still unknown whether there is an increase in the number of intrinsically bursting neurons in PMG. It is likely, however, that additional cellular anomalies exist beyond the hyperinnervation of pyramidal neurons by excitatory afferents. We believe this is particularly true because on P10 there is a significant increase in mEPSC frequency, yet field epileptiform activity is not observed at this age (Jacobs et al. 1999a).
The polymicrogyral region in human epilepsy patients has been shown to be functional during visual, motor, and language tasks (Araujo et al. 2006; Innocenti et al. 2001; Janszky et al. 2003; Staudt et al. 2004; Zesiger et al. 2002). Despite this, white matter and tract abnormalities identified with MRI suggest that there may still be abnormal connectivity (Bonilha et al. 2007; Munakata et al. 2006; Staudt et al. 2004; Trivedi et al. 2006). Development of mirror-movement abnormalities in patients with polymicrogyria also suggests abnormal connectivity in cortical tracts (RamachandranNair et al. 2006; Sahin et al. 2006). Magnetoencephalography studies further suggest abnormalities in organization and a high degree of variation between polymicrogyria patients (Ishitobi et al. 2005). Focal regions of abnormal connectivity, particularly in the spatial location of inputs, could explain these results as well as those associated with various forms of dyslexia (Galaburda and Eidelberg 1982).
Currently identified polymicrogyria genes GPR56 and PAX6 are suggested to have a role in migration and cortical patterning (Chapouton et al. 1999; Fukuda et al. 2000; Jansen and Andermann 2005; Jimenez et al. 2002; Mitchell et al. 2003; Piao et al. 2004). Pax6 is also involved in determining the proportion of specific neuronal cell types (Caric et al. 1997; Stoykova et al. 2003). How these genes affect cortical connectivity is currently unknown, but the loss or abnormal positioning of specific cortical cell types is likely to affect molecular cues necessary for afferent targeting. An example of this comes from MAM rats, in which the heterotopic neurons within the hippocampus have characteristics of superficial layer neocortical neurons (Castro et al. 2002; Chevassus-Au-Louis et al. 1998). Hippocampal heterotopia show abnormal connectivity to the overlying neocortex (Chevassus-Au-Louis et al. 1998; Colacitti et al. 1998; Jacobs et al. 1999b; Tschuluun et al. 2005). Additional connectional abnormalities are found in both hippocampus and neocortex as the result of both loss of normal targets and abnormal positioning (Chevassus-Au-Louis et al. 1999; Jones et al. 1981).
It is also possible that the polymicrogyria genes are important for functions beyond the time of development, as suggested in two genetic mutants that show spontaneous seizures. An autosomal recessive mutation causes the telencephalic structural heterotopia (TISH) rat that mimics human heterotopia associated with epilepsy (Lee et al. 1997). The seizures are delayed until approximately P30, suggesting that the affected gene may play a role in maintenance of GABAergic populations (Trotter et al. 2006). Inactivation of the urokinase plasminogen activator receptor (uPAR) gene in mice produces a structurally normal cortex, but with a substantial decrease in the numbers of GABAergic parvalbumin-immunopositive cells (Powell et al. 2001, 2003). Both the decrease in interneurons and the onset of seizures is delayed beyond the time of cortical development (Bae et al. 2005, 2006), again suggesting that both development and maintenance of GABAergic neurons are modulated by uPAR. Seizures are often delayed in humans with polymicrogyria (Bartolomei et al. 1999; Kobayashi et al. 2001) because they are in the freeze-lesion model, again suggesting that multiple factors, including hyperinnervation by excitatory cortical afferents, are at play to cause seizure onset.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. M. Jacobs, Dept. of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23298 (E-mail: kmjacobs{at}vcu.edu)
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2 events occurring with an interevent interval of <10 ms. E: coefficient of variation (CV) for interevent intervals in mEPSC recordings from control (gray) and PMG (black) cells. Data in C and D for sEPSCs from 65 control and 55 PMG cells, for mEPSCs from 32 control and 32 PMG cells. ***P < 0.005, t-test; *P < 0.05, t-test.
