J Neurophysiol 111: 323–335, 2014. First published October 23, 2013; doi:10.1152/jn.00652.2012.

Targeted disruption of layer 4 during development increases GABAA receptor neurotransmission in the neocortex J. Abbah,1 Maria F. M. Braga,1,2 and S. L. Juliano1,2 1 Program in Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, Maryland; and 2Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland

Submitted 26 July 2012; accepted in final form 15 October 2013

Abbah J, Braga MF, Juliano SL. Targeted disruption of layer 4 during development increases GABAA receptor neurotransmission in the neocortex. J Neurophysiol 111: 323–335, 2014. First published October 23, 2013; doi:10.1152/jn.00652.2012.—Cortical dysplasia (CD) associates with clinical pathologies, including epilepsy and mental retardation. CD results from impaired migration of immature neurons to their cortical targets, leading to clustering of neural cells and changes in cortical properties. We developed a CD model by administering methylazoxymethanol (MAM), an anti-mitotic, to pregnant ferrets on embryonic day 33; this leads to reduction in cortical thickness in addition to redistribution and increased expression of GABAA receptors (GABAAR). We evaluated the impact of MAM treatment on GABAAR-mediated synaptic transmission in postnatal day 0 –1 neurons, leaving the ganglionic eminence (GE) and in layer 2/3 pyramidal cells of postnatal day 28 –38 ferrets. Embryonic day 33 MAM treatment significantly increases the amplitude and frequency of spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs) in the cells leaving the GE. In older MAM-treated animals, the amplitude and frequency of GABAAR-mediated spontaneous IPSCs in layer 2/3 pyramidal cells is increased, as are the amplitude and frequency of miniature IPSCs. The kinetics of GABAAR opening also altered following treatment with MAM. Western blot analysis shows that the expression of the GABAA␣3R and GABAA␥2R subunits amplified in our model animals. We did not observe any significant change in the passive properties of either the layer 2/3 pyramidal cells or cells leaving the GE after MAM treatment. These observations reinforce the idea that synaptic neurotransmission through GABAAR enhances following treatment with MAM and coincides with our finding of increased GABAA␣R expression within the upper cortical layers. Overall, we demonstrate that small amounts of toxins delivered during corticogenesis can result in long-lasting changes in ambient expression of GABAAR that influence intrinsic neuronal properties. cortical dysplasia; neuronal migration; patch-clamp; ferret; Western blot

mammalian neocortex, immature neurons generated in the germinal zones of the neocortex and ganglionic eminences (GE) migrate in an inside-out pattern to create the six-layered neocortex (Angevine and Sidman 1961; Rakic 1974). This laminar organization is crucial for establishing a balance between excitation and inhibition, leading to proper overall function of the neocortex. Events with adverse impact on neuronal migration lead to cortical dysplasia (CD), a developmental abnormality characterized by aberrant distribution and clustering of neocortical cells, resulting in a pleth-

DURING DEVELOPMENT OF THE

Address for reprint requests and other correspondence: S. L. Juliano, Anatomy, Physiology and Genetics, Uniformed Services Univ. of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799 (e-mail: [email protected]). www.jn.org

ora of neuropathological disorders, such as epilepsy, schizophrenia, and mental retardation (Calcagnotto and Baraban 2003; Calcagnotto et al. 2002; Choi and Mathias 1987; Colombo et al. 2003; Gleeson and Walsh 2000; Guerrini et al. 2003; Moroni et al. 2005, 2008; Palmini et al. 1991; Ross and Walsh 2001; Tassi et al. 2002; Taylor et al. 1971; Zhu and Roper 2000). A common pathological finding in clinical cases and animal models of CD is an abnormal distribution and grouping of neurons within the neocortex, which alters the microcircuitry and cortical architecture (Benardete and Kriegstein 2002; Colacitti et al. 1999; Ferrer 1993; Jacobs et al. 1999; Moroni et al. 2008; Roper 1998). In addition, abnormal GABA signaling frequently associates with CD, leading to consequent alteration in the balance of excitation and inhibition and disruption of normal cortical function (Benardete and Kriegstein 2002; Brill and Huguenard 2010; Moroni et al. 2008; Roper et al. 1999; Xiang et al. 2006; Zhou and Roper 2010, 2011; Zhu and Roper 2000, Zhu et al. 2009). We developed a model of CD in ferrets using methylazoxymethanol (MAM; also see Johnston et al. 1982 for initial description). Taking advantage of the pharmacokinetic property of MAM as a short-acting anti-mitotic and the long developmental time course of ferret corticogenesis, we selectively disrupted the birth of layer 4 by delivery on embryonic day 33 (E33) (Noctor et al. 1997, 1999; Palmer et al. 2001). The ferret, as the smallest mammal with a gyrencephalic cortex, is crucial for the study of neocortical development. Because ferrets possess an expanded neocortex with sulci, gyri, and large amounts of white matter, it is distinctive as a research subject. The protracted period of corticogenesis and long period of cell proliferation in the large outer subventricular zone may contribute to the expanded cerebral cortex in the ferret compared with the rodent (Fietz et al. 2010; MartinezCerdeño et al. 2006). Distinctive features in the ferret related to neocortical development make it essential to study developmental processes in this animal, since aspects of neurodevelopment differ in mammals with a convoluted vs. lissencephalic cortex (Kriegstein et al. 2006; Poluch et al. 2008). As a likely result of the loss of layer 4 in our model, thalamic afferents that normally synapse directly in this layer become redistributed to upper and lower cortical layers; the capacity for entrainment and information transfer is also lost within the somatosensory cortex (McLaughlin and Juliano 2005; Noctor et al. 2001; Palmer et al. 2001). Ferrets treated with MAM on E33 additionally show an increased expression of GABAA receptors (GABAAR), which expands to upper cortical layers, and interneurons are disorganized in their laminar positions (Jablonska et al. 2004; Poluch et al. 2008). Changes in the 323

324

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

migratory behavior of GE-derived cells also occur (Abbah and Juliano 2013). These changes are specific to MAM delivery on E33, as administration on different embryonic dates leads to dramatically different results (Gierdalski and Juliano 2003, 2005; Noctor et al. 1999; Poluch and Juliano 2007, 2010). An important question emerging from these observations is whether the loss of layer 4 and subsequent redistribution of GABAAR and interneurons alters GABA signaling within upper cortical layers. We also assessed whether the emergence of these properties influenced the functional properties of migrating interneurons. Our laboratory’s earlier studies in MAM-treated (MAM-Tx) animals show increased expression of GABAAR within the neocortex, as well as an abnormal pattern of migration and distribution of interneurons leaving the GE (Abbah and Juliano 2013; Jablonska et al. 2004; Poluch et al. 2008). These features prompted us to characterize GABAergic synaptic transmission in interneurons migrating toward the neocortex and evaluate the functional impact of the altered neocortical environment on pyramidal cell responses. We applied whole-cell patch-clamp recording to measure spontaneous GABAAR-mediated inhibitory postsynaptic currents (sIPSCs) in two types of cells. We studied cells migrating away from the GE relatively early during ferret cortical development postnatal day (P) 0 to P1 and pyramidal cells of layer 2/3 in juvenile ferrets (P28 –P38). Choosing these two types of cells for functional analysis allows us to determine the effect of early environmental changes 1) on GABAergic cells as they migrate into the neocortex; and 2) on inhibitory neurotransmission in the somatosensory region of the resulting neocortex. Our results indicate that after E33 MAM treatment, GABAergic synaptic neurotransmission is increased significantly in cells migrating into the cortex from the GE and on cells populating the upper neocortical layers. MATERIALS AND METHODS

Animals. Handling of animals complied with the Animal Care and Use Committee of the Uniformed Services University of the Health Sciences (USUHS). All procedures involving animals were approved by the USUHS Animal Care and Use Committee. Pregnant ferrets were obtained from Marshal Farms (New Rose, NY) and maintained in the animal facilities of USUHS. On E33, ferrets were anesthetized with isofluorane (1–2%) and injected intraperitoneally with 14 mg/kg of MAM (Midwest Research Institute, Kansas City, MO). Ferrets were closely monitored and allowed to recover from anesthesia before returning to the animal facility to await delivery of their kits. Ferret kits were used for experiments at P0 –P1 or P28 –P38. Organotypic slices. Preparation of organotypic cultures followed the method described by Palmer et al. (2001). Briefly, P0 ferret kits were anesthetized with pentobarbital sodium (50 mg/kg), and after observing insensitivity to pain, brains were removed and maintained in ice-cold artificial cerebrospinal fluid (aCSF) bubbled with 95% O2 and 5% CO2 under aseptic conditions in a laminar flow hood. The aCSF is composed of the following (in mM): 124 NaCl, 3.2 KCl, 2.4 CaCl2, 1.2 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 glucose. Coronal slices of the neocortex (350- to 500-␮m thick) were generated using a tissue chopper (Stoelting, Wood Dale, IL). Each slice was plated into a 0.4-␮m culture plate insert (Millicell-CM, Bedford, MA) and placed into six-well plates containing Neurobasal media with B27, N2, and G1.2 (containing gentamycin and glutamine) supplements. Slices were incubated at 37°C with 95% O2 and 5% CO2. Cell label. Migrating GE cells were labeled through electroporation using a modification of the method described by Flames et al. (2004). Transfection was achieved using a plasmid that codes for red fluo-

rescent protein (RFP), which was cloned into pCAGGS expression vector (a gift from Dr. Tarik Haydar). We applied ⬃1 ␮l of the plasmid DNA (5.3 ␮g/␮l) to the ventricular region of the GE of organotypic slices of ferrets. The cathode of a gene paddle electrode (Harvard Apparatus, Holliston, MA) was placed within the lateral ventricle close to the GE and the anode situated close to the pia in an appropriate location; plasmid DNA was driven across the organotypic tissue wall by electric pulse generated by a BTX ECM830 pulse generator (Harvard Apparatus, Holliston, MA). A pulse of 60-V current was delivered four times, each lasting for 50 ms at intervals of 950 ms (Gal et al. 2006). Preparation of acute slices. P28 –P38 ferrets were anesthetized with pentobarbital sodium (50 mg/kg ip) and then decapitated. Brains were quickly removed and placed in ice-cold aCSF bubbled with 95% O2 and 5% CO2. The parietal cortex was blocked, and serial coronal sections (400-␮m thick) were cut on a vibratome (LEICA VT1000S, Buffalo Grove, IL). Slices were rapidly transferred into a holding chamber with aerated aCSF maintained at room temperature for 30 min to 1 h after dissection and before electrophysiological recordings. The aCSF was composed of the following (in mM): 125 NaCl, 2.5 KCl, 1.0 CaCl2, 2.0 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 25 glucose. Recordings. Whole-cell patch-clamp recordings were obtained from 1) neocortical pyramidal cells within layer 2/3 of the parietal cortex in acute slices; and 2) GE-derived cells labeled with RFP in organotypic slices after 14 –20 days in culture. Pyramidal cells were identified by morphology; GE-derived cells were identified under fluorescent microscopy using an upright microscope with ⫻40 objective (ZEISS Axioscope 2FS PLUS). Differential interference optics were used for visualizing pyramidal cells. Similarly, cells migrating away from the GE were initially identified using fluorescent microscopy but subsequently visualized under differential interference optics. Only cells leaving the GE that were transfected with RFP and crossed the corticostriatal junction toward the neocortex and displayed a characteristic elongated leading process were selected for recording. All recordings were performed in a submersion type recording chamber, where slices were continuously perfused with aerated aCSF composed of the following (in mM): 125 NaCl, 2.5 KCl, 2.0 CaCl2, 1.0 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 25 glucose maintained at 28°C for pyramidal cells and 37°C for cells migrating from the GE. Slices were perfused at a rate of 3– 4 ml/min. Recordings used a pulled borosilicate pipette filled with an internal solution composed of the following (in mM): 135 Cs-gluconate, 10 MgCl2, 0.1 CaCl2, 1 EGTA, 10 HEPES, 2 Na-ATP, 0.2 Na3 GTP, pH 7.3 (280 –290 mosM). Each cell was recorded for at least 5 min; 1 min toward the end of the 5 min session was used for analysis. In some experiments, neurobiotin at a final concentration of 0.2% was included in the internal solution for post hoc identification. Whole-cell recordings from the soma of neuronal cells occurred in voltage-clamp mode using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) after a tight seal (1 G⍀) was formed between the recording electrode (with resistance 5–7 M⍀ for pyramidal cells and 7–11 M⍀ for GE-derived cells) and the cell body. For all recordings, a threshold of 2 kHz was applied to filter signals, which were digitized (Digidata1322A, Axon Instruments) and recorded using pClamp software (Axon Instruments). IPSCs were analyzed offline using the Mini Analysis Program (Synaptosoft, Leonia, NJ) and detected by manually setting the IPSC threshold (⬃1.5 times the baseline noise amplitude) after visual inspection. The data were tested for significance on both the mean values presented in bar graphs (using a Student’s t-test) and the cumulative probability distributions using the KomolgorovSmirnov test. The access resistance was constantly monitored throughout the recording, and cells that showed fluctuations beyond 15% were excluded from analysis. Morphology. After recording, acute slices were immediately fixed in 4% paraformaldehyde overnight at 4°C. After several washes with phosphate-buffered saline (PBS), slices were incubated with a solu-

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

tion containing 10% methanol and 0.6% hydrogen peroxide in PBS for 30 min at room temperature. After further washes with PBS, slices were incubated in a solution containing 2% bovine serum albumin and 0.75% Triton X-100 in PBS for 1 h, and then in 2% bovine serum albumin and 0.1% Triton X-100 for 15 min. Slices were then placed in a dilute solution of ABC from an Elite kit overnight at 4°C. After multiple washes, cells were visualized with peroxidase staining using 3,3=-diaminobenzidine (Immpact DAB, Vector Laboratories, Burlingame, CA), and mounted in moviol. Western blotting. Pieces of the neocortex cut from blocks of the parietal cortex prepared as described above were frozen on dry ice and preserved at ⫺80°C prior to use. Protein samples were prepared first by homogenizing the tissues using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) followed by centrifugation at 14,000 g at 4°C. An estimate of the concentration of protein was obtained through colorimetric assay using the Pierce BCA assay kit. Protein concentration was determined after 30-min incubation at 60°C, followed by spectrophotometry. Separation of proteins in the samples was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% Bis-Tris precast gel, and separated proteins were transferred to a polyvinylidene fluoride membranes. A loading volume of 10 –15 ␮l containing 20 – 40 ␮g of protein was used for each analysis. To limit nonspecific binding of antibodies, membranes were initially incubated with casein blocking buffer [PBS (0.5 M NaCl) ⫹ 3% casein ⫹ 0.5% Tween 20] for at least 2 h, followed by affinity purified rabbit polyclonal antibodies directed against GABAA␣2 (1:200; ProSci, Poway, CA), GABAA␣3 (1:2000; Sigma, St. Louis, MO), GABAA␥2 (1:400, Sigma-Aldrich, St. Louis, MO), and monoclonal anti-GAPDH (1:6,000, Abcam, Cambridge, MA) for 24 h. After several washes with PBS, protein bands were detected using horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:1,000, Jackson Laboratories, West Grove, PA) and horseradish peroxidase-conjugated anti-mouse (1:6,000, Thermo Scientific, Rockford, IL) and visualized using enhanced chemiluminescence detection. Signal intensities were quantified using ImageJ software (http://rsb.info.nih.gov/ij/). The neocortices of five different brains of ages P28 –P35 were used for this analysis. Each brain was run separately on the gel, and each was normalized to the density of GAPDH. The density of the five brains was used to obtain an average and to conduct statistical analysis. Statistics. The Student’s t-test or Kolmogorov-Smirnov test was applied for all statistical analyses, and differences evaluated at P ⬍ 0.05. Drugs. The following drugs were added to the recording buffer to isolate sIPSCs and/or miniature IPSCs (mIPSCs): 50 ␮M D-(-)-2amino-5-phosphonopentanoic acid (AP-V), 20 ␮M 5-methyl-10,11dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, both N-methyl-Daspartate receptor antagonists, 20 ␮M (2S)-(⫹)-5,5-dimethyl-2-morpholineacetic acid (SCH50911), GABAB antagonist; 10 ␮M bicuculline methiodide, a GABAAR antagonist; 10 ␮M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist (all from Tocris Cookson, Ballwin, MO), 1 ␮M tetrodotoxin (TTX), a sodium channel blocker (Sigma, St. Louis, MO).

325

RESULTS

Experimental population. In this study, two populations of cells from the parietal cortex were used. 1) We assessed cells leaving the GE and migrating toward the neocortex in normal and MAM-Tx organotypic cultures. These were labeled by electroporation at P0 and allowed to migrate for several days. At this point we identified the migrating cells and recorded responses. We previously determined that MAM-Tx brains have a specific deficit in interneuron migration (Abbah and Juliano 2013; Poluch et al. 2008); it is important to understand distinctions of the functional properties of MAM-Tx interneurons as they migrate into the neocortex. 2) We additionally recorded from layer 2–3 pyramidal cells of parietal cortex from animals 4 –5 wk old. We also know that the distribution of interneurons in the mature cortex is altered, most likely reflecting the migratory deficits. We concluded that the most effective way of determining the effect of the altered distribution was to record from the targets of these cells, the pyramidal cells. We also know from our previous studies that MAM treatment leads to increased GABAAR expression; it is important to determine the effect of this increased expression in relation to the migrating interneuron and also in relation to the more mature pyramidal cells that function in the environment of increased GABAAR. The passive properties of the layer 2/3 pyramidal cells at 4 –5 wk of age and the cells migrating away from the GE were determined and are presented in Table 1. No significant differences between the normal and MAM-Tx cells in either condition were observed that could account for altered response properties. Exposure to MAM increases GABAAR neurotransmission in cells originating from the GE. Migrating cells leaving the GE are influenced by a variety of signaling molecules including GABA. Our laboratory previously demonstrated that the normal pattern of migration and orientation of GE-derived neurons is altered in MAM-Tx animals, in part due to altered GABAAR-mediated activity (Poluch et al. 2008). To determine whether migrating interneurons are influenced by increased GABAAR activity, we evaluated GABAAR-mediated synaptic neurotransmission in cells leaving the GE by recording sIPSCs. Migrating cells were prior labeled with a plasmid that codes for RFP using electroporation directed into the GE and identified under fluorescent microscopy. Labeled cells were presumed to be migrating if they expressed RFP, crossed the corticostriatal junction, and displayed the morphology of a migrating cell (exhibited a leading process). An example can be seen in Fig. 1E. Whole-cell patch-clamp recording occurred on cells leaving the GE after they crossed the corticostriatal boundary and were

Table 1. Passive properties of layer 2/3 pyramidal cells and cells migrating from the GE Treatment Pyramidal cells Normal MAM-Tx GE cells Normal MAM-Tx

Capacitance, pF

Rm, M⍀

Ra, M⍀

Vm

90.97 ⫾ 5.06 84.87 ⫾ 10.55

110.10 ⫾ 6.65 118.23 ⫾ 16.00

19.79 ⫾ 1.69 23.14 ⫾ 2.79

⫺62.63 ⫾ 1.26 ⫺65.28 ⫾ 1.00

31.76 ⫾ 4.32 27.15 ⫾ 2.21

645.68 ⫾ 131.00 608.62 ⫾ 79.75

21.17 ⫾ 3.12 24.29 ⫾ 2.03

⫺28.00 ⫾ 1.13 ⫺31.39 ⫾ 2.45

Values are means ⫾ SE. Rm, membrane resistance; Ra, access resistance; Vm, membrane potential; MAM-Tx, methylazoxymethanol-treated; GE, ganglionic eminence. J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

326

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

Fig. 1. In methylazoxymethanol (MAM)-treated (Tx) animals, both amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) are increased in migrating ganglionic eminence (GE) cells within the developing cortex. sIPSCs were recorded in a voltage-clamp mode at a holding potential of ⫺70 mV and isolated using 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 ␮M), (2S)-(⫹)-5,5-dimethyl-2-morpholineacetic acid (SCH50911; 20 ␮M), and D-(-)-2-amino-5-phosphonopentanoic acid (AP-V; 50 ␮M). A: representative traces of sIPSCs recorded in a cell leaving the GE in normal cortex shown over different periods of time and when blocked with bicuculline. B: sIPSCs in a representative MAM-Tx cell leaving the GE, which were also blocked by bath application of 10 ␮M bicuculline. C: the cumulative probability of the amplitude shows a shift to the right that is significantly greater in the cells leaving the GE in the MAM-Tx animals (red line) compared with the controls from untreated animals (black line). D: the cumulative probability plot of sIPSC frequency shows a leftward shift following treatment with MAM (red line), indicating a reduction in interevent interval and significant increase in frequency. E: a schematic diagram of an organotypic slice illustrating the positioning of the red fluorescent protein construct in a pipette and the migratory path of the cells exiting the GE. The small black dots in the left diagram indicate the site of electroporation, while the larger black dot in the right diagram indicates the approximate position of cells at the time of recording. The white dotted line indicates the position of the corticostriatal junction. Also shown in E is an image of a cell migrating away from the GE during whole-cell patch-clamp recording. CTX, cortex. F: the mean amplitude of the sIPSCs is significantly increased in MAM-Tx animals relative to control. G: the mean frequency is also significantly increased in the MAM-Tx cells. **P ⬍ 0.0001, Komolgorov-Smirnov test. *P ⬍ 0.05, Student’s t-test. Error bars ⫽ standard error of mean. See Table 2 for sample sizes.

positioned close to the parietal neocortex (Fig. 1E). sIPSCs were isolated using CNQX (10 ␮M) to block AMPA/kainite receptors, SCH50911 (20 ␮M) to block GABABR, and AP-V (50 ␮M) to block N-methyl-D-aspartate receptors and were recorded at a holding potential of ⫺70 mV. The amplitude (MAM-Tx: 31.26 ⫾ 3.09 pA; n ⫽ 21 cells or slices/8 animals; Fig. 1, A, B, C, F) and frequency (76.86 ⫾ 14.27 Hz; Fig. 1, D and G) of sIPSCs are significantly increased in migrating MAM-Tx GE cells relative to the control (control amplitude: 21.54 ⫾ 1.99 pA; frequency: 34.31 ⫾ 6.75 Hz; n ⫽ 14 cells or slices/8 animals), indicating a heightened state of GABAergic neurotransmission in this population of cells (Fig. 1). The controls in all instances were taken from animals that were not treated with MAM. See Table 2 for sample numbers.

Treatment with MAM increases the amplitude of sIPSCs in layer 2/3 pyramidal cells of parietal cortex. Following treatment with MAM on E33, the width of layer 4 in ferret somatosensory cortex diminishes, leading to reduction in the overall cortical thickness (Noctor et al. 2001). Additionally, the distribution of GABAA␣R subunits is expanded and increased in the upper cortical layers; several subtypes of interneurons are also abnormally positioned (Jablonska et al. 2004; Poluch et al. 2008). To examine the functional implication of the increased expression of GABAA␣R subunits on the inhibitory tone within the upper cortical layers, we performed whole-cell patch-clamp recording on pyramidal cells that resided in the parietal cortex. sIPSCs were isolated using CNQX (10 ␮M), SCH50911 (20 ␮M) and AP-V (50 ␮M) and recorded at a

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

327

Table 2. Sample sizes for all experiments Treatment Pyramidal cells: sIPSCs Normal MAM-Tx Pyramidal cells: mIPSCs Normal MAM-Tx GE cells: sIPSCs Normal MAM-Tx

Cells, no.

Slices, no.

Animals, no.

Litters, no.

Events for Cumulative Probability Analysis, no.

40 25

40 25

7 6

4 3

4,066 2,011

20 25

20 25

5 6

2 4

348 806

16 21

14 21

8 8

3 4

474 1,474

sIPSC, spontaneous inhibitory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current.

holding potential of ⫺70 mV. In P28 –P38 MAM-Tx animals, the amplitude of sIPSCs was significantly increased (40.08 ⫾ 5.61 Pa; n ⫽ 22 cells/6 animals) relative to control animals (29.45 ⫾ 2.31 pA; n ⫽ 40 cells/7 animals) (Fig. 2, A–C). Our data also indicate that the frequency of sIPSCs was significantly altered following treatment with MAM when tested on the cumulative frequency curves, and a nonsignificant trend toward an increase occurred when tested on the mean values (MAM-Tx animals: 125.09 ⫾ 18.01 Hz; control: 101.65 ⫾ 10.69 Hz; P ⱕ 0.05, Fig. 2, D and H). The increased amplitude of sIPSCs (Fig. 2, C and G) in MAM-Tx animals with less change in frequency (Fig. 2, D and H) suggests that the altered responsivity results from enhanced GABAAR expression in the postsynaptic membrane of layer 2/3 in the parietal cortex. See Table 2 for sample numbers. Several cells were injected with neurobiotin and reacted to visualize their morphology. An example of the types of cells from which we recorded can be seen in Fig. 2, E and F. All of the cells reported here were similar in morphology and position within the cortex. In MAM-Tx animals, both amplitude and frequency of mIPSCs were increased relative to control. The increase in amplitude of sIPSCs indicates that GABAAR-mediated activity is augmented following treatment with MAM. The change in sIPSC amplitude may be due to changes in postsynaptic response. To further determine the underlying mechanism for increased GABAAR-mediated activity, we studied action potential independent IPSCs. mIPSCs were recorded in the presence of CNQX (10 ␮M), SCH50911 (20 ␮M), AP-V (50 ␮M), and TTX (1 ␮M). The amplitude of mIPSCs increased in MAM-Tx animals (Fig. 3, A, B, C, and E). Quantitative analysis revealed rightward shift in the normal cumulative probability of the amplitude (Fig. 3C) and a leftward shift (Fig. 3D) in the interevent interval, indicating a significantly increased frequency of mIPSC generation. The addition of bicuculline eliminated this response. The mean values of the amplitude (MAM-Tx: 17.71 ⫾ 1.61 pA; control: 11.78 ⫾ 0.67 pA, Fig. 3E) and frequency (MAM-Tx: 40.30 ⫾ 6.18 Hz; control: 26.77 ⫾ 4.39 Hz, Fig. 3F) of the mIPSCs observed in the MAM-Tx animals were both significantly increased from normal (control: n ⫽ 20 cells or slices/5 animals; MAM-Tx: 25 cells or slices/6 animals). This result provides further indication of increased postsynaptic GABAAR synaptic transmission in MAM-Tx animals. See Table 2 for sample numbers. The kinetics of GABAAR are altered in MAM-Tx animals. An increased frequency of mIPSCs in MAM-Tx animals suggests that alterations in presynaptic mechanisms may occur, such as

a facilitation of quantal release of GABA and the number and configuration of interneurons, whereas changes in the amplitude of mIPSC suggest changes in the sensitivity of the postsynaptic membrane, probably due to the observed increase in expression of GABAAR. To further evaluate factors responsible for these alterations, we evaluated the kinetics of GABAAR. First, we analyzed the 10 –90% rise time and compared control with MAM-Tx values. The 10 –90% rise time provides spatial information about key synaptic elements and potential alterations due to changes in presynaptic terminal number and positioning (Alberto and Hirasawa 2010; Kobayashi and Buckmaster 2003; Wierenga and Wadman 1999; Xie et al. 1997). In our study, the 10 –90% rise time of the sIPSCs (MAM-Tx: 6.00 ⫾ 0.25 ms; control: 6.82 ⫾ 1.27 ms) and mIPSCs (MAM-Tx: 6.34 ⫾ 0.44 ms; control: 5.66 ⫾ 0.24 ms) was not significantly different between normal and MAM-Tx animals (Fig. 4, AI, BI, DI, and EI). This parameter was significantly reduced in cells migrating from the GE in our model animals during the recording of these cells in organotypic cultures (4.94 ⫾ 0.41 ms and 6.86 ⫾ 0.86 ms, MAM-Tx and control, respectively, Fig. 4, CI and FI). We next evaluated the decay time constant. In MAM-Tx animals, the decay time constant of sIPSCs (MAM-Tx: 19.20 ⫾ 1.00; control: 9.04 ⫾ 0.90) and mIPSCs (MAM-Tx: 4.17 ⫾ 0.29; control: 3.54 ⫾ 0.24) in the pyramidal cells was significantly prolonged (Fig. 4, AII, BII, DII, and EII). This is also true for the migrating cells originating from the GE (9.08 ⫾ 0.82 ms and 6.53 ⫾ 0.72 ms, MAM-Tx and control, respectively, Fig. 4, CII and FII). Analysis of the charge transfer (measured as area) also reveals a significant increase in this parameter for the sIPSCs of the pyramidal cells in layer 2–3 (537.34 ⫾ 77.38 pA ⫻ ms and 345.95 ⫾ 42.09 pA ⫻ ms MAM-Tx and control, Fig. 4, AIII and DIII) and the cells originating in the GE (371.14 ⫾ 61.44 pA ⫻ ms and 226.11 ⫾ 28.07 pA ⫻ ms MAM-Tx and control, respectively, Fig. 4, CIII and FIII). This was also true for the recorded mIPSCs (MAM-Tx: 133.40 ⫾ 16.62 pA ⫻ ms; control: 72.76 ⫾ 4.57 pA ⫻ ms; Fig. 4, BIII and EIII). Treatment with MAM increases the expression of GABAAR within the parietal cortex of ferrets. The electrophysiological results presented so far support the idea that GABAAR-mediated activity is increased following treatment with MAM. To further validate the aberrant elevation of GABAAR activity in our model animals, we performed Western blot analysis to measure the level of expression of different subtypes of the GABAAR within the somatosensory cortex. We tested several receptor subtypes based on the likelihood of their expression in neocortex at this stage of development in ferrets. Although we

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

328

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

Fig. 2. Amplitude of sIPSCs are increased in layer 2/3 pyramidal cells of ferret parietal cortex after MAM treatment. sIPSCs were recorded in voltage clamp mode at a holding potential of ⫺70 mV in the presence of CNQX (10 ␮M), SCH50911 (20 ␮M), AP-V (50 ␮M). A: representative traces of recorded sIPSCs in layer 2/3 pyramidal cells of normal cortex at different speeds. B: representative traces of recorded sIPSCs in layer 2/3 pyramidal cells of MAM-Tx cortex. sIPSCs in both normal and MAM-Tx pyramidal cells were blocked by bath application of 10 ␮M of bicuculline. C: the cumulative probability of the amplitude shows that the MAM-Tx cells (red curve) show greater amplitude. D: the cumulative probability curve of the frequency of sIPSCs indicates that the interevent interval was significantly decreased following treatment with MAM. E: example of pyramidal cells in parietal cortex during whole-cell patch-clamp recording. F: post hoc identification of pyramidal cell in layer 2/3 of the parietal cortex after whole-cell patch-clamp recording. G: the mean amplitude of the sIPSCs is significantly increased in MAM-Tx animals relative to control. H: the mean frequency of the sIPSCs in MAM-Tx cells shows a trend toward an increase, but is not significantly different from the normal cells. *P ⬍ 0.05, Student’s t-test. Error bars ⫽ standard error of mean. **P ⬍ 0.0001, Komolgorov-Smirnov test. See Table 2 for sample sizes.

expected that GABAA␣1R would be present at this age in ferret cortex, we were not able to visualize them. There were no obvious differences in the GABAA␣2R between normal and MAM-Tx neocortex (Fig. 5, top). The GABAA␣3R and the GABAA␥2R subunits, however, were increased in the parietal cortex after MAM treatment (Fig. 5, middle and bottom). These results indicate that GABAAR expression is increased, underlying the enhanced GABAAR-mediated synaptic transmission in MAM-Tx animals. DISCUSSION

The present study uses a ferret model of CD to investigate the alterations of GABAergic synaptic transmission recorded in layer 2–3 pyramidal cells of the somatosensory cortex and on migrating GE-derived cells. We show that the basal activity mediated by GABAAR is enhanced within layer 2/3 and in

cells leaving the GE in our model animals. An increased expression of GABAAR after MAM treatment is likely to underlie the changes in synaptic transmission. In our model, layer 4 formation is selectively disrupted by in utero administration of MAM on E33, leading to its profound reduction in size, a redistribution of interneurons, thalamic afferents, and GABAAR (Jablonska et al. 2004; Noctor et al. 1999; Palmer et al. 2001; Poluch et al. 2008). We also observed that cells leaving the GE and traveling to the neocortex after MAM treatment display abnormal dynamic properties while en route and are abnormally positioned upon reaching their neocortical target (Abbah and Juliano 2013; Poluch et al. 2008). The present study evaluated the functional implications of these findings in two populations of cells: GABAergic neurons leaving the GE, and pyramidal cells in layer 2–3 of neocortex, presumably influenced by inhibitory synapses.

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

329

Fig. 3. In MAM-Tx animals, both amplitude and frequency of miniature IPSC (mIPSC) is increased in pyramidal cells of layer 2/3 of ferret parietal cortex. mIPSCs were recorded in voltage clamp mode at a holding potential of ⫺70 mV and isolated using CNQX (10 ␮M), SCH50911 (20 ␮M), AP-V (50 ␮M), and TTX (1 ␮M). Representative traces are shown of mIPSCs recorded in pyramidal cells of layer 2/3 normal cortex (A) or MAM-Tx cortex (B). mIPSCs in both normal and MAM-Tx pyramidal cells were blocked by application of 10 ␮M bicuculline. C: cumulative probability plots of the amplitude shows a shift to the right, indicating an overall increase in amplitude of the mIPSCs in the MAM-Tx cells. D: the cumulative probability plot of the mIPSC frequency shows a left-ward shift following treatment with MAM indicating a reduction in interevent interval and increase in frequency. The mean amplitude (E) and frequency (F) of the mIPSCs are both significantly increased in MAM-Tx animals relative to control. *P ⬍ 0.05, Student’s t-test. Error bars ⫽ standard error of mean. **P ⬍ 0.0001, Komolgorov-Smirnov test.

Treatment with MAM increases the amplitude of sIPSCs in layer 2/3 pyramidal cells. The increased amplitude of sIPSCs in pyramidal cells of layer 2/3 occurred without any significant change in frequency. This indicates that synaptic transmission through GABAAR is upregulated in MAM-Tx animals, which is likely due to the increased GABAA␣R density on the postsynaptic membrane previously observed in MAM-Tx animals (Jablonska et al. 2004). To further investigate the mechanism underlying the increase in sIPSC amplitude, we evaluated the mIPSCs in the same layer 2/3 pyramidal cells. mIPSCs are caused by the quantal release of GABA from presynaptic terminals of GABAergic interneurons in the absence of action potentials (Edwards et al. 1990). The number of presynaptic terminals of interneurons and/or the postsynaptic GABAAR density on pyramidal cells determines the frequency of mIPSCs (Shao and Dudek 2005). In our study, both amplitude and frequency of mIPSCs were significantly increased in MAM-Tx animals relative to control. The increase in mIPSC frequency and amplitude signifies that both pre- and postsynaptic mechanisms are involved in altering GABAergic neurotransmission in our model animals. Presynaptic factors, such as alterations in the number of GABAergic interneurons and the quantal size of release, determine the frequency of response. It is possible that the GABAergic neurons in our model are altered in some way to induce a greater number of synapses or greater quantal release. We also know that after MAM treatment, subpopulations of interneurons show altered distributions (Poluch et al. 2008). Such redistributions may result in ectopic projections or altered synapses with upper cortical layer pyramidal cells.

Region-specific alterations in local circuit connections occur in other models of CD (Brill and Huguenard 2010). Postsynaptic mechanisms, such as the density and internalization of postsynaptic protein, modulate the size of GABAergic neurotransmission contributing to the change in mIPSC amplitude (DeFazio and Hablitz 1999; Goodkin et al. 2007; Hartmann et al. 2008; Kittler et al. 2000; Loup et al. 2006; Michels and Moss 2007). The frequency of sIPSCs, which is the summation of both action potential-dependent and -independent events, was altered to a lesser degree than the frequency of mIPSC in MAM-Tx animals. A number of factors may be responsible for this, such as the nature of the network of interneurons within layer 2/3 (Bacci et al. 2003; Tamas et al. 1998). Bacci and colleagues (2005) suggest that intrinsic modulation of selective populations of interneurons may have a strong effect on the excitatory activity of pyramidal cells. A number of extrinsic and intrinsic modulatory mechanisms are available, including autaptic transmission impacting on GABAergic cells, which subsequently influence the global signaling of excitatory pyramidal cells. Since we know that interneurons are abnormally placed in juvenile MAM-Tx ferrets, this may cause an abnormal influence on pyramidal cells. Although reduction in amplitude and frequency of IPSCs frequently associate with epilepsy and CD (Buckmaster and Dudek 1997; Hablitz and Defazio 1998; Henry et al. 1993; Jacobs et al. 1999; McDonald et al. 1991; Rocha et al. 2007), the converse occurs in other models of these diseases. Our finding of increased GABAergic neurotransmission in layer 2/3 pyramidal cells corresponds with a recent finding of enhanced

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

330

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

GABAergic activity in pediatric CD (Cepeda et al. 2012). Other studies demonstrate a similar increase in GABAergic activity. A freeze lesion model of CD reports increased IPSCs (Brill and Huguenard 2010; DeFazio and Hablitz 1999; Prince

et al. 1997). In other models of CD, however, a reduction in inhibitory tone occurs, such as in utero exposure to carmustine, MAM, or radiation in rats (Karlsson et al. 2011; Xiang et al. 2006; Zhou and Roper 2010; Zhou et al. 2009). In these

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

331

Fig. 5. Expression of GABAA receptor subunits following treatment with MAM. Treatment with MAM results in a significant increase in the expression of GABAA␣3 and the GABAA␥2 receptor subunits. No obvious differences were seen for the GABAA␣2 receptor subunits in the neocortex, as demonstrated by Western blots. Protein bands were detected through enhanced chemiluminescence and quantitative analysis of the bands performed by densitometric analysis using imageJ software for both normal and MAM-Tx cortex. The quantitative analysis used 5 samples (i.e., the parietal cortex of 5 animals), which were run simultaneously on the gel for both normal and MAM-Tx animals. *P ⬍ 0.05, Student’s t-test.

models, the development of seizures is believed to be a consequence of reduced inhibitory drive similar to epilepitogenic foci reported in animal models of epilepsy (Cossart et al. 2001; Hirsch et al. 1999; Kobayashi and Buckmaster 2003; Rice et al. 1996; Shao and Dudek 2005; Sun et al. 2007). This analysis is complicated further by evidence of elevated GABAergic signaling in other seizure models (Cossart et al. 2001; Gibbs et al. 1997; Nusser et al. 1998; Shao and Dudek 2005; Zhan and Nadler 2009). Overall this suggests that abnormal GABAergic signaling is a feature of abnormal cortical development, and that both increased and decreased GABA receptor neurotransmission contribute to impaired neural function. GABAAR-mediated neurotransmission enhances in migrating GE-derived neurons after MAM treatment. The orientation and distribution of interneurons is altered in E33 MAM-Tx animals, in part due to elevated GABAAR-mediated activity (Poluch et al. 2008). Recently, we found that high levels of GABAAR alter the dynamic behavior of migrating cells leaving the GE (Abbah and Juliano 2013). To determine whether GE-derived cells receive abnormally increased GABAAR signaling after MAM treatment, we measured the sIPSCs. Both amplitude and frequency of sIPSCs were increased in MAM-Tx animals, suggesting that GABAAR synaptic transmission enhances in MAM-Tx animals. GABA signaling plays an important role in promoting migration of GE cells, and alterations in the basal level of GABAAR activity are likely to be deleterious

in the overall migration of GE-derived interneurons. This was also demonstrated in a study by Heck and colleagues (2007), who used GABAA agonists and antagonists to show altered migration after disrupting these receptors. Elevated GABAARmediated activity, while initially promoting migration of cells derived from the GE, triggers early maturation of these cells by inducing precocious expression of the potassium chloride cotransporter (Abbah and Juliano 2013; Ganguly et al. 2001). During development, potassium chloride cotransporter maintains a high extracellular Cl⫺ gradient and induces hyperpolarization of cells in response to GABA, playing a role in the developmental switch from the GABA-induced depolarization to hyperpolarization, which may also terminate cell migration (Bortone and Polleux 2009; Rivera et al. 1999). Thus the elevated functional responses to GABAAR synaptic transmission in cells migrating away from the GE supports the idea that an aberrant GABAergic environment contributes to both abnormal migration and disrupted processing of information. The GE-derived cells that we studied were still en route to their target destination in the neocortex. Consequently, synaptic contacts with these cells are transient, and different regions of the neocortical environment during transit influence the level of GABA signaling. In our model, loss of layer 4 leads to redistribution of thalamic afferents and associated GABAAR; this may also alter the neocortical environment with respect to GABA signaling to influence the migration and positioning of

Fig. 4. Kinetics of GABAA receptors are altered following treatment with MAM. A, I: in pyramidal cells, the cumulative probability of the 10 –90% rise time of the sIPSCs was not altered following treatment with MAM. II: the red curves represent the MAM-Tx events. However, the cumulative probability of the decay time constant was prolonged (II); the cumulative probability of the charge transfer was also increased in MAM-Tx animals (red curves; III). B, I: analysis of GABA receptor kinetics of the mIPSCs (Minis) indicates that the cumulative probability of the 10 –90% rise time was unchanged in MAM-Tx animals. The cumulative probability of the decay time constant, however, was significantly prolonged (II), and the charge transfer also significantly decreased (III). C, I: the kinetics of GABA receptors in migrating GE cells showed that the cumulative probability of the 10 –90% rise time of recorded sIPSC in migrating GE cells was shortened in MAM-Tx animals relative to control. The cumulative probability of the decay time constant (II) and charge transfer (III), however, increased. D–F: the mean values of the 10 –90% rise time, the decay time constant, and the charge transfer also show the same changes as the normal cumulative probability curves for each category of response studied. *P ⬍ 0.05, Student’s t-test. ##P ⬍ 0.02 and **P ⬍ 0.0001, Kolmogorov-Smirnov test. Error bars ⫽ standard error of mean. J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

332

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

migrating GE cells. The increase in both amplitude and frequency of sIPSCs suggests that both presynaptic mechanisms and postsynaptic GABAAR expression contribute to the elevated GABAAR-mediated increase in inhibitory synaptic neurotransmission. Kinetics of GABAA signaling are altered in MAM-Tx animals. The measure of 10 –90% rise time gives relative spatial information with respect to postsynaptic sites; changes in rise time deduce the locus of change in presynaptic terminal number. It is widely accepted that receptors located in the soma of postsynaptic membrane give a faster rise time compared with those located on the dendrites (Shao and Dudek 2005). In our study, there is no significant change in the rise time in either the sIPSCs or the mIPSCs of pyramidal cells in layer 2/3, indicating that there is no significant positional change in the active zones and receptor clusters between normal and MAM-Tx animals (Alberto and Hirasawa 2010; Xie et al. 1997). In the cells migrating from the GE, however, we observed a significant reduction in rise time after MAM treatment compared with normal. Although the reason for this change is unclear, one factor that may be responsible is the relative position of the migrating cells in the developing neocortex, which may affect the nature of synaptic contact they receive. The decay time constant is influenced by the density of postsynaptic receptors and receptor subtype composition (Shao and Dudek 2005; Xie et al. 1997); prolongation of decay time constant in both the sIPSCs and mIPSCs in MAM-Tx animals corresponds with increased expression of GABAAR. In this study and others by our group, we demonstrated increased GABAAR in this model of MAM treatment (Abbah and Juliano 2013; Jablonska et al. 2004). We also show an altered distribution in the composition of receptor subtypes, which vary with maturity, in this study and in previous work (Abbah and Juliano 2013; Jablonska et al. 2004). The amplitude of the IPSCs and decay time constant determines the charge transfer. In our study, the charge transfer was significantly increased for both sIPSC and mIPSC in cells leaving the GE and layer 2/3 pyramidal cells following treatment with MAM. This agrees with the increased amplitude and decay time reported here. Expression of GABAAR subunits is altered in MAM-Tx animals. Our results show that GABAergic neurotransmission is enhanced in the neocortex of MAM-Tx animals, probably as a result of increased expression of GABAAR. We also demonstrated that GABAAR increase after E33 MAM treatment, primarily in the upper cortical layers using receptor binding and immunohistochemistry (Jablonska et al. 2004). Here we find an increase in the amplitude of sIPSCs and mIPSCs in pyramidal cells of layer 2/3 and a prolonged decay time. To further confirm that increased GABAAR expression is responsible for the enhanced GABAergic neurotransmission, we used Western blotting to measure the level of various subunits of GABAAR; GABAA␣3 expression is increased in dysplastic cortex compared with control. The mammalian GABAAR is made of five subunits generated from 19 possible subunits (␣1– 6, ␤1–3, ␥1–3, ␦, ⑀, ␪, ␳, and ␳1–3) (Olsen and Sieghart 2008). Compelling evidence indicates that these subunits have differences in temporal expression and regional distribution during development (Araki et al. 1992; Carlson et al. 2010; Fritschy et al. 1994; Garrett et al. 1990; Hashimoto et al. 2009; Laurie et al. 1992; McKernan et al. 1991; Pirker et al.

2000; Yu et al. 2006). The GABAA␣3 subunit is expressed early in development and mediates a number of developmental activities (Carlson et al. 2010; Fritschy et al. 1994; Laurie et al. 1992; Poulter et al. 1992). The increased expression of GABAA␣3R and GABAA␥2R appears to underlie the enhanced GABAergic neurotransmission reported here. Our laboratory also observed increased expression of GABAA␣2 and GABAA␣3 subunits in the neocortex of MAM-Tx animals at P0 (Abbah and Juliano 2013). A possible explanation for increased GABAAR expression in layer 2/3 of parietal cortex in MAM-Tx animals is the redistribution of thalamic afferents, which associate closely with GABAAR during development (Jablonska et al. 2004; Noctor et al. 2001; Palmer et al. 2001). In normal animals, thalamic afferents make synaptic contacts primarily in layer 4 (Jones and Burton 1976). This ensures that afferent information to the neocortex is first received in layer 4 before being transferred to the upper layers and lower layers 5 and 6 (Johnson and Alloway 1996; Senft and Woolsey 1991; White 1989). When layer 4 diminishes, the thalamic afferents and associated GABAAR become redistributed to upper cortical layers; this thalamic redistribution may account for the increase in GABAergic signaling in this region. Others report a close correlation between the expression of GABAAR and projection of thalamic afferents in the somatosensory and visual cortexes of rodents (Broide et al. 1996; Schlaggar and O’Leary 1994). Thalamic afferents play a significant role in the expression and regional distribution of GABAAR subunits within the visual and somatosensory cortical areas of rodents (Paysan et al. 1997). In rats, the expression of the ␣1-subunit, the predominant subunit of GABAAR in adult animals, is highest within the primary somatosensory (S1) and visual (V1) cortical areas; these regions also receive substantial terminations of thalamic afferents (Fritschy et al. 1994; Paysan et al. 1994). Huntsman and Jones (1998) reported that the mRNA of various GABAAR subtypes diminish in visual cortex after enucleation, also implicating thalamic influence on GABAAR expression. GABAA␣3 and GABAA␥2 expression significantly increase in our model animals. The increase in these two receptor subunits represents an alteration in the stoichiometry of the GABAAR and may underscore the elevated GABA transmission in pyramidal cells of MAM-Tx juvenile ferrets. A change in the composition of the channels could easily lead to a change in the kinetics of response. The GABAA␥2R subunit is presumed to be important for synaptic transmission and for the postsynaptic clustering of GABAAR during the development of synapses (Heck et al. 2003). In this regard, it could clearly play a role in the abnormal synaptic transmission observed here. It is not clear why we did not observe an increase or change in the ␣1-receptors in either the P0 –P1 or P28 –P38 animals, as we would expect these to increase as the animal matures. It may be that the ferret neocortex is still relatively immature, even at P28 –P38, and does not express high levels of this receptor until a later date. In any case, the obvious increase and probable change in the composition of at least two GABAAR subtypes can clearly underlie the change in kinetics that we observed in our recordings.

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT ACKNOWLEDGMENTS We thank Drs. Nathan Cramer, Volodymyr Pidoplichko, and Sylvie Poluch for helpful discussions and scientific assistance. We also thank Latoya Hyson for excellent animal care and technical assistance.

GRANTS This work was supported by funding from National Institute of Neurological Disorders and Stroke Grant PHS RO1 24014 (S. L. Juliano).

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS Author contributions: J.A., M.F.M.B., and S.L.J. conception and design of research; J.A. and S.L.J. performed experiments; J.A. analyzed data; J.A., M.F.M.B., and S.L.J. interpreted results of experiments; J.A. and S.L.J. prepared figures; J.A. drafted manuscript; J.A., M.F.M.B., and S.L.J. edited and revised manuscript; J.A., M.F.M.B., and S.L.J. approved final version of manuscript.

REFERENCES Abbah J, Juliano SL. Altered migratory behavior of interneurons in a model of cortical dysplasia: the influence of elevated GABAA activity. Cereb Cortex. First published April 9, 2013; doi:10.1093/cercor/bht073. Alberto CO, Hirasawa M. AMPA receptor-mediated miniature EPSCs have heterogenous time courses in orexin neurons. Biochem Biophys Res Commun 400: 707–712, 2010. Angevine JB, Sidman RL. Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192: 766 –768, 1961. Araki T, Kiyama H, Tohyama M. GABAA receptor subunit messenger RNAs show differential expression during cortical development in the rat brain. Neurosci 51: 583–591, 1992. Bacci A, Huguenard JR, Prince DA. Modulation of neocortical interneurons: extrinsic influences and exercises in self-control. Trends Neurosci 28: 602– 610, 2005. Bacci A, Rudolph U, Huguenard JR, Prince DA. Major differences in inhibitory synaptic transmission onto two neocortical interneuron subclasses. J Neurosci 23: 9664 –9674, 2003. Benardete EA, Kriegstein AR. Increased excitability and decreased sensitivity to GABA in an animal model of dysplastic cortex. Epilepsia 43: 970 –982, 2002. Bortone D, Polleux F. KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner. Neuron 62: 53–71, 2009. Brill J, Huguenard JR. Enhanced infragranular and supragranular input onto layer 5 pyramidal neurons in a rat model of cortical dysplasia. Cereb Cortex 20: 2926 –2938, 2010. Broide RS, Robertson RT, Leslie FM. Regulation of ␣7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents. J Neurosci 16: 2956 –2971, 1996. Buckmaster PS, 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. Calcagnotto ME, Baraban SC. An examination of calcium current function on heterotopic neurons in hippocampal slices from rats exposed to methylazoxymethanol. Epilepsia 44: 315–321, 2003. Calcagnotto ME, Paredes MF, Scott C, Baraban SC. Heterotopic neurons with altered inhibitory synaptic function in an animal model of malformation associated epilepsy. J Neurosci 22: 7596 –7605, 2002. Carlson VCC, Yeh HH. GABAA receptor subunit profiles of tangentially migrating neurons derived from the medial ganglionic eminence. Cereb Cortex 21: 1792–1802, 2010. Cepeda C, Andre VM, Hauptman JS, Yamazaki I, Huynh MN, Chang JW, Chen JY, Fisher RS, Vinters HV, Levine MS, Mathern GW. Enhanced GABAergic network and receptor function in pediatric cortical dysplasia TypIIB compared with tuberous sclerosis complex. Neurobiol Dis 45: 310 –321, 2012.

333

Choi BH, Matthias SC. Cortical dysplasia associated with massive ectopia of neurons and glial cells within the subarachnoid space. Acta Neuropathol (Berl) 73: 105–109, 1987. Colacitti C, Sancini G, DeBiasi S, Franceschetti S, Caputi A, Frassoni C, Cattabeni F, Avanzini G, Spreafico R, Di Luca M, Battaglia G. Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses. J Neuropathol Exp Neurol 58: 92–106, 1999. Colombo N, Tassi L, Galli C, Citterio A, Lo Russo G, Scialfa G, Spreafico R. Focal cortical dysplasias: MR imaging, histopathologic and clinical correlations in surgically treated patients with epilepsy. Am J Neuroradiol 24: 724 –733, 2003. Cossart R, Dinocourt C, Hirsch JC, Merchan-Perez A, De Felipe J, Ben-Ari Y, Esclapez M, Bernard C. Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy. Nat Neurosci 4: 52– 62, 2001. DeFazio RA, Hablitz JJ. Reduction of zolpidem sensitivity in a freeze lesion model of neocortical dysgenesis. J Neurophysiol 81: 404 – 407, 1999. Edwards FA, Konnerth A, 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. Ferrer I. Experimentally induced cortical malformations in rats. Childs Nerv Syst 9: 403– 407, 1993. Fietz SA, Kelava I, Vogt J, Wilsch-Brauninger M, Stenzel D, Fish JL, Corbeil D, Riehn A, Distler W, Nitsch R, Huttner WB. oSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling. Nat Neurosci 13: 690 – 699, 2010. Flames N, Long JE, Garrat AN, Fsicher TM, Gassmann M, Birchmeier C, Lai C, Rubenstein JLR, Marin O. Short- and long-range attraction of cortical GABAergic interneurons by neuregullin-1. Neuron 44: 251–261, 2004. Fritschy JM, Paysan J, Enna A, Mohler H. Switch in the expression of rat GABAA-receptor subtypes during postnatal development: an immunohistochemical study. J Neurosci 14: 5302–5324, 1994. Gal JS, Morozov YM, Ayoub AE, Chatterjee M, Rakic P, Haydar TF. Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones. J Neurosci 26: 1045–1056, 2006. Ganguly K, Schinder AF, Wong ST, Poo M. GABA itself promotes the developmental-switch of neuronal GABAergic responses from excitation to inhibition. Cell 105: 521–532, 2001. Garrett KM, Saito N, Duman RS, Abel MS, Ashton RA, Fujimori S, Beer B, Tallmann JF, Vitek MP, Blume AJ. Differential expression of ␥-aminobutyric acidA receptor subunits. Mol Pharmacol 371: 652– 657, 1990. Gibbs JW III, Shumate MD, Coulter DA. Differential epilepsy associated alterations in postsynaptic GABA(A) receptor function in dentate granule and CA1 neurons. J Neurophysiol 77: 1924 –1938, 1997. Gierdalski M, Juliano SL. Endogenous neuregulin restores radial glia in a (ferret) model of cortical dysplasia. J Neurosci 25: 8498 – 84504, 2005. Gierdalski M, Juliano SL. Factors affecting the morphology of radial glia. Cereb Cortex 13: 572–579, 2003. Gleeson GJ, Walsh CA. Neuronal migration disorders: from genetic diseases to developmental mechanisms. Trends Neurosci 23: 352–359, 2000. Goodkin HP, Sun CS, Yeh JL, Mangan PS, Kapur J. GABA(A) receptor internalization during seizures. Epilepsia 48: 109 –113, 2007. Guerrini R, Sicca F, Parmeggiani L. Epilepsy and malformations of the cerebral cortex. Epileptic Disord 5: S9 –S26, 2003. Hablitz JJ, Defazio T. Excitability changes in freeze-induced neo-cortical microgyria. Epilepsy Res 32: 75– 82, 1998. Hartmann K, Bruehl C, Golovko T, Draguhn A. Fast homeostatic plasticity of inhibition via activity-dependent vesicular filling. PLos One 3: e2979, 2008. Hashimoto T, Nguyen QL, Rotaru D, Keenan T, Arion D, Beneyto M, Gonzalez-Burgos G, Lewis DA. Protracted developmental trajectories of GABAA receptor alpha1 and alpha2 subunit expression in primate prefrontal cortex. Biol Psychiatry 65: 1015–1023, 2009. Heck N, Kilb W, Reiprich P, Kubota H, Furukawa T, Fukuda A, Luhmann HJ. GABA-A receptors regulate neocortical neuronal migration in vitro and in vivo. Cereb Cortex 17: 138 –148, 2007. Heck WL, Slusarczyk BA, Schweitzer L. Early GABAA receptor clustering during development of the rostral nucleus of the solitary tract. J Anat 202: 387–396, 2003. Henry TR, Frey KA, Sackellares JC, Gilman S, Koeppe RA, Brunberg JA, Ross DA, Berent S, Young AB, Kuhl DE. In vivo cerebral metabolism and

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

334

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT

central benzodiazepine receptor binding in temporal lobe epilepsy. Neurology 43: 1998 –2006, 1993. Hirsch JC, Agassandian C, Merchán-Pérez A, Ben-Ari Y, DeFelipe J, Esclapez M, Bernard C. Deficit of quantal release of GABA in experimental models of temporal lobe epilepsy. Nat Neurosci 2: 499 –500, 1999. Huntsman MM, Jones EG. Expression of alpha3, beta3 and gamma1 GABA(A) receptor subunit messenger RNAs in visual cortex and lateral geniculate nucleus of normal and monocularly deprived monkeys. Neuroscience 87: 385– 400, 1998. Jablonska B, Smith AL, Palmer SL, Noctor SC, Juliano SL. GABAA receptors reorganize when layer 4 in ferret somatosensory cortex is disrupted by methylazoxymethanol (MAM). Cereb Cortex 14: 432– 440, 2004. Jacobs KM, Kharazia VN, Prince DA. Mechanisms underlying epileptogenesis in cortical malformations. Epilepsy Res 36: 165–188, 1999. Johnson MJ, Alloway KD. Cross-correlation analysis reveals laminar differences in thalamocortical interactions in the somatosensory system. J Neurophysiol 75: 1444 –1457, 1996. Johnston MV, Haddad R, Carman-Young A, Coyle JT. Neurotransmitter chemistry of lissencephalic cortex induced in ferrets by fetal treatment with methylazoxymethanol acetate. Brain Res 256: 285–291, 1982. Jones EG, Burton H. Areal differences in the laminar distribution of thalamic afferents in cortical fields of the insular, parietal and temporal regions of primates. J Comp Neurol 168: 197–247, 1976. Karlsson A, Lindquist C, Malmegren K, Asztely F. Altered spontaneous synaptic inhibition in an animal model of cerebral heterotopias. Brain Res 1383: 54 – 61, 2011. Kittler JT, Delmas P, Jovanovic JN, Brown DA, Smart TG, Moss SJ. Constitutive endocytosis of GABA(A) receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci 20: 7972–7977, 2000. Kobayashi M, Buckmaster PS. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. J Neurosci 23: 2440 –2452, 2003. Kriegstein A, Noctor S, Martinez-Cerdeno. Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion. Nat Rev Neurosci 7: 883– 890, 2006. Laurie DJ, Wisden W, Seeburg PH. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci 12: 4151– 4172, 1992. Loup F, Picard F, Andre VM, Kehrli P, Yonekawa Y, Wieser HG, Fritschy JM. Altered expression of alpha3-containing GABA(A) receptors in the neocortex of patients with focal eoileosy. Brain 129: 3277–3289, 2006. Martinez-Cerdeño V, Noctor SC, Kriegstein AR. The role of intermediate progenitor cells in the evolutionary expansion of the cerebral cortex. Cereb Cortex 16, Suppl 1: i152–i161, 2006. McDonald JW, Garofalo EA, Hood T, Sackellares JC, Gilman S, McKeever PE, Troncoso JC, Johnston MV. Altered excitatory and inhibitory amino acid receptor binding in hippocampus of patients with temporal lobe epilepsy. Ann Neurol 29: 529 –524, 1991. McKernan RM, Quirk K, Prince R, Cox PA, Gillard NP, Ragan CL, Whiting P. GABAA receptor subtypes immunopurified from rat brain with a subunit-specific antibodies have unique pharmacological properties. Neuron 7: 667– 676, 1991. McLaughlin DF, Juliano S. Disruption of layer 4 development alters laminar processing in ferret somatosensory cortex. Cereb Cortex 15: 1791–1803, 2005. Michels G, Moss SJ. GABA(A) receptors: properties and trafficking. Crit Rev Biochem Mol Biol 42: 3–14, 2007. Moroni R, Regondi MC, Inverardi F, Garbelli R, Spreafico R, Frassoni C. BCNU-treated rat as a model of cortical dysplasia. In: Proceedings of the National Congress of the Italian Society for Neuroscience, October 1– 4, 2005, Ischia, Italy, 2005, p. 647. Moroni RF, Inverardi F, Regondi MC, Panzica F, Spreafico R, Frassoni C. Altered spatial distribution of PV-cortical cells and dysmorphic neurons in the somatosensory cortex of BCNU-treated rat model of cortical dysplasia. Epilepsia 49: 872– 887, 2008. Noctor SC, Palmer SL, Hasling T, Juliano SL. Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers. Cereb Cortex 9: 121–136, 1999. Noctor SC, Palmer SL, McLaughlin DF, Juliano SL. Disruption of layers 3 and 4 during development results in altered thalamocortical projections in ferret somatosensory cortex. J Neurosci 21: 3184 –3195, 2001.

Noctor SC, Scholnicoff NJ, Juliano SL. Histogenesis of ferret somatosensory cortex. J Comp Neurol 387: 179 –193, 1997. Nusser Z, Hajos N, Somogyi P, Mody I. Increased number of synaptic GABA(A) receptors underlies potentiation at hippocampal inhibitory synapses. Nature 395: 172–177, 1998. Olsen RW, Sieghart W. International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacol Rev 60: 243–260, 2008. Palmer SL, Noctor SC, Jablonska B, Juliano SL. Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex. Eur J Neurosci 13: 1559 –1571, 2001. Palmini A, Andermann F, Olivier A, Tampieri D, Robitaille Y, Andermann E, Wright G. Focal neuronal migration disorders and intractable partial epilepsy: a study of 30 patients. Ann Neurol 30: 741–749, 1991. Paysan J, Bolz J, Mohler H, Fritschy JM. GABAA receptor ␣1 subunit, an early marker for area specification in developing rat cerebral cortex. J Comp Neurol 350: 133–149, 1994. Paysan J, Kossel A, Bolz J, Fritschy JM. Area specific regulation of ␥-aminobutyric acid type A receptor subtypes by thalamic afferents in developing rat cortex. Proc Natl Acad Sci USA 94: 6995–7000, 1997. Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101: 815– 850, 2000. Poluch S, Jablonska B, Juliano SL. Alteration of interneuron migration in a ferret model of cortical dysplasia. Cereb Cortex 18: 78 –92, 2008. Poluch S, Juliano SL. A normal radial glial scaffold is necessary for migration of interneurons during neocortical development. Glia 55: 822– 830, 2007. Poluch S, Juliano SL. Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia. PLos One 5: 1–17, 2010. Poulter MO, Barker JL, O’Carroll AM, Lolait SJ, Mahan LC. Differential and transient expression of GABAA receptor ␣-subunit mRNAs in the developing rat CNS. J Neurosci 12: 2888 –2900, 1992. Prince DA, Jacobs KM, Salin PA, Hoffman S, Parada I. Chronic focal neocortical epileptogenesis: does disinhibition play a role? Can J Physiol Pharmacol 75: 500 –507, 1997. Rakic P. Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183: 425– 427, 1974. Rice A, Rafiq A, Shapiro SM, Jakoi ER, Coulter DA, DeLorenzo RJ. Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy. Proc Natl Acad Sci U S A 93: 9665–9669, 1996. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtenin H, Lamsa K, Pirvola U, Saarma M, Kaila K. The K⫹/Cl⫺ co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251–255, 1999. Rocha L, Suchomelová L, Mares P, Kubová H. Effects of LiCl/pilocarpineinduced status epilepticus on brain mu and benzodiazepine receptor binding: regional and ontogenetic studies. Brain Res 1181: 104 –117, 2007. Roper SN. In-utero irradiation of rats as a model of human cerebrocortical dysgenesis: a review. Epilepsy Res 32: 63–74, 1998. Roper SN, Eisenschenk S, King MA. Reduced density of parvalbumin- and calbindin D28-immunoreactive neurons in experimental cortical dysplasia. Epilepsy Res 37: 63–71, 1999. Ross EM, Walsh CA. Human brain malformations and their lessons for neuronal migration. Annu Rev Neurosci 24: 1041–1070, 2001. Schlaggar BL, O’Leary DDM. Early development of the somatotopic map and barrel patterning in rat romatosensory cortex. J Comp Neurol 346: 80 –96, 1994. Senft SL, Woolsey TA. Growth of thalamic afferents into mouse barrel cortex. Cereb Cortex 1: 308 –335, 1991. Shao LR, Dudek FE. Changes in mIPSCs and sIPSCs after kainate treatment: evidence for loss of inhibitory input to dentate granule cells and possible compensatory responses. J Neurophysiol 94: 952–960, 2005. Sun C, Mtchedlishvili Z, Bertram EH, Erisir A, Kapur J. Selective loss of dentate hilar interneurons contributes to reduced synaptic inhibition of granule cells in an electrical stimulation-based animal model of temporal lobe epilepsy. J Comp Neurol 500: 876 – 893, 2007. Tamas G, Somogyi P, Buhl EH. Differentially interconnected networks of GABAergic interneurons in the visual cortex of the cat. J Neurosci 18: 4255– 4270, 1998.

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org

GABAA TRANSMISSION AFTER DISRUPTED CORTICAL DEVELOPMENT Tassi L, Colombo N, Garbelli R, Francione S, Lo Russo G, Mai R, Cossu M, Ferrario A, Galli C, Bramerio M, Citterio A, Spreafico R. Focal cortical dysplasia: neuropathological subtypes, EEG, neuroimaging and surgical outcome. Brain 125: 1719 –1732, 2002. Taylor DC, Falconer MA, Bruton CJ, Corsellis JAN. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiatry 34: 369–387, 1971. White EL. Cortical Circuits: Synaptic Organization of the Cerebral Cortex– Structure, Function and Theory. Boston, MA: Birkhauser, 1989. Wierenga CJ, Wadman WJ. Miniature inhibitory postsynaptic currents in CA1 pyramidal neurons after kindling epileptogenesis. J Neurophysiol 82: 1352–1362, 1999. Xiang H, Chen HX, Yu XX, King MA, Roper SN. Reduced excitatory drive in interneurons in an animal model of cortical dysplasia. J Neurophysiol 96: 569 –578, 2006. Xie X, Liaw JS, Baudry M, Berger TW. Novel expression mechanism for synaptic potentiation: alignment of presynaptic release site and postsynaptic receptor. Proc Natl Acad Sci U S A 94: 6983– 6988, 1997.

335

Yu ZY, Wang W, Fritschy JM, Witte OW, Redecker C. Changes in neocortical and hippocampal GABAA receptor subunit distribution during brain maturation and aging. Brain Res 1099: 73– 81, 2006. Zhan RZ, Nadler V. Enhanced tonic GABA current in normotopic and hilar ctopic dentate granule cells after pilocarpine-induced status epilepticus. J Neurophysiol 102: 670 – 681, 2009. Zhou FW, Chen HX, Roper SN. Balance of inhibitory and excitatory synaptic activity is altered in fast-spiking interneurons in experimental cortical dysplasia. J Neurophysiol 102: 2514 –2525, 2009. Zhou FW, Roper SN. Densities of glutamatergic and GABAergic presynaptic terminals are altered in experimental cortical dysplasia. Epilepsia 51: 1468 – 1476, 2010. Zhou FW, Roper SN. Altered firing rates and patterns in interneurons in experimental cortical dysplasia. Cereb Cortex 21: 1645–1658, 2011. Zhu WJ, Roper SN. Reduced inhibition in an animal model of cortical dysplasia. J Neurosci 20: 8925– 8931, 2000.

J Neurophysiol • doi:10.1152/jn.00652.2012 • www.jn.org