Slow endogenous activity transients and developmental expression of K + Cl ) cotransporter 2 in the immature human cortex

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1 European Journal of Neuroscience, Vol. 22, pp , 2005 ª Federation of European Neuroscience Societies Slow endogenous activity transients and developmental expression of K + Cl ) cotransporter 2 in the immature human cortex Sampsa Vanhatalo, 1,2 J. Matias Palva, 1 Sture Andersson, 3 Claudio Rivera, 1,4 Juha Voipio 1 and Kai Kaila 1,5 1 Department of Biological and Environmental Sciences, P.O. Box 65, University of Helsinki, Finland 2 Department of Clinical Neurophysiology, 3 Department of Pediatrics, Hospital for Children and Adolescents, 4 Institute of Biotechnology and 5 Neuroscience Center, University of Helsinki, Finland Keywords: cortical development, DC-EEG, intracellular chloride, KCC2, preterm EEG, trace alternant Abstract Spontaneous transients of correlated activity are a characteristic feature of immature brain structures, where they are thought to be crucial for the establishment of precise neuronal connectivity. Studies on experimental animals have shown that this kind of early activity in cortical structures is composed of long-lasting, intermittent network events, which undergo a developmental decline that is closely paralleled by the maturation of GABAergic inhibition. In order to examine whether similar events occur in the immature human cortex, we performed direct current-coupled electroencephalography (EEG) recordings from sleeping preterm babies. We show now that much of the preterm EEG activity is confined to spontaneous, slow activity transients. These transients are characterized by a large voltage deflection that nests prominent oscillatory activity in several frequency bands covering the whole frequency spectrum of the preterm EEG (< Hz). The slow voltage deflections had an amplitude of up to 800 lv. Most of these giant events originated in the temporo-occipital areas, with a maximum rate of about 8 min, and their occurrence as well as amplitude showed a decline by the time of normal birth. In age-matched fetal brain tissue, this decrease in the spontaneous activity transients was associated with a developmental up-regulation of the neuronal chloride extruder K + Cl ) cotransporter 2, a crucial molecule for the generation of inhibitory GABAergic Cl currents. Our work indicates that slow endogenous activity transients in the immature human neocortex are mostly confined to the prenatal stage and appear to be terminated in parallel with the maturation of functional GABAergic inhibition. Introduction Slow, intermittent bursts of spontaneous network activity are a characteristic feature of immature structures of the central nervous system, including the cortex, hippocampus, retina and spinal cord (Katz & Shatz, 1996; Feller, 1999; O Donovan, 1999; Sernagor et al., 2001; Ben-Ari, 2002). There is much evidence indicating that the early endogenous activity is crucial for the establishment and maintenance of the precise connectivity of cortical networks at a developmental stage where sensory inputs are yet to become functional. The cellular and network mechanisms of endogenous activity in immature cortical structures (hippocampus and neocortex) have been extensively studied in rodents during the first 2 weeks of postnatal life (Ben-Ari, 2002; Rivera et al., 2005), which corresponds roughly to the human developmental time window that spans the last trimester of pregnancy and an early postnatal stage (Clancy et al., 2001). In rodents, the disappearance of the early spontaneous activity is closely paralleled by Correspondence: Dr Sampsa Vanhatalo, 1 Department of Biological and Environmental Sciences, as above. sampsa.vanhatalo@helsinki.fi Received 2 August 2005, revised 13 September 2005, accepted 22 September 2005 the maturation of functional GABAergic inhibition (Garaschuk et al., 2000; Ben-Ari, 2002; Khazipov et al., 2004a). This, in turn, is governed by the developmental expression of the neuron-specific K + Cl ) cotransporter 2 (KCC2), a molecule that is mandatory in the generation of the driving force of GABA A receptor-mediated hyperpolarizing Cl currents (Khirug et al., 2005; Rivera et al., 1999; Payne et al., 2003; Lee et al., 2005). A wealth of studies on pre- and full-term babies, starting from the work of Dreyfus-Brisac (1975) (reviewed in Lamblin et al., 1999), have shown that, at a stage of early prematurity (beginning at a conceptional age of about 24 weeks), the electroencephalography (EEG) is markedly discontinuous and undergoes thereafter a progressive change resulting in continuous activity at full-term and later. However, the conventional EEG techniques used in all previous studies set the lower limit of the recording bandwidth at around 0.5 Hz, which effectively precludes the detection of infraslow events (Vanhatalo et al., 2002, 2004a). In the present study, we have used direct current (DC) coupled EEG recordings (Vanhatalo et al., 2002, 2004a) to study whether slow endogenous activity with a developmental profile akin to those seen in animal experiments can be detected in pre- and full-term human babies. doi: /j x

2 2800 S. Vanhatalo et al. Materials and methods Subjects and electroencephalography recording We recorded cortical activity during sleep from 20 neurologically healthy babies [conceptional age weeks; mean postnatal age 2.1 (range ) weeks] using DC-coupled EEG amplifier with four to eight Ag AgCl electrodes (type LP220, In Vivo Metric, CA, USA) placed according to standard electrode locations with the acquisition reference on the ipsilateral mastoid (for further details, see Vanhatalo et al., 2002, 2004b). This study conforms to The Code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the Ethics Committee of the Helsinki University Central Hospital. Informed consent was obtained from the parents. Electroencephalography analysis The EEG was categorized into discontinuous (trace discontinu and trace alternant) and continuous EEG epochs using conventionally filtered signal, i.e. high-pass filter at 0.5 Hz (Lamblin et al., 1999). Periods of slow wave activity in full-term infants were categorized into discontinuous EEG because, at this age, trace alternant gradually develops into slow-wave activity during quiet sleep (Lamblin et al., 1999). Most of the EEG analyses were performed on the full bandwidth (Vanhatalo et al., 2004a) signals re-referenced to Cz derivation. Closer examination of slow activity transient (SAT) characteristics was performed from the derivation where the SAT waveform amplitude was largest (occipital in 17 neonates, temporal in three neonates). Morlet-wavelet-based time-frequency (TF) analysis was performed for continuous data (Fig. 2A) as well as on epochs consisting of 14-s EEG segments centred at the point of SAT where the amplitude of both slow (<1 Hz) and faster (>5 Hz) activity was at its maximum. These TF representations of SAT epochs were averaged first within each individual and then within the two youngest (32.8 and 33.3 weeks), two intermediate age (35.1 and 35.3 weeks) and two older (38.6 and 39.6 weeks; full-term) neonates (see Fig. 2B). The amplitude modulation of fast activities during the slow voltage deflections during the SATs, i.e. the presence of nested oscillations, was quantified as 1 : 1 phase synchrony (Palva et al., 2005) of the fast oscillations amplitude envelope with the slower oscillations phase (Vanhatalo et al., 2004b). These slow oscillations were extracted with combined finite impulse response high- and low-pass filters. Faster activities and their amplitude envelopes were analysed in three frequency bands: 1 3, 4 35 and Hz. To quantify the frequency of SAT occurrence, SATs were identified visually from data from 20 babies (1231 SATs in s of continuous EEG; 1448 SATs in s of discontinuous EEG; see Fig. 1D). SATs were typically associated with simultaneous oscillations at multiple frequency bands between 1 and 30 Hz (see Fig. 2). Hence, we also performed an automated detection of multiband activity transients (MBATs). We used a finite impulse response filter family (from f 1 ¼ 1Hztof 9 ¼ 30 Hz) that had no frequency-domain overlap between adjacent filters. Each filter, i, was characterized by a center frequency of f i and comprised of a finite impulse response highpass filter (stop band, 0.85f i ; pass band, f i ) and a finite impulse response low-pass filter (pass band, f i ; stop band, 1.3f i ). The center frequencies of successive filters were separated so that f i+1 ¼ 1.53f i. The infraslow deflection of SATs was extracted by filtering with the pass-band from 0.1 to 0.5 Hz (stop bands 0.2 and 0.85 Hz, respectively). The amplitude envelope of each frequency band was estimated by using the Hilbert transform and then z-transformed (normalized with SD after the subtraction of the mean). MBAT onsets Fig. 1. Developmental profile of slow activity transients (SATs) in the immature human cortex. (A and B) Electroencephalography (EEG) recording (with the full bandwidth, 0 50 Hz) of a premature neonate (33 weeks; derivation O1 Cz) demonstrates that SATs are present during both continuous and discontinuous EEG activity. SATs are also observed in full-term neonates (39 weeks; derivation O1 Cz). Representative SATs are indicated with asterisks (*33 weeks; **39 weeks) and shown on an expanded scale in the four insets. Traces high-pass filtered at 0.5 Hz illustrate the EEG at the conventional clinical bandwidth. (C) Spectral analysis demonstrates a major, very low frequency (<0.5 Hz) component that is attenuated during development. Amplitude spectra are averages of three spectra (each computed from 3-min artifact-free epochs during discontinuous EEG, 60-s Hanning window) for each individual and across seven, nine and three individuals in each age group (32 36, and weeks, respectively). Statistically significant differences (anova) are indicated with black (P <0.01) and grey (P <0.05) bars. (D) Developmental profile of SATs and multiband activity transients (MBATs). Left, rate of SAT occurrence based on visual inspection of the data. Right, rate of occurrence of those MBATs that involved a significant, very slow deflection. The error bars show ± SEM. (E) Spatiotemporal properties of SATs. The specimen recordings in E1 and E2 are from the same individual. In some instances (e.g. E2) the SAT is only seen in the occipital or temporal derivations. Data in E3 are based on 106 SATs from six individuals (32 39 weeks). and ends were defined to take place when the amplitude in four or more frequency bands transiently exceeded a threshold of 1.5 SD. The results were similar for thresholds from 1 to 2 SD. To estimate the contribution of false positives, we generated 200 realizations of surrogate data by randomly shifting the time courses of different frequency bands relative to each other. The rate of occurrence of MBATs, detected as described above, exceeded in all babies the mean +2 SDs of that calculated for surrogate data.

3 Slow endogenous activity in preterm cortex 2801 Fetal human brain tissue and in situ hybridization Histological sections (15 lm thick) of fetal human brain tissue were obtained from two brain banks (Prof. I. Kostovic, University of Zagreb, Croatia, see Kostovic et al., 2002; Prof. N. Ulfig, University of Rostock, Germany, see Ulfig et al., 2000). Tissues were immersion-fixed in paraformaldehyde and processed for cryostat sections (sections from Prof. Ulfig) or paraffin sections (from Prof. Kostovic). A 1039-bp mouse KCC2 EST clone (AA982489), corresponding to nucleotides of the full-length rat KCC2 cdna was used for the synthesis of digoxigenin-labeled crna KCC2 probe. Sections were deparaffinated by graded dehydration, microwaved in Tris EDTA buffer (1 mm EDTA, 1 mm sodium citrate, 2 mm Tris- HCl, ph 9.0) for 12 min at 800 W and processed for in situ hybridization (Lazarov et al., 1998). Labeled crna was monitored with alkaline phosphatase-conjugated anti-digoxigenin antibody (1 : 200; Boehringer Mannheim). The sections were then reacted with BM purple (Boehringer Mannheim) containing 2 mm levamisole, fixed in 4% paraformaldehyde, 0.1% glutaraldehyde and mounted in mowiol. The optical density of the KCC2 in situ signal was analysed using the program TINA (TAMRO, Finland) from 16-bit TIF grayscale files. The regions of interest covered all (and only) the cortical layers, and white matter was used as background level for subtraction. Results In a pilot DC-EEG study (Vanhatalo et al., 2002) we found that deltafrequency band (1 4 Hz) bursts, a hallmark of the preterm EEG (Lamblin et al., 1999), were associated with long-lasting, highamplitude voltage deflections. Such slow waves are undetectable with conventional alternating current (AC)-coupled EEG and hence have remained unnoticed (Vanhatalo et al., 2004a). To elucidate the developmental profile of these spontaneous SATs, we carried out DC-coupled EEG recordings on 20 pre- and full-term babies with conceptional ages from 32 to 46 weeks. In all preterm babies, SATs with an amplitude of up to 800 lv (Fig. 1A and B) were clearly Fig. 2. Time-frequency (TF) characteristics of slow activity transients (SATs). (A) Wavelet analysis of ongoing EEG activity shows that SATs typically consist of a slow wave (duration >1 s) with nested faster activities. Top trace shows the same recording at the conventional clinical bandwidth (>0.5 Hz). (B) TF representations of SATs at different ages and during continuous and discontinuous EEG activity show that the TF characteristics of SATs remain similar during development. Each TF representation and the corresponding group of superimposed traces are based on SATs from two individuals (total n ¼ 724; SATs from each individual). Fig. 3. Amplitude modulation of faster oscillations by the phase of ongoing slow activity associated with slow activity transients (SATs). (A) For analysis of amplitude modulation, slow (red trace, Hz) and fast (upper gray trace, 4 35 Hz) frequency oscillations were extracted and the phase of low frequency oscillation (blue middle trace) as well as the amplitude envelope of the faster oscillations (a.e.; blue trace on top) were obtained with the Hilbert transform. The 100-lV calibration refers to the 4 35-Hz trace. In both discontinuous (B) and continuous (C) electroencephalography (EEG) the amplitude of the fast oscillations is correlated with the phase of SATs. Highly significant phase locking was observed between SATs and all higher frequency bands studied here (the slow voltage deflections associated with SATs are schematically indicated by broken line; see also Fig. 2B). k kl (± SEM) values give the ratio of the phase-locking factors of original and surrogate data (k kl > 2.94 corresponds to P < 0.001). Each phase-locking histogram represents an average of six babies (ages weeks), where the values for each baby are computed from one to three 10-min epochs of sleep. Error bars give the SEM.

4 2802 S. Vanhatalo et al. observable during both continuous and discontinuous EEG. At nearand full-term age, the SATs were smaller, with maximum amplitudes of around 300 lv. The temporal properties of the SATs will be described in detail below. Amplitude spectra of discontinuous EEG epochs revealed that the amplitude at Hz declined significantly in the course of maturation (Fig. 1C). There was also a clear peak at about Hz that was seen throughout the developmental period examined here. This activity may constitute a predecessor of the <1-Hz oscillations described in adult cortex (Steriade et al., 1993). Based on data from 20 babies, visually identified SATs had a rate of occurrence of about 3 8 SATs min at weeks at temporo-occipital locations (Fig. 1D, left). In the overall data, a developmental decrease in SAT frequency was highly significant for both continuous and discontinuous EEG (P <0.001, one-way anova). However, SATs were still present at term age. To corroborate the developmental decline in SAT occurrence that was based on visual identification of the events, we exploited the multiband nature of the SATs (for details, see Materials and methods). There was a significant number of events during which the amplitude of multiple (four or more) frequency bands simultaneously exceeded a fixed threshold set at 1.5 SD above the mean. Moreover, in a considerable portion of these MBATs, an infraslow deflection also exceeded the amplitude threshold (Fig. 1D, right). The frequencies of visually identified SATs and automatically detected MBATs associated with the slow deflection (Fig. 2B) were highly correlated within individual babies (R 2 ¼ 0.66, P < 0.03). In order to examine interareal differences in SAT generation, we analysed those SATs that could be identified in all electrode locations within a time window of 5 s (as in Fig. 1, E1; see E2 for a nonpropagating SAT). The onset times (see vertical broken line Fig. 1E1) were determined separately and independently for each derivation (occipital, temporal and frontal). Closer analysis of six babies (age weeks) revealed that the vast majority of SATs (91%, n ¼ 106) were first observed in occipital or temporal EEG derivations (Fig. 1, E3) and they showed an apparent propagation to frontal areas with delays of up to 3 s (mean delay 0.9 ± 0.7 s). A Morlet-wavelet-based TF analysis of ongoing activity (Fig. 2A) indicated that SATs were associated with pronounced oscillatory activity that took place simultaneously in several frequency bands ( Hz, Fig. 2A). This observation was confirmed and extended by averaged TF representations of SATs (a total of 724 epochs in six babies at weeks) which showed that SATs were indeed characterized by oscillations at multiple frequencies ranging up to the highest observed in the neonatal EEG (30 Hz) (Fig. 2B). Notably, the mean TF profile of SATs was very similar during both continuous and discontinuous EEG as well as throughout the developmental period studied here. We then analysed prolonged sleep epochs (10 min each) to further quantify the correlation between the amplitude of higher frequency oscillations and the phase of the very low frequency oscillations comprising of the SATs. There was a very robust phase locking as seen in Fig. 3 during both continuous and discontinuous EEG activity. In the immature rodent brain, the disappearance of slow network activity is known to closely parallel the developmental up-regulation of KCC2 which acts as a developmental switch to convert immature depolarizing GABAergic responses into hyperpolarizing inhibitory postsynaptic potentials (Rivera et al., 1999, 2005; Payne et al., 2003). As shown by in situ hybridization (n ¼ 12 individuals, conceptional age weeks), cortical structures in fetal human brains at 29 weeks showed a rather low expression level of KCC2 mrna (Fig. 4A). The developmental decline of SAT activity (see Fig. 4. Developmental up-regulation of K + Cl ) cotransporter 2 (KCC2) mrna in the human neocortex. Non-radioactive in situ hybridization in representative sections at 29 (A and D), 31 (B and E) and 40 (C and F) weeks showing specific hybridization signals of KCC2 mrna in neocortical (A C) and hippocampal (D F) structures. Nissl staining (G) and sense signal (H) are shown in 40-week sections. Scale bar, 1 mm. The histogram (I) is based on optical density of the in situ hybridization signal for neocortical sections at 29 (n ¼ 6) and 40 weeks (n ¼ 7). Bars represent + SEM. Fig. 1D) was paralleled by a steep up-regulation of KCC2 expression in the neocortex and hippocampus (Fig. 4A F). The diencephalon, midbrain and pons showed already at 29 weeks of age an elevated KCC2 level (data not shown) which points to a brain region-specific developmental expression profile similar to that found in the rat (Rivera et al., 1999). Comparison of neocortical tissue at 29 weeks (n ¼ 6 sections) and 40 weeks (n ¼ 7 sections) showed a 3.3-fold increase in the expression of KCC2 mrna (Fig. 4I). This increase in KCC2 mrna expression is very similar to that recently reported in rats (Yamada et al., 2004) during a developmental time window that is roughly similar to that of the human fetal tissue studied here. Discussion The discontinuous, burst-like appearance of the preterm EEG was first described several decades ago (see Introduction). In this work, we demonstrate that these endogenous bursts are often nested in large and infraslow voltage deflections. Moreover, we observed that the bursts, in fact, were comprised of simultaneous oscillations in several distinct frequency bands from 1 to 30 Hz (see Fig. 2). The spectral pattern of these fast oscillations during SATs was essentially similar in both continuous and discontinuous EEG as well as throughout the developmental time window from 32 to 46 weeks. The frequency of occurrence of these SATs decreased towards full-term, although they remained detectable at least until week 44. Whether there is any

5 Slow endogenous activity in preterm cortex 2803 relationship between SATs in preterm babies and the phenomenologically similar infraslow oscillations and activity bursts (Steriade et al., 1996; Vanhatalo et al., 2004a,b) in the adult brain remains an issue for further studies. While the slow voltage shifts characteristic of SATs in the preterm scalp EEG are literally gigantic slow events, they are lost or strongly distorted by high-pass filtering in conventional (i.e. clinical) AC-coupled EEG (see Fig. 1A and B; Vanhatalo et al. 2002). The presence of this kind of intermittent, slow events in the immature human cortex suggests that they are homologous to the slow endogenous transients that have been extensively studied in the rodent cortex and hippocampus. At first sight, the developmental time course of SATs seems to contrast with observations in rodents where slow endogenous cortical activity is seen during the first two postnatal weeks (Garaschuk et al., 2000; Ben-Ari, 2002; Khazipov et al., 2004a; Adelsberger et al., 2005). However, rodents are born at a very immature stage (Clancy et al., 2001; Avishai-Eliner et al., 2002) and hence there is a difference in the timing of birth vs. developmental stage rather than in the temporal patterns of cortical development (Khazipov et al., 2001). In rodents, the gradual abolishment of the intermittent early slow activity during cortical maturation is closely paralleled by the developmental expression of the neuronal K + Cl ) cotransporter KCC2, which leads to a change in GABAergic transmission from excitatory (depolarizing) to inhibitory (hyperpolarizing) (Rivera et al., 1999, 2005; Payne et al., 2003). In line with this, we found that the developmental decrease in SAT occurrence was associated with a pronounced increase in the expression of KCC2 mrna as seen in age-matched fetal human tissue. When considering the presence of a causal link between the enhanced expression of KCC2 and the decline of SAT activity, it is important to note that, in native cortical neurons, the developmental increase in KCC2 mrna is directly reflected in an increase in the expression of functional KCC2 protein and enhanced Cl extrusion (Rivera et al., 1999; Yamada et al., 2004; Khirug et al., 2005). In line with a functional role in the shaping of human cortical networks, SATs appear at a time of intense development of short- and long-range intracortical connections and of thalamocortical synapses (Kostovic & Judas, 2002; Molliver et al., 1973; Schwartz & Goldman- Rakic, 1991; Mrzljak et al., 1992; Burkhalter, 1993). It is notable that not only the somatosensory but also the visual and auditory systems are capable of responding to modality-specific stimuli during human intrauterine development (Grubb & Thompson, 2004; Fulford et al., 2003; Schleussner & Schneider, 2004). Furthermore, recent data on the immature rat primary somatosensory area suggest an intriguing interaction between endogenous cortical network activity (primary somatosensory area spindle activity) and sensory-evoked responses, whereby somatosensory stimuli enhance the probability of the occurrence of the discrete endogenous events (Khazipov et al., 2004b). All these observations (see also Khazipov et al., 2001) point to highly conserved evolutionary mechanisms, whereby the immature neocortex generates slow endogenous large-scale activity that interacts with thalamic inputs before and during the maturation of sensorydriven signals. It is most likely that the human cortical SATs will turn out to have both diagnostic and prognostic value (Penn & Shatz, 1999). Moreover, the influence on SATs of substances of abuse as well as drugs routinely used in neonatal care and during pregnancy (Rennie & Boylan, 2003; Stanwood & Levitt, 2004; Bernard et al., 2005; Cohen et al., 2005), including compounds acting on GABAergic mechanisms (Rennie & Boylan, 2003; Crowther & Henderson-Smart, 2001), warrants attention. Acknowledgements We thank Prof. Ivica Kostovic and Norbert Ulfig for providing human fetal brain tissue and for expert advice and Prof. Kostovic and Arthur Konnerth for helpful comments on an early version of this work. This work was supported by the Academy of Finland and by the Sigrid Jusélius Foundation. Abbreviations AC, alternating current; DC, direct current; EEG, electroencephalography; KCC2, K + Cl ) cotransporter 2; MBAT, multiband activity transient; SAT, slow activity transient; TF, time-frequency. References Adelsberger, H., Garaschuk, O. & Konnerth, A. (2005) Cortical calcium waves in resting newborn mice. Nat. Neurosci., 8, Avishai-Eliner, S., Brunson, K.L., Sandman, C.A. & Baram, T.Z. (2002) Stressed-out, or in (utero)? Trends Neurosci., 25, Ben-Ari, Y. (2002) Excitatory actions of GABA during development: the nature of the nurture. Nat. Rev. Neurosci., 3, Bernard, C., Milh, M., Morozov, Y.M., Ben-Ari, Y., Freund, T.F. & Gozlan, H. (2005) Altering cannabinoid signaling during development disrupts neuronal activity. Proc. Natl Acad. Sci. U.S.A., 102, Burkhalter, A. (1993) Development of forward and feedback connections between areas V1 and V2 of human visual cortex. Cerebral Cortex, 3, Clancy, B., Darlington, R.B. & Finlay, B.L. (2001) Translating developmental time across mammalian species. Neuroscience, 105, Cohen, G., Roux, J.C., Grailhe, R., Malcolm, G., Changeux, J.P. & Lagercrantz, H. (2005) Perinatal exposure to nicotine causes deficits associated with a loss of nicotinic receptor function. Proc. Natl Acad. Sci. U.S.A., 102, Crowther, C.A. & Henderson-Smart, D.J. (2001) Phenobarbital prior to preterm birth for preventing neonatal periventricular haemorrhage. Cochrane Database Syst. Rev., 2, CD Dreyfus-Brisac, C. (1975) Neurophysiological studies in human premature and full-term newborns. Biol. Psychiat., 10, Feller, M.B. (1999) Spontaneous correlated activity in developing neural circuits. Neuron, 22, Fulford, J., Vadeyar, S.H., Dodampahala, S.H., Moore, R.J., Young, P., Baker, P.N., James, D.K. & Gowland, P.A. (2003) Fetal brain activity in response to a visual stimulus. Hum. Brain Mapp., 20, Garaschuk, O., Linn, J., Eilers, J. & Konnerth, A. (2000) Large-scale oscillatory calcium waves in the immature cortex. Nat. Neurosci., 3, Grubb, M.S. & Thompson, I.D. (2004) The influence of early experience on the development of sensory systems. Curr. Opin. Neurobiol., 14, Katz, L.C. & Shatz, C.J. (1996) Synaptic activity and the construction of cortical circuits. Science, 274, Khazipov, R., Esclapez, M., Caillard, O., Bernard, C., Khalilov, I., Tyzio, R., Hirsch, J., Dzhala, V., Berger, B. & Ben-Ari, Y. (2001) Early development of neuronal activity in the primate hippocampus in utero. J. Neurosci., 21, Khazipov, R., Khalilov, I., Tyzio, R., Morozova, E., Ben-Ari, Y. & Holmes, G.L. (2004a) Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus. Eur. J. Neurosci., 19, Khazipov, R., Sirota, A., Leinekugel, X., Holmes, G.L., Ben-Ari, Y. & Buzsaki, G. (2004b) Early motor activity drives spindle bursts in the developing somatosensory cortex. Nature, 432, Khirug, S., Huttu, K., Ludwig, A., Smirnov, S., Voipio, J., Rivera, C., Kaila, K. & Khiroug, L. (2005) Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices. Eur. J. Neurosci., 21, Kostovic, I. & Judas, M. (2002) Correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants. Anat. Rec., 267, 1 6. Kostovic, I., Judas, M., Rados, M. & Hrabac, P. (2002) Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging. Cerebral Cortex, 12, Lamblin, M.D., Andre, M., Challamel, M.J., Curzi-Dascalova, L., d Allest, A.M., De Giovanni, E., Moussalli-Salefranque, F., Navelet, Y., Plouin, P., Radvanyi-Bouvet, M.F., Samson-Dollfus, D. & Vecchierini-Blineau, M.F. (1999) Electroencephalography of the premature and term newborn. Maturational aspects and glossary. Neurophysiol. Clin., 29,

6 2804 S. Vanhatalo et al. Lazarov, N., Schmidt, U., Wanner, I. & Pilgrim, C. (1998) Mapping of D1 dopamine receptor mrna by non-radioactive in situ hybridization. Histochem. Cell. Biol., 109, Lee, H., Chen, C.X., Liu, Y.J., Aizenman, E. & Kandler, K. (2005) KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses. Eur. J. Neurosci., 21, Molliver, M.E., Kostovic, I. & van der Loos, H. (1973) The development of synapses in cerebral cortex of the human fetus. Brain Res., 50, Mrzljak, L., Uylings, H.B., Kostovic, I. & van Eden, C.G. (1992) Prenatal development of neurons in the human prefrontal cortex. II. A quantitative Golgi study. J. Comp. Neurol., 316, O Donovan, M.J. (1999) The origin of spontaneous activity in developing networks of the vertebrate nervous system. Curr. Opin. Neurobiol., 9, Palva, J.M., Palva, S. & Kaila, K. (2005) Phase synchrony among neuronal oscillations in the human cortex. J. Neurosci., 25, Payne, J.A., Rivera, C., Voipio, J. & Kaila, K. (2003) Cation-chloride cotransporters in neuronal communication, development and trauma. Trends Neurosci., 26, Penn, A.A. & Shatz, C.J. (1999) Brain waves and brain wiring: the role of endogenous and sensory-driven neural activity in development. Pediatr. Res., 45, Rennie, J.M. & Boylan, G.B. (2003) Neonatal seizures and their treatment. Curr. Opin. Neurol., 16, Rivera, C., Voipio, J., Payne, J.A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., Pirvola, U., Saarma, M. & Kaila, K. (1999) The K+ Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature, 397, Rivera, C., Voipio, J. & Kaila, K. (2005) Two developmental switches in GABAergic signalling: the K+-Cl cotransporter KCC2 and carbonic anhydrase CAVII. J. Physiol., 562, Schleussner, E. & Schneider, U. (2004) Developmental changes of auditoryevoked fields in fetuses. Exp. Neurol., 190 (Suppl. 1), S59 S64. Schwartz, M.L. & Goldman-Rakic, P.S. (1991) Prenatal specification of callosal connections in rhesus monkey. J. Comp. Neurol., 307, Sernagor, E., Eglen, S.J. & Wong, R.O.L. (2001) Development of retinal ganglion cell structure and function. Prog. Retinal Eye Res., 20, Stanwood, G.D. & Levitt, P. (2004) Drug exposure early in life: functional repercussions of changing neuropharmacology during sensitive periods of brain development. Curr. Opin. Pharmacol., 4, Steriade, M., Nunez, A. & Amzica, F. (1993) A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components. J. Neurosci., 13, Steriade, M., Amzica, F. & Contreras, D. (1996) Synchronization of fast (30 40 Hz) spontaneous cortical rhythms during brain activation. J. Neurosci., 16, Ulfig, N., Feldhaus, C. & Bohl, J. (2000) Transient expression of synaptogyrin in the ganglionic eminence of the human fetal brain. Ann. Anat., 182, Vanhatalo, S., Tallgren, P., Andersson, S., Sainio, K., Voipio, J. & Kaila, K. (2002) DC-EEG discloses prominent, very slow activity patterns during sleep in preterm infants. Clin. Neurophysiol., 113, Vanhatalo, S., Voipio, J. & Kaila, K. (2004a) Infraslow EEG activity. In Niedermeyer, E. & Lopes Da Silva, F. (Eds), Electroencephalography: Basic Principles, Clinical Applications, and Related Fields, 5th Edn. Lippincott, Williams & Wilkins, Baltimore, pp Vanhatalo, S., Palva, J.M., Holmes, M.D., Miller, J.W., Voipio, J. & Kaila, K. (2004b) Infraslow oscillations modulate excitability and interictal epileptic activity in the human cortex during sleep. Proc. Natl Acad. Sci. U.S.A., 101, Yamada, J., Okabe, A., Toyoda, H., Kilb, W., Luhmann, H.J. & Fukuda, A. (2004) Cl uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J. Physiol., 557,