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Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California
Submitted 22 March 2004; accepted in final form 15 September 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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); however interest in this correlation has increased in the last 15 years. This is due to improvements in imaging techniques that have demonstrated a higher incidence of cortical malformations than previously diagnosed, particularly in patients with refractory epilepsy (Farrell et al. 1992; Hauser 1995; Lagae 2000; Palmini et al. 1991a, b). It is not currently known whether mechanisms underlying hyperexcitability in malformed cortex are unique among the epilepsies or why seizures associated with cortical malformations are particularly difficult to treat with currently available anti-epileptic medications.
A number of animal models of developmental malformations associated with epilepsy have been characterized in recent years (Chevassus-Au-Louis et al. 1999; Jacobs et al. 1999b). These models mimic various aspects of the histopathology as well as the increased propensity for cortical hyperexcitability. The form of structural defect or type of malformation created depends on the timing of the insult. Most malformations occur after the failure of a critical process in neurogenesis (resulting in microcephaly), migration (resulting in double cortex, or other heterotopias), or neuronal differentiation (resulting in focal cortical dysplasia). The malformation studied here, focal unilateral 4-layered microgyria, is distinct from others because the structural abnormality is usually formed as a result of cell death rather than an error in a developmental program. A correlation between prenatal traumatic events and the subsequent detection of polymicrogyria has been demonstrated in a number of cases (Barkovich et al. 1995; Montenegro et al. 2002; Richman et al. 1974). Because neurons of the deep layers migrate into place first, these neurons will be lost (Rosen et al. 1996), while cells of the superficial layers migrate through the area of damage, resulting in a region of dyslamination bordered by normal six-layered cortex (Dvorak and Feit 1977).
Animals with microgyral cortex show enhanced cfos expression long after the freeze lesion is placed (Jacobs et al. 2001) and greater propensity for seizures after systemic kainic acid injections (unpublished observations). Epileptiform activity can be evoked without the addition of seizure-inducing agents in neocortical slices containing microgyri (Jacobs et al. 1996; Luhmann and Raabe 1996) after a 10- to 11-day latency after the freeze lesion (Jacobs et al. 1999a). We have previously determined that epileptiform activity is most readily evoked by stimulation within the normally laminated cortical region surrounding the microgyrus (the "paramicrogyral" zone) and that severing the connections between the microgyrus and surrounding cortex does not eliminate this hyperexcitability (Jacobs et al. 1999b). Therefore we have focused efforts on identifying the epileptogenic mechanisms in the paramicrogyral cortex.
Previous findings indicate that both inhibitory and excitatory synaptic transmission may be altered (Jacobs et al. 1999b). For example, immunocytochemical experiments showed that there were decreased numbers of parvalbumin-immunoreactive neurons adjacent to the microgyrus, suggesting that at least one population of inhibitory interneurons might be reduced in this region (Rosen et al. 1998). Also, enhanced excitatory connectivity likely results from substantial rewiring of excitatory circuits, presumably because the lesion-induced loss of neurons occurs prior to maturation of axonal projection patterns (Holopainen and Lauren 2003; Jacobs et al. 1999b; Poulter et al. 1997; Redecker et al. 2000; Rosen et al. 2000).
To test the hypothesis that enhanced excitatory and/or reduced inhibitory synaptic activity contributes to a malformation model of partial epileptogenesis, we recorded spontaneous and evoked postsynaptic currents from layer V pyramidal neurons within the region of hyperexcitability adjacent to the malformation just after the onset of epileptogenesis (13–16 days postnatally). These cells are known to have particular intrinsic characteristics and axonal projection patterns that make them likely to be involved in initiation and spread of epileptiform activity (Chagnac-Amitai et al. 1990; Connors 1984; Hoffman et al. 1994). Results suggest that there is enhanced glutamatergic excitatory innervation of both layer V pyramidal cells and interneurons in the paramicrogyral zone resulting in an increased amplitude of spontaneous and evoked inhibitory currents as well as a shift in the balance of synaptic activation of pyramidal neurons toward excitation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Briefly, Sprague Dawley rat pups <48 h old were covered with ice for 
4 min until movements and responses to tail pinch were absent. The skull was exposed through a scalp incision and a freezing probe with a rectangular tip, 5 x 2 mm, cooled to –50 to –60°C, was placed on the skull over somatosensory cortex for 5–6 s. The scalp was then sutured, and the pup warmed and returned to the dam. Thirteen to 16 days later, rats were anesthetized with pentobarbital (55 mg/kg ip) and decapitated and brains were removed. The site of the previous freeze lesion could be located as a small depression in the pial surface that corresponded to the site of the microsulcus (Jacobs et al. 1996). Standard techniques were used for preparing and maintaining neocortical slices. After removal, the brain was immediately placed in a sucrose-modified artificial cerebral spinal fluid (ACSF) containing (in mM) 2.5 KCl, 10 MgSO4, 3.4 CaCl2, 1.25 NaH2PO4, 234 sucrose, 11 glucose, and 26 NaHCO3; pH 7.4 when saturated with 95% O2-5% CO2. Coronal 300-µm-thick slices through the microgyrus and adjacent area or homologous control cortex were cut in the modified ACSF with a vibratome. After cutting, slices were immediately placed in normal oxygenated ACSF containing (in mM) 126 NaCl, 3.0 KCl, 2.0 MgCl2, 2.0 CaCl2, 1.25 NaH2PO4, 10 glucose, and 26 NaHCO3. Slices were maintained in this solution at 34°C for 45 min and thereafter at room temperature. Recordings were made in normal ACSF at 31–32°C.
Whole cell patch-clamp recordings were made with an AxoClamp 2B amplifier (Axon Instruments) from layer V pyramidal neurons visually identified with infrared differential interference contrast (DIC) microscopy. The intracellular patch solution consisted of (in mM) 117 CsGluconate, 11 CsCl, 10 HEPES, 11 EGTA, 1 MgCl2, 1 CaCl2, 4 Na-ATP, 0.2 Na-GTP, and 0.5 QX-314, pH adjusted to 7.3 with CsOH and osmolarity to 280–290 mosmol. With this internal solution, estimated ECl- was –51 mV, and inhibitory postsynaptic currents (IPSCs) were recorded as outward currents at a holding potential of 0 or +10 mV (e.g., Fig. 1). In some cases, 0.5% biocytin was included in the patch pipette solution to verify that the labeled morphology was consistent with that observed with DIC microscopy, prior to patching the cell. Standard immunocytochemical techniques used for subsequent identification of neuronal morphology (Xiang et al. 1998). Access resistance was compensated 50–70% and assessed every 20 s during voltage-clamp recordings. Only recordings with an access resistance <20 M
that varied <20% were accepted for analysis. Input resistances were typically between 100 and 400 M with a mean of 251.9 ± 11 (SE) M (n = 255). Data were either stored on magnetic tape and later digitized or digitized on-line (2–5 kHz) using software from Axon Instruments. During recordings, slices were perfused with normal ACSF (above). For experiments involving blockade of ionotropic glutamate receptors, 20 µM 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) and 50 µM D-2-amino-5-phosphonopentanoic acid (APV) were included in the normal ACSF. Miniature synaptic currents were recorded in ACSF containing 1 µM tetrodotoxin (TTX). Effectiveness of these drugs was determined during wash-in by examining evoked synaptic currents every 30 s. Recordings of spontaneous currents for analysis were obtained only after the changed response became stable or was eliminated (in TTX), and 
5 min after beginning wash-in.
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) and at an age when nearly every slice from every animal generated evoked epileptiform activity in previous experiments (Jacobs et al. 1999a). For most, but not all neurons, the response to nearby electrical stimulation was recorded. Stimulus parameters were similar to those that evoked epileptiform activities in our previous experiments. Monopolar square pulses were applied within layer V, 50–300 µM from the recorded cell with a glass micropipette filled with 1 M NaCl. A 20-µs-duration pulse was applied at threshold intensity for evoking a just detectable IPSC. A series of graded intensity stimuli was then applied by increasing the pulse duration to 40, 80, 160, and 320 µs (2x, 4x, 8x, and 16x threshold). Digitized waveforms were imported into Microsoft Excel and macros used to calculate peak and area of evoked currents. Measurements of peak current were made over a 0.5-ms time window from short-latency evoked responses.
Spontaneous events were recorded for a period of 3–10 min with 50 and typically >500 events for each recording condition. Currents were analyzed with event detection software (Detector, by John Huguenard). For amplitude measurements, currents uncontaminated by subsequent events were isolated and peak current over a 1-ms window was determined. Ratios of event frequencies were made in individual cells, as follows: EPSC frequency/(EPSC frequency + IPSC frequency). Measurements are reported as means ± SE. Student’s t-test were used to test for significance.
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RESULTS |
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In slices from control animals, two types of response were evoked by stimuli delivered within 300 µm of the recorded layer V pyramidal neuron in normal ACSF. Short-latency outward IPSCs that were graded in amplitude with stimulus intensity, were evoked in 65% of control cells ("standard" responses, n = 83, Fig. 1A1). In the other 35% of control cells, variable form, variable latency multiphasic events were evoked in an all-or-none manner, in addition to the graded short latency IPSC (see 
, Fig. 1A2). This multiphasic activity had characteristics similar to those seen in the field potentialsevoked in unlesioned immature animals (Luhmann and Prince 1990) and the paramicrogyral cortex of mature animals (Jacobs et al. 1996; Luhmann and Raabe 1996). Parameters of sIPSCs were not significantly different in neurons that had standard versus polysynaptic responses to stimulation (Fig. 1B, 1 and 2; Table 1) and all control cells were therefore considered as a single group.
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). In terms of their long and shifting latency, variable amplitude and all-or-none appearance, these responses closely resembled the evoked epileptiform field potentials and neuronal responses seen in this and other models of lesion-induced chronic focal epileptogenesis (Hoffman et al. 1994; Jacobs et al. 1996; Prince and Tseng 1993). For this reason, we have termed the neurons showing such abnormal evoked responses PMGE cells. A smaller population of PMG neurons had standard responses consisting of short-latency, smoothly decaying evoked IPSCs (termed PMGS cells in the following text). Both experimental cell groups (PMGE and PMGS) were found throughout the depth of layer V and the entire PMG cortex examined (0.3–2.0 mm from the microsulcus). The type of response in a given cell could not be altered by changes in stimulus parameters or stimulus position. In some cases, these two different response types were seen in neurons close to one another within the same slice (Fig. 1C). There was no difference in mean input resistance for the two PMG cell groups (250 ± 21 and 263 ± 23 M for 70 PMGE and 49 PMGS, respectively). For measures in which the two PMG groups were not significantly different quantitatively, they are compared as a single group to the control population. On measures for which PMGE cells were significantly different from PMGS cells, these experimental groups were compared individually to the entire control cell group.
As previously reported (Salin and Prince 1996), characteristics of sIPSCs varied greatly between individual layer V pyramidal neurons, even within the control population. The frequency of sIPSCs ranged from 0.3 to 21.5 Hz and mean peak conductance from 0.18 to 0.59 nS. When control and PMG cell populations were compared, there was no significant difference in sIPSC rise time (1.3 ± 0.04 ms control and 1.3 ± 0.03 ms PMG) or mean frequency (see Table 1). However, as suggested from examination of raw traces (e.g., Fig. 1B, 1 and 2, vs. 3 and 4), the peak conductance of sIPSCs was significantly larger in the PMG group (0.38 ± 0.02 nS, n = 80) than in control cells (0.32 ± 0.01 nS, n = 72; P < 0.05; Fig. 2). This increase in the average peak conductance of sIPSCs was mainly due to a greater number of large events rather than an increase in the maximal amplitude recorded (Fig. 2C). For PMG cells, most sIPSCs (51%) had an peak conductance >0.45 nS, whereas this was true for only 33% of sIPSCs recorded in control cells (Fig. 2C, inset). The largest event recorded from control cells had a peak conductance of 3.70 nS. Only 2 of 6,000 sIPSCs recorded from PMG cells were larger than this, having peak conductances of 3.75 and 4.39 nS. When the two groups of PMG cells were separately compared with controls, only the PMGE group showed a significant increase in average sIPSC peak conductance (Table 1). IPSCs evoked in control ACSF were also significantly larger in PMG neurons, at all intensities tested (Fig. 2D). One potential mechanism for these differences in sIPSC and evoked (e)IPSC amplitude would be an increased excitatory input onto GABAergic interneurons (see DISCUSSION). To examine this possibility, we recorded sIPSCs and eIPSCs after perfusion of slices with bathing medium containing ionotropic glutamate receptor antagonists DNQX and APV to block AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors.
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Addition of glutamate antagonists decreased both the frequency and mean amplitude of spontaneous sIPSCs (Fig. 3, A–C). Under these conditions, there was no longer any difference in peak conductance between control and PMG cells for either the spontaneous (Table 1) or evoked IPSCs (Fig. 3F). The mean amplitude of sIPSCs is affected by the relative proportions of smaller miniature (m)IPSCs and larger action potential-driven IPSCs (Edwards et al. 1990). Thus the difference in IPSC peak conductance between these cell groups found in normal bathing medium was most likely due to a greater proportion of the large, action potential-related IPSCs resulting from enhanced glutamatergic excitatory activation of inhibitory interneurons synapsing on PMGE cells. We further assessed the proportion of glutamate-driven IPSCs within individual cells by calculating changes in the sIPSC frequency after addition of glutamate antagonists. In most cells, the direction of change in sIPSC frequency was a decrease. The average decrease in frequency was significantly greater for PMGE cells than for controls (48 vs. 18%, respectively; Fig. 3D). PMGS cells were not significantly different from controls on this measure (Fig. 3, D and E). PMGE and PMGS cells were therefore clearly separate populations by this measure because nearly all PMGS cells showed less than a 30% decrease in IPSC frequency, while nearly all PMGE cells showed more than a 30% decrease in IPSC frequency (upper dashed line in Fig. 3E marks 30% decrease). The amount of decrease in sIPSC frequency after addition of DNQX/APV and was not simply dependent on the initial sIPSC frequency recorded in normal slice bathing medium (Fig. 3E).
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sEPSCs
Characteristics of sEPSCs also varied greatly between cells within the control population, with frequencies of 0.3–19.5 Hz and peak conductances of 0.17–1.31 nS for 26 layer V neurons. There was no significant difference between control and PMG cells in sEPSC rise time (1.3 ± 0.09 and 1.3 ± 0.06 ms, respectively) or peak conductance (see Table 1). The mean frequency of sEPSCs was significantly greater for PMG cells than for controls (Fig. 4). Because the frequency of both sIPSCs and sEPSCs varied greatly between cells, we compared sIPSCs and sEPSCs within individual cells for 21 control and 23 PMG neurons. Control cells typically had a higher frequency of inhibitory currents than excitatory currents (e.g., 4.9 and 4.1 Hz, respectively, in cell of Fig. 4A). For PMG cells, sEPSCs were more frequent than sIPSCs (e.g., 3.6 and 5.1 Hz for sIPSCs and sEPSCs, respectively, in neuron of Fig. 4B). We used a ratio of EPSC and IPSC frequencies (E ratio, see METHODS) to compare these events in individual cells. The mean E ratio was slightly below 0.5 for control cells (Fig. 4E) and significantly higher for PMG cells (0.6, P < 0.05). This indicates that the relative frequencies of EPSCs and IPSCs are shifted toward excitation in individual pyramidal neurons in layer V of the PMG zone. Ratios were not significantly different for PMGE versus PMGS neurons (0.56 + 0.05 and 0.63 + 0.06, respectively, N.S.).
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To estimate relative numbers of functional synapses, miniature events were recorded with 1 µM TTX in the bathing medium. There was no difference in mIPSC frequency (Fig. 5, A–D), amplitude (Table 1), or rise time (1.1 ± 0.04 and 1.2 ± 0.04 ms, respectively) for 34 control versus 45 PMG cells. Assuming no significant differences in the probability of transmitter release at inhibitory terminals between these cell groups, results suggest that control and PMG layer V pyramidal neurons receive approximately equivalent numbers of inhibitory synapses. In contrast to this, miniature (m)EPSCs were nearly twice as frequent in PMGS cells as in control cells (Fig. 5E, Table 1), suggesting that PMGS neurons have more functional excitatory synapses. Amplitude and rise times of mEPSCs were not significantly different between groups for mEPSCs (Table 1; rise times: 1.1 ± 0.1, 1.4 ± 0.1, and 1.1 ± 0.1 ms for control, PMGE, and PMGS, respectively). The mean ratio of mEPSC and mIPSC frequencies, calculated in individual cells as in the preceding text, was also significantly higher for PMGS than for either control or PMGE neurons (Fig. 5F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Response types in PMG neurons
The two subgroups of PMG neurons were distinguished not only by their response to stimulation, but also by measures of change in sIPSC frequency and eIPSC amplitude after DNQX/APV application, ratio of sEPSC frequency to the sum of sEPSC and sIPSC frequency (E ratio), and mEPSC frequency. The fact that these measures are not directly related suggests that the PMG subgroups reflect distinct neuronal types rather than simply different responses due to changes in stimulus location or methodological differences in the recordings. Neocortical layer V pyramidal neurons can be separated into a number of different groups based on dendritic and axonal morphology, intrinsic firing pattern, and postsynaptic targets (Chagnac-Amitai et al. 1990; Kasper et al. 1994; Larkman 1991; Mason and Larkman 1990; Tseng and Prince 1993). These characteristics have particular depth correlations within layer V. It is not clear whether the two groups of PMG cells are also differentiated by any of these other factors; however, the PMG subgroups are not segregated by depth. In fact, these response types were sometimes seen in cells within the same slice. This makes it less likely that PMG subgroups will correlate with the previously described layer V cell types.
The response of PMGE cells to stimulation suggests that they receive input from inhibitory neurons that are activated during epileptiform activity; however, we do not know whether the PMGE cells typically also participate in initiation of that epileptiform activity by firing action potentials. Similarly, the absence of epileptiform-like activity in the evoked IPSCs of PMGS neurons does not indicate that these cells are silent during abnormal hypersynchronous activity, in fact the opposite may be true because less inhibition occurs at longer latencies when epileptiform activity is typically observed in field potential recordings. Current-clamp recordings in which action potentials are not blocked could resolve this issue. Consistent differences in the amount of inhibition during epileptiform discharges have previously been observed in specific layer V cell types (Chagnac-Amitai and Connors 1989). Although we do not currently know whether these cell types have any relation to our PMGE and PMGS cells, this finding does suggest that inhibitory cells do not innervate layer V pyramidal neurons in a random fashion. In addition, these two cell types likely receive different amounts of excitatory innervation because significantly higher mEPSC frequencies were recorded in the PMGS cells relative to both controls and PMGE cells.
Changes in cortical inhibition
A reduction in functional inhibition has been observed in some epilepsy models, using measures of peak inhibitory postsynaptic potential (IPSP) conductance (Luhmann et al. 1995), IPSC amplitude (Isokawa 1996), sIPSC frequency (Li and Prince 2002), mIPSC frequency (Mangan and Bertram 1998), and response to GABA application (Whittington et al. 1995). Recent work has also suggested that function of GABAB receptors may be reduced in slices from human dysplastic cortex (D’Antuono et al. 2004). In addition, previous results in the microgyrus model have shown a decrease in the numbers of parvalbumin immunohistochemically stained interneurons throughout the hemisphere containing the microgyrus in young animals (P13-15) and focally within layers IV and V of the PMG zone (0.5 mm outside of the malformed region, but not 2.0 mm distant) in older (P21-60) animals (Rosen et al. 1998; but see Schwarz et al. 2000). While parvalbumin might be downregulated independently of loss of GABAergic neurons (Wittner et al. 2001), our previous finding raised the possibility that a reduction in numbers of inhibitory neurons and synapses in the PMG zone would lead to decreases in inhibition. Inhibitory currents due to GABA release from parvalbumin-containing interneurons that target somata and proximal dendrites of pyramidal cells should be detectable in somatic voltage-clamp recordings (Soltesz et al. 1995). However, the lack of reduction in frequency of mIPSCs in the current experiments suggests that the overall number of GABAergic synapses is unchanged adjacent to the malformation, assuming that the probability of release is not altered. The idea that inhibition is functional in this model is supported by others’ findings as well (Hagemann et al. 2000). One possible explanation for these seemingly discrepant findings is compensatory sprouting of axonal arbors of surviving inhibitory interneurons with establishment of new inhibitory synapses that may be an important consequence of cortical injury (Davenport et al. 1990; Katsumaru et al. 1986; Nieoullon and Dusticier 1981). It is important to emphasize that monosynaptic eIPSCs are not altered in the PMG neurons perhaps because the degree of interneuronal loss and compensatory new inhibitory connectivity are roughly balanced. It is also possible that subtypes of GABAergic neurons in PMG cortex are differentially affected (Buckmaster and Dudek 1997; Dinocourt et al. 2003; Morin et al. 1998), leading to an increase in the contacts provided by one subgroup and a decrease in those from another group (Cossart et al. 2001). Recent demonstration of separate inhibitory cortical networks for different physiologically-defined types of GABAergic cells (Galarreta and Hestrin 1999; Gibson et al. 1999) suggests that these function independently and therefore their synapses are likely to be differentially regulated. GABAergic cell types distinguished by their content of peptides and calcium-binding proteins display differential changes in cell numbers after kainate-induced epilepsy (Buckmaster and Dudek 1997; Magloczky and Freund 1993). Changes in one subtype without changes in the total GABAergic cell population have also been described for human temporal lobe epilepsy patients (Mathern et al. 1995). The possibility that similar selective changes in inhibitory cell types occur in malformed cortex is currently being investigated.
A second compensatory mechanism that might maintain the strength of inhibition in the PMG zone is an increased afferent excitatory input onto inhibitory neurons. This appears to be relatively selective for inhibitory neurons that synapse on a subgroup of layer V pyramidal neurons (PMGE cells). The increase in sIPSC amplitude for the population of PMG cells is mainly due to PMGE cells (see Table 1). The significantly greater decrease in IPSC frequency after addition of glutamate antagonists is also specific to the PMGE group and supports our conclusion that increased sIPSC amplitude in this cell group is due to a greater number of large events from impulse-related GABA release (Otis et al. 1991). It is possible that the increased afferent drive onto inhibitory neurons is a compensatory change in response to hyperactivity of this cortical region; however, the timing of these alterations in sIPSC and eIPSC amplitude relative to the onset of epileptogenesis (at P12) is not currently known. Apparent increases in cortical inhibition have been described in other epilepsy models (Buhl et al. 1996; Prince et al. 1997) as well as in tissue from human epilepsy patients (Wittner et al. 2001). Enhanced inhibition may also contribute to the synchronization of excitatory cortical activity that is necessary to initiate the spread of epileptiform activity (Avoli et al. 2002; Khazipov and Holmes 2003; Michelson and Wong 1994; Troyer et al. 1992).
Hyperinnervation of PMG region
It is well known that specific thalamic afferents target layer IV neocortical neurons, even when their normal placement is altered (see for review Bolz et al. 1993). We have hypothesized that, for this reason, thalamic afferents originally destined to synapse in the malformed region may instead synapse mainly in the adjacent cortex, where layer IV neurons are present. Anatomical studies tracing thalamocortical afferents have supported this idea (Jacobs et al. 1999b; Rosen et al. 2000). Increased frequency of mEPSCs seen in the current study may reflect an increased number of excitatory synapses onto layer V pyramidal cells from these or other redirected afferents. We also cannot rule out the possibilities that the increase in mEPSC frequency is caused by an increase in release probability or a redistribution of the same number of excitatory synapses, causing more mEPSCs to be detected. Altered dendritic morphologies shown to occur in this model include longer basal dendrites of layer V neurons (Di Rocco et al. 2002), which may specifically be associated with redistribution of excitatory synapses. These dendrites may also provide increased synaptic "space" for hyperinnervation. Whatever the mechanism, the ratio of numbers of EPSCs to IPSCs was significantly increased, suggesting that these changes may help to maintain the membrane potential near the threshold for action potential firing (Bernander et al. 1991; Destexhe and Pare 1999; Fellous and Sejnowski 2003; Ho and Destexhe 2000; McCormick et al. 2003), thus increasing the overall excitability of the cortex.
The increase in mEPSC frequency was specific to the cell group that did not show changes in inhibition (PMGS). Our data suggest that if hyperinnervation occurs, it does so on selective postsynaptic targets rather than in a random fashion. One possible explanation for these differences may be in the main cortical afferents that contact these two cell groups. Thalamic afferents invade single whisker representations in somatosensory cortex (barrels) (Agmon et al. 1993), whereas callosal afferents avoid these regions and synapse mainly in the surrounding zones (septa) (Hayama and Ogawa 1997). Callosal axons have not lost all targets within the malformation because superficial layer neurons remain. We would expect then that there would be less aberrant connectivity of callosal afferents than of thalamic afferents. Rosen and colleagues indeed found that callosal axons did invade the malformed cortex, presumably finding appropriate targets there (Rosen et al. 2000). In addition, a study of cortical-cortical association projections suggests that these may be reduced rather than enhanced (Giannetti et al. 2000). Thus if the apical dendrites of PMGS cells passed through layer IV barrels, while those of PMGE cells were passed through layer IV septal regions, an increase in excitatory afferent contacts would be expected for the reorganized thalamocortical afferents synapsing in the barrels. This increase might not be observed in PMGE cells if they receive mainly callosal axons and few thalamocortical afferents. Although we have no direct evidence on changes in innervation relative to barrel location, our results do show PMGS cells receive different levels of excitatory afferent innervation than do PMGE cells.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Present address and 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Avoli M, D’Antuono M, Louvel J, Kohling R, Biagini G, Pumain R, D’Arcangelo G, and Tancredi V. Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro. Prog Neurobiol 68: 167–207, 2002.[CrossRef][Web of Science][Medline]
Barkovich AJ, Gressens P, and Evrard P. Formation, maturation, and disorders of brain neocortex. Am J Neuroradiol 13: 423–446, 1992.[Abstract]
Barkovich AJ, Rowley H, and Bollen A. Correlation of prenatal events with the development of polymicrogyria. Am J Neuroradiol 16: 822–827, 1995.[Abstract]
Bernander O, Douglas RJ, Martin KA, and Koch C. Synaptic background activity influences spatiotemporal integration in single pyramidal cells. Proc Natl Acad Sci USA 88: 11569–11573, 1991.
Bolz J, Gotz M, Hübener M, and Novak N. Reconstructing cortical connections in a dish. Trends Neurosci 16: 310–316, 1993.[CrossRef][Web of Science][Medline]
Buckmaster PS and Dudek FE. Neuron loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 385: 385–404, 1997.[CrossRef][Web of Science][Medline]
Buhl EH, Otis TS, and Mody I. Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science 271: 369–373, 1996.[Abstract]
Castro PA, Cooper EC, Lowenstein DH, and Baraban SC. Hippocampal heterotopia lack functional Kv4.2 potassium channels in the methylazoxymethanol model of cortical malformations and epilepsy. J Neurosci 21: 6626–6634, 2001.
Chagnac-Amitai Y and Connors BW. Synchronized excitation and inhibition driven by intrinsically bursting neurons in neocortex. J Neurophysiol 62: 1149–1162, 1989.
Chagnac-Amitai Y, Luhmann HJ, and Prince DA. Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features. J Comp Neurol 296: 598–613, 1990.[CrossRef][Web of Science][Medline]
Chevassus-Au-Louis N, Baraban SC, Gaïarsa JL, and Ben-Ari Y. Cortical malformations and epilepsy: new insights from animal models. Epilepsia 40: 811–821, 1999.[CrossRef][Web of Science][Medline]
Clark GD. Cerebral gyral dysplasias: molecular genetics and cell biology. Curr Opin Neurol 14: 157–162, 2001.[CrossRef][Web of Science][Medline]
Connors BW. Initiation of synchronized neuronal bursting in neocortex. Nature 310: 685–687, 1984.[CrossRef][Medline]
Cossart R, Dinocourt C, Hirsch JC, Merchan-Perez A, De Felipe J, Esclapez M, Bernard C, and Ben Ari Y. Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy. Nat Neurosci 4: 52–62, 2001.[CrossRef][Web of Science][Medline]
Couillard-Despres S, Winkler J, Uyanik G, and Aigner L. Molecular mechanisms of neuronal migration disorders, quo vadis? Curr Mol Med 1: 677–688, 2001.[CrossRef][Medline]
Crome L. Microgyria. J Path Bact 64: 479–495, 1952.
D’Antuono, M. Louvel J, Kohling R, Mattia D, Bernasconi A, Olivier A, Turak B, Devaux A, Pumain R, and Avoli, M. GABAA receptor-dependent synchronization leads to ictogenesis in the human dysplastic cortex. Brain 127: 1626–1640, 2004.
Davenport CJ, Brown WJ, and Babb TL. Sprouting of GABAergic and mossy fiber axons in dentate gyrus following intrahippocampal kainate in the rat. Exp Neurol 109: 180–190, 1990.[CrossRef][Web of Science][Medline]
Destexhe A and Pare D. Impact of network activity on the integrative properties of neocortical pyramidal neurons in vivo. J Neurophysiol 81: 1531–1547, 1999.
Di Rocco F, Giannetti S, Gaglini P, Di Rocco C, and Granato A. Dendritic architecture of corticothalamic neurons in a rat model of microgyria. Childs Nerv Syst 18: 690–693, 2002.[CrossRef][Web of Science][Medline]
Dinocourt C, Petanjek Z, Freund TF, Ben Ari Y, and Esclapez M. Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures. J Comp Neurol 459: 407–425, 2003.[CrossRef][Web of Science][Medline]
Dvorak K and Feit J. Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats. Acta Neuropathol (Berl) 38: 203–212, 1977.[CrossRef][Medline]
Edwards FA, Konnerth A, and Sakmann B. Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J Physiol 430: 213–249, 1990.
Farrell MA, DeRosa MJ, Curran JG, Lenard Secor D, Cornford ME, Comair YG, Peacock WJ, Shields WD, and Vinters HV. Neuropathologic findings in cortical resections (including hemispherectomies) performed for the treatment of intractable childhood epilepsy. Acta Neuropathol (Berl) 83: 246–259, 1992.[CrossRef][Medline]
Fellous JM and Sejnowski TJ. Regulation of persistent activity by background inhibition in an in vitro model of a cortical microcircuit. Cereb Cortex 13: 1232–1241, 2003.
Galarreta M and Hestrin S. A network of fast-spiking cells in the neocortex connected by electrical synapses. Nature 402: 72–75, 1999.[CrossRef][Medline]
Giannetti S, Gaglini P, Di Rocco F, Di Rocco C, and Granato A. Organization of cortico-cortical associative projections in a rat model of microgyria. Neuroreport 11: 2185–2189, 2000.[Web of Science][Medline]
Gibson JR, Beierlein M, and Connors BW. Two networks of electrically coupled inhibitory neurons in neocortex. Nature 402: 75–79, 1999.[CrossRef][Medline]
Hagemann G, Redecker C, and Witte OW. Intact functional inhibition in the surround of experimentally induced focal cortical dysplasias in rats. J Neurophysiol 84: 600–603, 2000.
Hauser, WA. Recent developments in the epidemiology of epilepsy. Acta Neurol Scand Suppl 162: 17–21, 1995.[Medline]
Hayama T and Ogawa H. Regional differences of callosal connections in the granular zones of the primary somatosensory cortex in rats. Brain Res Bull 43: 341–347, 1997.[CrossRef][Web of Science][Medline]
Ho N and Destexhe A. Synaptic background activity enhances the responsiveness of neocortical pyramidal neurons. J Neurophysiol 84: 1488–1496, 2000.
Hoffman SN, Salin PA, and Prince DA. Chronic neocortical epileptogenesis in vitro. J Neurophysiol 71: 1762–1773, 1994.
Holopainen IE and Lauren HB. Neuronal activity regulates GABAA receptor subunit expression in organotypic hippocampal slice cultures. Neuroscience 118: 967–974, 2003.[CrossRef][Web of Science][Medline]
Isokawa M. Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus. J Neurophysiol 75: 1901–1908, 1996.
Jacobs KM, Gutnick MJ, and Prince DA. Hyperexcitability in a model of cortical maldevelopment. Cereb Cortex 6: 514–523, 1996.
Jacobs KM, Hwang BJ, and Prince DA. Focal epileptogenesis in a rat model of polymicrogyria. J Neurophysiol 81: 159–173, 1999a.
Jacobs KM, Kharazia VN, and Prince DA. Mechanisms underlying epileptogenesis in cortical malformations. Epilepsy Res 36: 165–188, 1999b.[CrossRef][Web of Science][Medline]
Jacobs KM, Mogensen M, Warren L, and Prince DA. Experimental microgyri disrupt the barrel field pattern in rat somatosensory cortex. Cereb Cortex 9: 733–744, 1999c.
Jacobs KM, Parada I, and Prince DA. Enhanced c-fos staining in two post-lesional models of cortical hyperexcitability: neonatal freeze lesions and partial cortical isolations (Abstract). Epilepsia 42: 221. 2001.
Kasper EM, Lubke J, Larkman AU, and Blakemore C. Pyramidal neurons in layer 5 of the rat visual cortex. III. Differential maturation of axon targeting, dendritic morphology, and electrophysiological properties. J Comp Neurol 339: 495–518, 1994.[CrossRef][Web of Science][Medline]
Katsumaru H, Murakami F, Wu JY, and Tsukahara N. Sprouting of GABAergic synapses in the red nucleus after lesions of the nucleus interpositus in the cat. J Neurosci 6: 2864–2874, 1986.[Abstract]
Khazipov R and Holmes GL. Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo. J Neurosci 23: 5337–5341, 2003.
Lagae L. Cortical malformations: a frequent cause of epilepsy in children. Eur J Pediatr 159: 555–562, 2000.[CrossRef][Web of Science][Medline]
Larkman AU. Dendritic morphology of pyramidal neurones of the visual cortex of the rat. I. Branching patterns. J Comp Neurol 306: 307–319, 1991.[CrossRef][Web of Science][Medline]
Li H and Prince DA. Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex. J Neurophysiol 88: 2–12, 2002.
Luhmann HJ, Mittmann T, van Luijtelaar G, and Heinemann U. Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy. Epilepsy Res 22: 43–51, 1995.[CrossRef][Web of Science][Medline]
Luhmann HJ and Prince DA. Transient expression of polysynaptic NMDA receptor-mediated activity during neocortical development. Neurosci Lett 111: 109–115, 1990.[CrossRef][Web of Science][Medline]
Luhmann HJ and Raabe K. Characterization of neuronal migration disorders in neocortical structures. I. Expression of epileptiform activity in an animal model. Epilepsy Res 26: 67–74, 1996.[CrossRef][Web of Science][Medline]
Magloczky Z and Freund TF. Selective neuronal death in the contralateral hippocampus following unilateral kainate injections into the CA3 subfield. Neuroscience 56: 317–335, 1993.[CrossRef][Web of Science][Medline]
Mangan PS and Bertram EH. Ontogeny of altered synaptic function in a rat model of chronic temporal lobe epilepsy. Brain Res 799: 183–196, 1998.[CrossRef][Web of Science][Medline]
Mason A and Larkman A. Correlations between morphology and electrophysiology of pyramidal neurons in slices of rat visual cortex. II. Electrophysiology. J Neurosci 10: 1415–1428, 1990.[Abstract]
Mathern GW, Babb TL, Pretorius JK, and Leite JP. Reactive synaptogenesis and neuron densities for neuropeptide Y, somatostatin, and glutamate decarboxylase immunoreactivity in the epileptogenic human fascia dentata. J Neurosci 15: 3990–4004, 1995.[Abstract]
McCormick DA, Shu Y, Hasenstaub A, Sanchez-Vives M, Badoual M, and Bal T. Persistent cortical activity: mechanisms of generation and effects on neuronal excitability. Cereb Cortex 13: 1219–1231, 2003.
Michelson HB and Wong RK. Synchronization of inhibitory neurons in the guinea-pig hippocampus in vitro. J Physiol 477: 35–45, 1994.
Montenegro MA, Guerreiro MM, Lopes-Cendes I, Guerreiro CA, and Cendes F. Interrelationship of genetics and prenatal injury in the genesis of malformations of cortical development. Arch Neurol 59: 1147–1153, 2002.
Morin F, Beaulieu C, and Lacaille JC. Selective loss of GABA neurons in area CA1 of the rat hippocampus after intraventricular kainate. Epilepsy Res 32: 363–369, 1998.[CrossRef][Web of Science][Medline]
Nieoullon A and Dusticier N. Increased glutamate decarboxylase activity in the red nucleus of the adult cat after cerebellar lesions. Brain Res 224: 129–139, 1981.[CrossRef][Web of Science][Medline]
Otis TS, Staley KJ, and Mody I. Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545: 142–150, 1991.[CrossRef][Web of Science][Medline]
Palmini A, Andermann F, Olivier A, Tampieri D, Robitaille Y, Andermann E, and Wright G. Focal neuronal migration disorders and intractable partial epilepsy: a study of 30 patients. Ann Neurol 30: 741–749, 1991a.[CrossRef][Web of Science][Medline]
Palmini A, Andermann F, Olivier A, Tampieri D, Robitaille Y, Melanson D, and Ethier R. Neuronal migration disorders: a contribution of modern neuroimaging to the etiologic diagnosis of epilepsy. Can J Neurol Sci 18: 580–587, 1991b.[Web of Science][Medline]
Piao X, Basel-Vanagaite L, Straussberg R, Grant PE, Pugh EW, Doheny K, Doan B, Hong SE, Shugart YY, and Walsh CA. An autosomal recessive form of bilateral frontoparietal polymicrogyria maps to chromosome 16q12.2–21. Am J Hum Genet 70: 1028–1033, 2002.[CrossRef][Web of Science][Medline]
Poulter MO, Ohannesian L, Larmet Y, and Feltz P. Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation. J Neurochem 68: 631–639, 1997.[Web of Science][Medline]
Prince DA, Jacobs KM, Salin PA, Hoffman S, and Parada I. Chronic focal neocortical epileptogenesis: does disinhibition play a role? Can J Physiol Pharmacol 75: 500–507, 1997.[CrossRef][Web of Science][Medline]
Prince DA and Tseng GF. Epileptogenesis in chronically injured cortex: in vitro studies. J Neurophysiol 69: 1276–1291, 1993.
Redecker C, Luhmann HJ, Hagemann G, Fritschy JM, and Witte OW. Differential downregulation of GABAA receptor subunits in widespread brain regions in the freeze-lesion model of focal cortical malformations. J Neurosci 20: 5045–5053, 2000.
Richman DP, Stewart RM, and Caviness VSJ. Cerebral microgyria in a 27-week fetus: an architectonic and topographic analysis. J Neuropathol Exp Neurol 33: 374–384, 1974.[Web of Science][Medline]
Rosen GD, Burstein D, and Galaburda AM. Changes in efferent and afferent connectivity in rats with induced cerebrocortical microgyria. J Comp Neurol 418: 423–440, 2000.[CrossRef][Web of Science][Medline]
Rosen GD, Jacobs KM, and Prince DA. Effects of neonatal freeze lesions on expression of parvalbumin in rat neocortex. Cereb Cortex 8: 753–761, 1998.
Rosen GD, Sherman GF, and Galaburda AM. Birthdates of neurons in induced microgyria. Brain Res 727: 71–78, 1996.[CrossRef][Web of Science][Medline]
Salin PA and Prince DA. Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75: 1573–1588, 1996.
Schwarz P, Stichel CC, and Luhmann HJ. Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons? Epilepsia 41: 781–787, 2000.[CrossRef][Web of Science][Medline]
Soltesz I, Smetters DK, and Mody I. Tonic inhibition originates from synapses close to the soma. Neuron 14: 1273–1283, 1995.[CrossRef][Web of Science][Medline]
Tachibana Y. Special etiologies in the classification of epilepsy—with special reference to brain malformations. Jpn J Psychiatry Neurol 44: 335–339, 1990.[Medline]
Tanaka T and Gleeson JG. Genetics of brain development and malformation syndromes. Curr Opin Pediatr 12: 523–528, 2000.[CrossRef][Web of Science][Medline]
Troyer MD, Blanton MG, and Kriegstein AR. Abnormal action-potential bursts and synchronized, GABA-mediated inhibitory potentials in an in vitro model of focal epilepsy. Epilepsia 33: 199–212, 1992.[CrossRef][Web of Science][Medline]
Tseng GF and Prince DA. Heterogeneity of rat corticospinal neurons. J Comp Neurol 335: 92–108, 1993.[CrossRef][Web of Science][Medline]
Villard L, Nguyen K, Cardoso C, Martin CL, Weiss AM, Platt M, Grix AW, Graham JM Jr, Winter RM, Leventer RJ, and Dobyns WB. A locus for bilateral perisylvian polymicrogyria maps to Xq28. Am J Hum Genet 70: 1003–1008, 2002.[CrossRef][Web of Science][Medline]
Whittington MA, Traub RD, and Jefferys JG. Erosion of inhibition contributes to the progression of low magnesium bursts in rat hippocampal slices. J Physiol 486: 723–734, 1995.
Wittner L, Magloczky Z, Borhegyi Z, Halasz P, Toth S, Eross L, Szabo Z, and Freund TF. Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus. Neuroscience 108: 587–600, 2001.[CrossRef][Web of Science][Medline]
Xiang Z, Huguenard JR, and Prince DA. GABAA receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex. J Physiol 506: 715–730, 1998.
Zhu WJ and Roper SN. Reduced inhibition in an animal model of cortical dysplasia. J Neurosci 20: 8925–8931, 2000.
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, the PMGE cell; 
, the PMGS cell. D: cell pairs from a single slice in which the same type of response was recorded in both cells. 
, the location of PMGE cell pairs; 
, PMGS cell pairs. ECl- = –51 mV and holding potential = 0 mV.
, controls;