Propagation of spontaneous synchronized activity in cortical slice cultures recorded by planar electrode arrays

Bioelectrochemistry 51 Ž2000. 107–115 www.elsevier.comrlocaterbioelechem

Propagation of spontaneous synchronized activity in cortical slice cultures recorded by planar electrode arrays Y. Jimbo a,) , H.P.C. Robinson b a

NTT Basic Research Laboratories, 3-1 Morinosato Wakamiya, Atsugi-shi, Kanagawa 243-0198, Japan b Physiological Laboratory, UniÕersity of Cambridge, Cambridge CB2 3EG, UK Accepted 30 November 1999

Abstract The spatial propagation of synchronized activity in cortical slice cultures was characterized by multi-site extracellular recording. Spontaneous activity was studied in normal culture medium, and in bicuculline- or kainic acid-containing media. A common feature in all these conditions was that activity was generated first in superficial layers Ži.e., layer IrII. before spreading over the whole area of the slice. In culture medium or bicuculline-containing medium, the initiation site of the activity was not constant and showed a large variety of patterns of horizontal propagation. Kainic acid induced epileptiform activity, consisting of intense initial bursts followed by repetitive after-discharges. Though the patterns of spatial propagation of the bursts were variable as in the other conditions, the after-discharges followed a constant path. Cross-correlation analysis indicated that the network moved in a graded fashion to a steady state during the sequence of after-discharges. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Planar electrode arrays; Synchronized activity; Cortical slice cultures

1. Introduction Correlated neuronal activity is believed to play a central role in information processing in the brain w1–3x. In the developing nervous system, the refinement of the pattern of synaptic connections is controlled by synchronized activity w4,5x. Amongst the major regions in the mammalian brain, the cerebral cortex, in particular, shows a strong tendency to fire spontaneous synchronized bursts in reconstituted networks w6–9x. The temporal characteristics of this type of activity change with development, in parallel with changes in the expression of transmitter receptors w10–13x. Blockage of activity during development disrupts the normal layout of visual cortical circuitry w14x. Taken together, these observations suggest that synchronized activity plays an important role in cortical network formation. In addition to these physiological roles of synchronized firing, epileptiform activity, the most characteristic feature of which is intense synchronized activity in a wide area of cortex w15x, is a major pathology of the brain. Thus, ) Corresponding author. Tel.: q81-46-240-3524; fax: q81-46-2702364. E-mail address: [email protected] ŽY. Jimbo..

synchronized bursting is a key phenomenon in cortical neuronal networks. Synchronized bursting propagates rapidly, at speeds of up to 10 cmrs through cortical tissue. The precision of synchronous firing between cells thus depends upon their separation, but is seen locally as an increased correlation of spike times spread over tens or hundreds of milliseconds. The origin of such cortical synchronized bursting is likely to be determined by the layered structure of the cortex. Some studies have been carried out on the source and pathways of propagation of certain types of synchronized activity. Connors reported that synchronized activity could be induced by application of bicuculline, the antagonist for GABAA receptors, in acutely isolated cortical slices w16x. His results suggested that the cells in deep layers, particularly pyramidal cells in layers IVrV, may be responsible for initiating this type of synchronized activity w17x. With recent progress in optical recording techniques, however, the spatial propagation patterns of this synchronized activity have been visualized in detail w18,19x, showing that cells in superficial layers are probably responsible for the transmission of synchronized activity. More recently, a study has appeared showing that the source of activity varies according to whether it is induced by bicu-

0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 9 . 0 0 0 8 3 - 5

108

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

culline or kainic acid w20x. One reason for these differences between studies is the limitations of the available methods for recording spatially propagating activity. Conventional microelectrodes can only be used at a few recording sites simultaneously. Optical methods, using voltage-sensitive dyes, despite excellent spatial recording resolution, suffer from low signal-to-noise ratios, requiring either ensemble or spatial averaging, and are invariably associated with some degree of photodynamic damage to cells, as well as having unknown pharmacological effects. These factors make it difficult or impossible to observe the propagation of single spontaneous events in detail. Using a substrate with an embedded electrode array is another possibility for recording spatial patterns of electrical activity. This method has been used for spike recording from dissociated neuronal cultures w21–23x and for hippocampal slice recording w24,25x. The advantages of this method are its high time resolution with reasonable spatial resolution, the capability to resolve spikes from individual cells, and its noninvasiveness. A serious drawback of the method has been the technical problem of recording large numbers of voltages simultaneously, with an adequate sampling rate Ž) 10 kHz. for each channel. In this work, we have developed a recording system with 50 kHzrchannel Žmaximum. sampling rate and 64 recording sites, a resolution which is sufficient for recording unitary action potentials from neurons. Using a culture of rat cortical slices on arrays of 64 electrodes arranged in an 8 = 8 grid, we describe the initiation and propagation of individual spontaneous synchronized bursts in developing cortical circuitry. 2. Materials and methods 2.1. Slice cultures Gahwiller’s rotating tube method w26x for organotypic slice cultures, in which slices are cultured on cover slips inside sealed tubes mounted on a slowly rotating wheel to promote oxygenation of the medium and thinning of the culture, was modified for use with electrode-array substrates. The details of this method are described elsewhere w27x. Briefly, cortical tissue was isolated from P2 Wistar rat pups ŽCharles River, Japan.. Transverse slices of occipital cortex, approximately 300 mm thick, were prepared using a mechanical tissue chopper ŽMickle Lab., Surrey. and stored in oxygenated ice-cold saline for 1 h. The slices were then transferred onto the electrode array substrates, which were coated with poly-D-lysine ŽSigma., and aligned relative to the electrode layout. After fixing down of slices by gentle centrifugation Ž150 rpm, 30 s., 300 ml of culture medium was added. The culture medium consisted of Dulbecco’s modified Eagle’s medium ŽDMEM, Gibco. containing 5% FBS ŽHy-clone., 5% heat inactivated horse serum ŽGibco., 2.5 mgrml insulin ŽSigma., 20 ngrml NGF Ž7S, Sigma. and penicillinrstreptomycin Ž5–40

Urml, Sigma., conditioned overnight in dissociated glial cell cultures w28x. The medium was exchanged twice a week. The slice culture was maintained on a slowly rocking platform Ž"308, 0.1 Hz. for up to 6 weeks. 2.2. Recording method Electrode arrays were fabricated by photolithography, using quartz substrates with a sputtered layer of indium tin oxide ŽITO, 150 nm. w29x. After wet etching of the ITO layer to form the electrode patterns, the surface was insulated with a silicon-based positive photoresist w30x. The insulation layer was then removed selectively at the electrode terminals using reactive ion etching. The array comprised 64 electrodes arranged in two areas separated by 500 mm, each of which contained a 4 = 8 square grid. The size of the recording terminals was 30 = 30 mm2 and the distance between adjacent terminals was 150 mm. The surfaces of the recording terminals were coated electrochemically with a thin layer of platinum black to reduce interface impedance to around 100 k V at 1 kHz. Dishes were used only once following platinizing. The extracellular voltages at the 64 electrodes were amplified using a custom 64-channel amplifier Žbandwidth 0.1–10 kHz, NF, Yokohama, Japan., ArD converted Ž16 bit, 50 kHz sampling rate per channel Žmaximum., System Design Service, Tokyo, Japan. and stored on hard disk. The noise level on each electrode was within the range 10–20 mV p–p. Photographs of the surface structure of the substrate and of a cortical slice culture on the substrate, and an example of the raw signals measured at 64 sites are shown in Fig. 1. The data were analyzed and visualized using PV-wave software ŽVisual Numerics, Colorado.. To estimate current flow between cortical layers, the current source density ŽCSD. w31,32x was calculated. To visualize spatial propagation patterns of the activity in a horizontal direction, the power of the data was used. Recording was first carried out in culture medium. In perfusion experiments, external medium was then exchanged after approximately 1–2 min by slow perfusion of the bath Ž1 mlrmin. with physiological saline. This procedure minimized damage to the slice, but allowed recording to be carried out outside the high CO 2 atmosphere necessary for culture, while maintaining pH buffering. The saline solution had a very similar ionic composition to that of the medium, and consisted of 148 mM NaCl, 2.8 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose, pH 7.2. To induce spontaneous activity in this physiological saline, 10 mM bicuculline or kainic acid ŽSigma. was added to this solution. 3. Results 3.1. Synchronized periodic bursts in cortical slice cultures After 2 days in vitro ŽDIV., it was possible to record evoked extracellular field-potential responses ŽFig. 1c.,

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

109

Fig. 1. Multi-site extracellular recording from a cultured cortical slice. Ža. Layout of an electrode array substrate and magnified views of the recording sites. The size of individual recording terminals is 30 = 30 mm, separation 150 mm. The locations of recording sites are referred to by row ŽR. and column ŽC.. Žb. A 16 DIV cortical slice on an electrode array. The bottom boundary of the slice corresponds to the pial side, and the top to the white matter ŽWM. side. All the data shown in this study were obtained from this slice. Žc. An example of a 64-channel voltage recording obtained from the slice shown in Žb..

which are caused primarily by summed synaptic activation in many cells simultaneously. Single-unit spike activity, representing action potentials in individual cells, could also, be resolved in the potential waveforms. Spontaneous activity was observed after 5 DIV in culture medium. In the second week in vitro, cultured slices produced more frequent and stronger spontaneous activity. The conclusions in this paper are based on recordings from 15 slice cultures, but detailed results and analysis are presented for one slice culture in which the most complete data were obtained. This 16 DIV slice, which is shown in Fig. 1b,

Fig. 2. Spontaneous activity in: Ža. culture medium; Žb. bicuculline-containing medium Ž10 mM.; and Žc. kainic acid-containing medium Ž10 mM.. Data were recorded from the slice shown in Fig. 1b at site ŽR7, C2.. For description, see text.

displayed spontaneous activity as shown in Fig. 2. In culture medium, spontaneous synchronized bursts at irregTable 1 Spontaneous activity patterns in different conditions of excitation. Recordings were carried out in 15 different cultured slices, first in culture medium, then in bicuculline-containing medium Ž10 mM., and finally in kainate-containing medium Ž10 mM.. Activity was classified as periodic synchronized ŽPS. with or without asynchronous spikes ŽAS., and epileptiform ŽE. Sample

DIV

Culture medium

Bicuculline

Kainate

A B C D E F G H I J K L M N O

12 12 12 13 13 14 16 16 16 19 20 21 22 27 33

PS PS with AS PS PS PS PS PS PS PS PS PS PS with AS PS PS with AS PS with AS

E PS E PS E E PS PS PS PS PS PS PS PS PS

PS PS PS PS E E E E E E E E E E E

110

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

ular intervals were observed, with an average frequency of 0.25 Hz. In bicuculline, the events were more intense and lower in frequency. In kainic acid-containing medium, a characteristically shaped waveform was observed, consisting of an initial sharp discharge, followed by repetitive after-discharges lasting for seconds. Such bursts occurred at intervals of over a minute. This type of activity closely resembles epileptic firing, and will be referred to as epileptiform. The activity pattern elicited in each of the 15 slices is shown in Table 1. 3.2. Horizontal propagation To visualize the propagation of excitation in the horizontal axis Žparallel to the boundaries between cortical layers., all 64 recorded traces were displayed together. Fig. 3 shows an example of the bicuculline-induced spontaneous activity shown in Fig. 2b. The three axes represent time, recording site, and signal intensity, respectively. The two-dimensional array of recording sites is mapped onto the position axis from left to right and bottom to top ŽŽR0, C0., ŽR1, C0., . . . ,ŽR7, C7... This procedure produces eight spatial peaks, which correspond to the eight columns of recording electrodes, C0, C1, . . . ,C7. Using this representation, the site of initiation of activity can be easily identified, and its pathway of propagation in the horizontal direction. In this example, the activity was generated in column C3 and propagated in both directions. Thirty-five bursts recorded in the bicuculline-containing medium were analyzed in this way. The distribution of the identified initiation sites is shown as a histogram in Fig. 3. This result suggests that the initiation sites of bursts are not unique but are distributed horizontally over a wide region of the slice. The same kind of results were also obtained for activity in culture medium and for the initial bursts in kainate-induced activity, which will be described in detail below.

3.3. Dependence of the actiÕity on the cortical layered structure The slice shown in Fig. 1b is aligned with the two central columns, C3 and C4, perpendicular to the cortical layers so that columns of electrodes correspond to cortical columns. To visualize the propagation of activity amongst different layers, we examined events initiated at C3 or C4. The eight rows of activity in C3 or C4 in the three different conditions are shown in Fig. 4. A common feature was that activity was always detected first in layers near the pial side of the slice. In culture medium, much stronger activity was detected in superficial layers than in deep layers, while in kainate, an initial peak of activity localized in superficial layers was observed prior to the main activity. Activity in deep layers was clearly increased in bicuculline-containing medium. In order to exclude passive conduction of the signals in the volume conductor, we carried out two-dimensional CSD analysis w30x. The current source at Ž x, y . was approximated by i Ž x , y . s yC Ž s Ž x , y q d . q s Ž x , y y d . q s Ž x q d, y . qs Ž x y d, y . y 4 s Ž x , y .

Ž 1.

where C is a constant, s the recorded signal, and d the interelectrode distance. Because of the array layout, with a 500 mm gap between 4 = 8 banks of electrodes, the right neighbors of the signal in C3 were approximated by s Ž C3X . s 350r530 s Ž C3 . q 180r530 s Ž C4 .

Ž 2.

where sŽC3. and sŽC4. are the signals at the site C3 and C4, respectively, and likewise for the left neighbors of column C4. The results of this procedure are shown in the right panel of Fig. 4, and confirm the above conclusions.

Fig. 3. Horizontal localization of initiation of synchronized bursts. An example recording is shown of the voltages at 64 sites during a single burst in the presence of bicuculline. The activity is detected first at C3 and propagates in both directions within a few tens of milliseconds. A total of 35 bursts were localized in this way, and the distribution of initiation sites is shown at the right.

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

111

Fig. 4. Vertical structure of spontaneous synchronized activity in cortical slices. Single bursts obtained in: Ža. culture medium; Žb. kainite-containing medium Ž10 mM.; and Žc. bicuculline-containing medium Ž10 mM. are shown. Bursts in which the earliest signals were detected at the central columns ŽC3 or C4. are displayed since these columns were almost perpendicular to the layered structure of the slice. Magnified views of firing in these columns illustrate the layer structure of the activity. The earliest activity was detected at or near the superficial layer in all of the conditions. The contribution of cells in deep layers was greatly enhanced by bicuculline application. A two-dimensional CSD analysis for these columns is shown at the right.

3.4. Kainate-induced epileptiform actiÕity We analyzed the structure of three consecutive bursts over a period of about 5 min, each lasting about 5 s, in kainate-containing medium. The detailed structure of these events at one channel is shown in Fig. 5a. Each seizure-like burst consisted of an initial discharge, followed by repeated after-discharges. The waveform of the first initial discharge is shown in the inset. In this figure, the initial bursts are indicated by capital letters ŽA, B, and C.. In the first ‘‘seizure’’, the total number of bursts was 20. The 19 bursts in the after-discharge are designated by small letters Ža, b, . . . , s.. Fig. 5b shows the spatial propagation patterns of the three initial bursts. The first burst ‘‘A’’ had an initiation

site near the center of the slice and activity propagated in both directions. For ‘‘B’’, the initiation site was near the right edge of the slice. In ‘‘C’’, activity appeared to initiate at many sites simultaneously. Thus, both the initiation site and propagation direction were variable from event to event. This result suggests that the slice did not have a unique initiation site but that at least several sites have the potential for generating propagating bursts. In the after-discharge, the situation was different. In the periods between each burst, the slice was not totally silent but showed a small level of ongoing activity, which complicated the identification of spatial patterns in the afterdischarge. Three examples of the spatial patterns of afterdischarges are shown in Fig. 5c. Unlike the initial bursts, these followed a relatively stable spatial pattern, initiating

112

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

Fig. 5. Spatial propagation of kainate-induced epileptiform activity. Kainate was applied at a concentration of 10 mM. Ža. A single episode shown in Fig. 2c magnified in the time domain. The inset shows the detailed wave form of the initial burst designated ‘‘A’’. Žb. Variation of spatial patterns of initial bursts. In ‘‘A’’, activity initiated near column C4, the center of the slice, and propagated in both directions. In ‘‘B’’, the initiation site was near the edge of the slice, while in ‘‘C’’, initiation seemed to occur simultaneously at many positions. Žc. Spatial propagation patterns of after-discharges. In this case, there were 19 after-discharge bursts. Three examples designated ‘‘f’’, ‘‘n’’, and ‘‘s’’ Žsee Fig. 6. show that the after-discharges are relatively constant in their propagation pattern.

around C3 and propagating in both directions. This observation will be addressed in more detail in the next section.

4. Discussion 4.1. Transition from initial burst to after discharge pattern in kainate-induced epileptiform actiÕity

follows: 20 equal-length segments s0, s1, . . . , s19 were taken from the first seizure episode shown in Fig. 5a, each of which was aligned with respect to the negative peak in the respective discharge A . . . s ŽFig. 6a.. The cross-correlation c i j (m) between si (n) and s j (n q m) was then calculated as Ny1

ci j Ž m . s

Ý si Ž n . s j Ž n q m .

Ž 3.

ns0

The stability of the after-discharges following initial bursts of variable profile raises the question of how the network makes the transition from one type of firing to the other. Is the change graded, or is there an abrupt switch from one behavior to the other? In order to visualize this transition, we examined the similarity of waveforms within the sequence of after-discharges. The procedure was as

and normalized as follows: cXi j Ž m . s

ci j Ž m .

(c

ii

Ž m. cj j Ž m.

.

Ž 4.

An example of the calculated set w cXi0 Ž m.,cXi1Ž m., . . . cXi19 Ž m.x is shown in Fig. 6b. The normalized cross-correlation

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

113

Fig. 6. Transition from initial burst to after-discharge bursts in kainate-induced epileptiform activity. Kainate was applied at a concentration of 10 mM. Twenty consecutive sections, one for each discharge, taken from the first episode in Fig. 5a, are displayed aligned in Ža.. Normalized cross-correlation traces were calculated for all 400 combinations of sections. An example of the cross-correlations of each section with section n is shown in Žb., and shows a clear single peak for each trace. The maximum values of these cross-correlations were calculated for all combinations of the 20 after-discharge bursts. This procedure yielded a 20 = 20 matrix Žc.. Žd. Shows the average of the rows, excluding the cross-correlations with the first four bursts.

matrix Ci j was calculated by the following equation: Ci j s Max

cXi j

Ž m. .

Ž 5.

Each element Ci j is a measure of the similarity between si Ž t . and s j Ž t ., and C is diagonally symmetric with elements on the diagonal equal to one. C is plotted in Fig. 6c, and shows lower values near its edges. In Fig. 6d, we have plotted for each trace in turn Žeach row in C., the average of the maximal correlation to traces 4–19, the period over which the form of the cross-correlation function was constant. The averaged maximal correlation was approximately 0.85 for the initial burst, but increased progressively to a stable value of about 0.95 by trace d. Thus, this result shows that the network makes a graded change towards a stable dynamics during the after-discharge period. What could explain this shift from variable initiation site to this steady pattern of firing? Since excitation occurs stochastically w8,9x, and is distributed throughout the network Že.g., spontaneous transmitter release., it is reasonable to expect a variable site of initiation when the network is relatively unexcited, as it is before initiation of an epileptiform episode. However, when the whole network is raised to an excited level, as during after-discharges, exci-

tation could initiate as soon as permitted by the time course of recovery at the site with the lowest threshold, which would presumably be at a fixed location in the slice. 4.2. Layered-structure dependent actiÕity The issue of to what extent the structure and synaptic circuitry of intact cortex is preserved in cultured slices is problematic. Connors described a double-peaked variation of field-potential amplitude in his experiments on acutely isolated cortical slices w16x, which was attributed to the layered structure of the slice. The same kind of distribution of activity was observed in the present experiments, as can be seen in the kainate or bicuculline-induced activity in Fig. 4. One peak was on the pial side and the other on the white matter side, suggesting that the cultured cortical slices maintain a layered structure. The boundary of each layer was, however, not as clear visually as in acute slices. Therefore the terms ‘‘superficial’’ and ‘‘deep’’ layers are used here. Our experimental results indicated that the initial activity was generated in superficial layers, in all three conditions used here. This result is consistent with the spatial

114

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

patterns of evoked responses reported in acutely isolated adult slices by Albowitz and Kuhnt w19x. However, the pharmacological sensitivity reported in Ref. w20x was not observed. This difference could be due to factors relating to the age of the preparation: our slices were isolated from P2 rat pups and used at a nominal equivalent age of P14–P35. However, another factor is the detailed structure of the activity. As shown in the inset of Fig. 5a, the time course of extracellular voltage had multiple phases — we have focused on the initial peak of the activity, but the CSD analysis shown in Fig. 4 suggests the possibility that the layer profile of later phases of the activity is different. This requires further analysis in the future.

5. Conclusions In this study, we have applied a 64-channel recording system with a 50-kHz sampling rate per channel to characterize the spatial features of synchronized spontaneous bursts in cortical slice cultures. In culture medium, activity was detected predominantly in superficial layers. The increase in the strength of activity in bicuculline-containing medium suggests that there is a widespread inhibition in deep layers, presumably by local inhibitory interneurons. Kainic acid, causing a general excitation of neurons, produced epileptiform bursting at long intervals, consisting of an initial strong burst followed by repetitive after-discharges. In all the three conditions, initial triggering of activity was in superficial layers at variable horizontal positions. The continuous after-discharge in kainate, however, converged to a relatively constant horizontal pattern, suggesting that at a high level of background excitation, repetitive initiation occurs at fixed sites of lowest threshold. Thus, the present technique as applied to cortical slices allows spatial mapping of fast synchronized events in tissue whose original structure and connections are relatively intact, without averaging or toxic side effects, and is well suited to long-term study of the pattern and roles of such activity in developing cortex.

Acknowledgements The authors would like to thank Dr. A. Kawana of NTT Basic Research Laboratories, Dr. P. Gogan of CNRS Marseille, and Prof. S. Korogod of Dniepropetrovsk State ´ University for useful discussions.

References w1x W. Singer, Synchronization of cortical activity and its putative role in information processing and learning, Annu. Rev. Physiol. 55 Ž1993. 349–374.

w2x C.M. Gray, D.A. McCormick, Chattering cells: superficial pyramidal neurons contributing to the generation of synchronous oscillations in visual cortex, Science 274 Ž1996. 109–113. w3x P.R. Roelfsema, A.K. Engel, P. Konig, W. Singer, Visuomotor integration is associated with zero time-lag synchronization among cortical areas, Nature 385 Ž1997. 157–161. w4x M. Meister, R.O.L. Wong, D.A. Baylor, C.J. Shatz, Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina, Science 17 Ž1991. 939–943. w5x R.O.L. Wong, M. Meister, C.J. Shatz, Transient period of correlated bursting activity during development of the mammalian retina, Neuron 11 Ž1993. 923–938. w6x T.H. Murphy, L.A. Blatter, W.G. Wier, J.M. Baraban, Spontaneous synchronous calcium transients in cultured cortical neurons, J. Neurosci. 12 Ž1992. 4834–4845. w7x Y. Jimbo, H.P.C. Robinson, A. Kawana, Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture, IEEE Trans. Biomed. Eng. 40 Ž1993. 804–810. w8x H.P.C. Robinson, M. Kawahara, Y. Jimbo, K. Torimitsu, Y. Kuroda, A. Kawana, Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons, J. Neurophysiol. 70 Ž1993. 1606–1616. w9x E. Maeda, H.P.C. Robinson, A. Kawana, The mechanism of generation and propagation of synchronized bursting in developing networks of cortical neurons, J. Neurosci. 15 Ž1995. 6834–6845. w10x H.J. Luhmann, D.A. Prince, Postnatal maturation of the GABAergic system in rat neocortex, J. Neurophysiol. 65 Ž1991. 247–263. w11x E.C. Burgard, J.J. Hablitz, Developmental changes in NMDA and non-NMDA receptor-mediated synaptic potentials in rat neocortex, J. Neurophysiol. 69 Ž1993. 230–240. w12x H. Kamioka, E. Maeda, Y. Jimbo, H.P.C. Robinson, A. Kawana, Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures, Neurosci. Lett. 206 Ž1996. 109–112. w13x S. Watanabe, Y. Jimbo, H. Kamioka, Y. Kirino, A. Kawana, Development of low magnesium-induced spontaneous synchronized bursting and GABAergic modulation in cultured rat neocortical neurons, Neurosci. Lett. 210 Ž1996. 41–44. w14x L.C. Katz, C.J. Shatz, Synaptic activity and the construction of cortical circuits, Science 274 Ž1996. 1133–1138. w15x E.J. Apeckmann, C.E. Elger, The neurophysiological basis of epileptic activity: a condensed overview, Epilepsy Res. Suppl. 2 Ž1991. 1–7. w16x B.W. Connors, Initiation of synchronized neuronal bursting in neocortex, Nature 310 Ž1984. 685–687. w17x B.W. Connors, Y. Amitai, in: M.J. Gutnick, I. Mody ŽEds.., The Cortical Neuron, Functions of Local Circuits in Neocortex: Synchrony and Laminae, Oxford Univ. Press, Oxford, UK, 1995, pp. 123–140. w18x B. Sutor, J.J. Hablitz, F. Rucker, G. Bruggencate, Spread of epileptiform activity in the immature rat neocortex studied with voltage-sensitive dyes and laser scanning microscopy, J. Neurophysiol. 72 Ž1994. 1756–1768. w19x B. Albowitz, U. Kuhnt, Epileptiform activity in the guinea-pig neocortical slice spreads preferentially along supragranular layers — recordings with voltage-sensitive dyes, Eur. J. Neurosci. 7 Ž1995. 1273–1284. w20x A.C. Flint, B.W. Connors, Two types of network oscillations in neocortex mediated by distinct glutamate receptor subtypes and neuronal populations, J. Neurophysiol. 75 Ž1996. 951–956. w21x G.W. Gross, Simultaneous single unit recording in vitro with a photoetched laser deinsulated gold multimicroelectrode surface, IEEE Trans. Biomed. Eng. 26 Ž1979. 273–279. w22x J. Pine, Recording action potentials from cultured neurons with extracellular microcircuit electrodes, J. Neurosci. Methods 2 Ž1980. 19–31. w23x Y. Jimbo, A. Kawana, Electrical stimulation and recording from

Y. Jimbo, H.P.C. Robinsonr Bioelectrochemistry 51 (2000) 107–115

w24x

w25x

w26x w27x

w28x

cultured neurons using a planar electrode array, Bioelectrochem. Bioenerg. 29 Ž1992. 193–204. J.L. Novak, B.C. Wheeler, Multisite hippocampal slice recording and stimulation using a 32 element microelectrode array, J. Neurosci. Methods 23 Ž1988. 149–159. U. Egert, B. Schlosshauer, S. Fennrich, W. Nisch, M. Fejtl, T. Knott, T. Muller, H. Hammerle, A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays, Brain Res. Protoc. 2 Ž1998. 229–242. B.H. Gahwiller, Organotypic cultures of neural tissue, Trends Neurosci. 11 Ž1988. 484–489. H. Kamioka, Y. Jimbo, P. Charlety, A. Kawana, Planar electrode arrays for long-term measurement of neuronal firings in cultured cortical slices, Cell. Eng. 2 Ž1997. 148–153. R.W. Baughman, J. Huettner, K.A. Jones, A.A. Khan, in: G. Banker,

w29x

w30x

w31x

w32x

115

J. Goslin ŽEds.., Cell Culture of Neocortex and Basal Forebrain from Postnatal Rats, Culturing Nerve Cells MIT Press, 1991, pp. 227–229. G.W. Gross, W.Y. Wen, J.W. Lin, Transparent indium–tin oxide electrode patterns for extracellular, multisite recording in neuronal cultures, J. Neurosci. Methods 15 Ž1985. 243–252. A. Tanaka, H. Ban, S. Imamura, K. Onose, Resolution characteristics of novel silicone-based positive photo resist, J. Vac. Sci. Technol., B 7 Ž1989. 572–575. B.C. Wheeler, J.L. Novak, Current source density estimation using microelectrode array data from the hippocampal slice preparation, IEEE Trans. Biomed. Eng. 33 Ž1986. 1204–1212. L.B. Haberly, G.M. Shepherd, Current–density analysis of summed evoked potentials in opossum prepyriform cortex, J. Neurophysiol. 36 Ž1973. 789–803.