Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa Chiba 277-8562 and , 1 Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, The University of Tokyo and SORST, Japan Science and Technology Corporation, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The N-methyl-D-aspartate (NMDA)-type glutamate receptor is one of the receptor types responsible for cortical plasticity during development, including synaptic formation and synaptic plasticity. In the somatosensory cortex of the rodent, there is an early critical period during which whisker removal prevents the formation of cortical barrels (Woolsey and Wann, 1976; OLeary et al., 1994
). Interestingly, whereas earlier work had suggested that activity might have a minimal role in barrel formation (Chiaia et al., 1992
; Henderson et al., 1992
), recent work with transgenic mice lacking cortical NMDA receptors indicates that NMDA receptors are indeed crucial for the formation of barrels (Iwasato et al., 2000
). Spontaneous neuronal activity in the prenatal stage mediated through NMDA receptors (LoTurco et al., 1991
) may result in the formation of cortical microcircuitry. Expression of NMDA receptors in the neocortex of prenatal rodents has been confirmed in a series of reports (LoTurco et al., 1991
; Watanabe et al., 1992
; Monyer et al., 1994
). LoTurco et al. (LoTurco et al., 1991
) demonstrated that NMDA receptor channels on cortical plate neurons in embryonic brain slices are activated in the absence of any exogenous source of NMDA receptor agonist. Even though the opening of NMDA channels is blocked by magnesium at the resting membrane potential, ~60mV (Mayer et al., 1984
; Nowak et al., 1984
), the membrane potential of neurons at this developmental stage could be elevated by the presence of GABA and glycine. In immature neurons, GABAA receptors and glycine receptors have been found to elicit a depolarizing neuronal membrane potential due to raised ECl (Luhmann and Prince, 1991
; Owens et al., 1996
; Flint et al., 1998
; Miyakawa et al., 2002
).
In this paper, we use the NMDA-type glutamate receptor GluR2-/- mouse model, which does not possess any functional NMDA receptors in the developing neocortex (Kutsuwada et al., 1996
), to investigate the relationship between neuronal domains and NMDA receptors. We assessed the frequency of the occurrence of neuronal domains in acute cortical slices from newborn mice. High-speed confocal imaging of fluo-4-stained cortical slices has enabled us to better determine the spatial and temporal resolution of the intracellular Ca2+ signal. We clearly demonstrate here the involvement of NMDA receptors in the functional formation of neuronal domains in developing neocortex of the rodent.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The NMDA-type glutamate receptor e2 (GluR2/NR2B) subunit gene null-mutant was generated in a previous study (Kutsuwada et al., 1996
). Because homozygous null-mutant mice (GluR
2-/-) die within 24 h of birth, they were generated by means of mating heterozygous (GluR
2±) males and females. Genotyping was performed just after pups were born. In some experiments, we also used ICR mice (Sankyo Laboratory, Tokyo, Japan). All experiments were carried out in accordance with the guidelines for Animal Experiments of the Faculty of Frontier Sciences, The University of Tokyo.
Genotype was checked using a PCR technique. The tail tissues of animals were treated with 0.1 mg/ml proteinase K (Takara, Shiga, Japan) solution at 55°C for 60 min followed by inactivation at 95°C for 15 min, from which supernatants were used as genomic DNA samples for PCR. Primers were 5'-AGA GTC GAC GAG CTG AAG ATG AAG CCC AGC-3' (E2P1), 5'-CGG GGA ACT ACT GAG AGA TGA TGG AAG TCA-3' (E2P2) and 5'-GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT-3' (NeoP1) as previously described (Wainai et al., 2001). Reactions were performed using a HotstarTaq DNA polymerase kit (Qiagen, Valencia, USA) as follows: hot start at 95°C for 15 min followed by 30 cycles of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C. Aliquots of the PCR products were isolated by electrophoresis on 2% agarose gels and stained with ethidium bromide. PCR reactions of wild-type and targeted alleles gave bands of 218 and 377 bp.
Dissection and Fluo-4 Staining
Newborn mice (postnatal day 0: P0) were anesthetized by hypothermia and their brains were removed in cold artificial cerebro-spinal fluid (ACSF) solution. The constituents of the ACSF were as follows: NaCl, 124 mM; KCl, 2.5 mM; NaHCO3, 26 mM; MgCl2, 1 mM; CaCl2, 2 mM; NaH2PO4, 1.25 mM; and D-glucose 10 mM. The ACSF was always saturated with a 95% O2/5% CO2 gas mixture. A coronal section (400 µm thickness) was prepared from the primary somatosensory area of the cerebral cortex using a vibratome (Dosaka, Kyoto, Japan) as described previously (Miyakawa et al., 2002). The section was incubated in ACSF containing 10 µM fluo-4AM (Molecular Probes, Eugene, OR) at 37°C for 90 min. After staining, the section was briefly washed and stored at room temperature until used for experiments.
Confocal Calcium Imaging and the Observation of Neuronal Domains
Fluo-4 fluorescence was detected using a Leica confocal laser scanning microscopy system (TCS-NT or TCS-SL; Leica, Heidelberg, Germany). This system consists of an inverted microscope, a laser scanning unit, a photomultiplier and PC-based software. All fluorescent images were obtained as digital data. A stained section was placed under the microscope and fluorescent images were sequentially obtained. A frame was created from the average of two scans (each scan taking 1.67 s to perform). The interval time between the end of the pre-frame and the end of the post-frame was 6.34 s. All drugs were dissolved in ACSF and bath-applied by switching with normal ACSF. To activate NMDA receptor channels, we used NMDA (100 mM) plus glycine (100 mM), as reported previously (Yamada et al., 1999; Miyakawa et al., 2002
). Several antagonists and inhibitors were also used in this study: tetrodotoxin (TTX, 5 µM); 2-amino-5-phosphonopentanoic acid (APV, 100 mM); picrotoxin (100 mM); strychnine (100 mM); halothane (10 mM); and thapsigargin (10 mM). TTX, strychnine and glycine were purchased from Wako (Osaka, Japan). The other compounds were all from Sigma (St Louis, MO).
After measurement, the tempo-spatial dynamics of [Ca2+]i in each neuron were analyzed. When a group of (more than five) neurons exhibited a simultaneously elevated [Ca2+]i, we defined this event as a neuronal domain, as reported elsewhere (Yuste et al., 1992).
High-speed Confocal Imaging with Microinjection of IP3
Microinjection of IP3 into single cells was done using a micro-electrode amplifier (MEZ-8301; Nihon Kohden, Tokyo, Japan) and an electronic stimulator (SEN-3301; Nihon Kohden). The tip radius of glass microelectrodes was <0.1 µm, giving a tip resistance > 100 M. Microelectrodes were filled with a 1 M LiCl (Wako) solution containing 100 mM IP3 (Sigma). A brain section stained with fluo-4 was positioned under the upright microscope (BX51WI; Olympus, Tokyo, Japan) and a neuron in the developing neocortex was visualized with the aid of an IR-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The neuron was impaled with the glass electrode and electrophoretically injected with IP3 by means of a hyperpolarizing current pulse (-50 nA, 100 ms). Fluorescent digital images were taken using a Nipkow disk type confocal laser-scanning unit (CSU-21; Yokogawa, Tokyo, Japan), fitted with a cooled CCD camera (HiSCA; Hamamatsu) and an Aquacosmos interface (Hamamatsu).
Immunohistochemistry
The telencephalons of P0 GluR2+/+ and GluR
2-/- mice were post-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C and subsequently subjected to cryoprotection in 30% sucrose (Wako) in PBS for 3 days at 4°C. Tissue samples were embedded in OCT (Sakura, Tokyo) and 40 µm coronal sections cut using a freezing microtome (Microm, Walldorf, Germany). Sections were air dried at 55°C for 15 min, washed in PBS for 5 min and blocked by incubation for 2 h at room temperature in PBS containing 0.1% Triton X-100 and 3% normal goat serum. Incubation was continued at 4°C overnight in fresh blocking solution supplemented with a 1/50 dilution of anti-mouse Cx26 polyclonal antibody (71-0500, Zymed Laboratories Inc., South San Francisco, CA). Sections were then washed in PBS (3 x 20 min) and incubated for 2 h at room temperature in PBS containing anti-rabbit IgG conjugated with Alexa488 (Molecular Probes), followed by washing in PBS (3 x 10 min). Samples were mounted in Immumount plus DABCO and analyzed by laser confocal microscopy.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We analyzed the dynamics of changes in [Ca2+]i in the developing cerebral neocortex of newborn mice (P0) by means of confocal calcium imaging. In the cortical plate of the primary somatosensory cortex, calcium waves could be observed in 550 neighboring neuronal cells as they synchronously increased their [Ca2+]i. These spontaneous coordinated actions of neuronal cells appeared to occur randomly across the cortical plate (Fig. 1). The mean area of calcium waves was 5350 ± 389 µm2 (n = 49, mean ± SEM). As seen in fluorescent images from fluo-4-stained slices, calcium waves frequently originated in neuronal cells in primary layer 2/3.
|
|
The NMDA receptor is a glutamate-gated ion channel and is associated with circuit formation at the developmental stage (Rabacchi et al., 1992; Simon et al., 1992
). There are several subunit molecules of the NMDA receptor and their expressions are spatially and temporally regulated (Monyer et al., 1994
; Mori and Mishina, 1995
). It is known that the GluR
1 and GluR
2 subunits are predominantly expressed in developing cerebral cortex, meaning that the NMDA receptor is presumably not functional in the neocortex of GluR
2 knockout mice (Kutsuwada et al., 1996
; Miyakawa et al., 2002
). Neurons in brain slices from wild-type (GluR
2+/+) mice strongly responded with increased [Ca2+]i in the presence of 100 µM NMDA (Fig. 3
), which was completely inhibited upon exposure to 100 mM APV. On the other hand, the NMDA-mediated increase in [Ca2+]i was not seen in brain slices from GluR
2-/- mice. This result indicates that calcium signals elicited via NMDA receptor activation are completely absent in GluR
2-/- mice.
|
The frequency of occurrence of neuronal domains in brain slices from GluR2-/- mice was compared to that in wild-type (GluR
2+/+) mice. The GluR
2-/- and GluR
2+/+ mice used in these experiments were littermates. Increased calcium activity in neuronal domains was frequently and spontaneously generated in developing cortical plates from GluR
2+/+ mice, whereas in GluR
2-/- mice the frequency of appearance of neuronal domains was significantly less at around just 10% of that seen in GluR
2+/+ mice (Fig. 4
). The size of the infrequent neuronal domains observed in GluR
2-/- mice (5167 ± 823 µm2, n = 20, mean ± SEM) was quite similar to that seen in GluR
2+/+ mice (4970 ± 477 µm2, n = 43). The location of the infrequent neuronal domains in GluR
2-/- mice was also at layer 2/3 of developing cortex, as it was in wild-type mice. Representative observations of wild-type and knockout mice are also shown in a movie file (Supplementary Material, movie 1). This result clearly demonstrates the critical role of NMDA receptors in the generation of neuronal domains in developing cerebral cortex.
|
|
It is already known that a neuronal domain can be induced by injecting IP3 into a single neuron (Kandler and Katz, 1998). For the injection of IP3, these authors used the natural diffusion of IP3 via a patch-clamp electrode. With a slight modification of this technique, we injected IP3 electrophoretically by means of a glass microelectrode of tip diameter <0.1 µm. This method allows one to produce neuronal domains at a point immediately after the precise injection of IP3. In addition, we used a Nipkow disk type confocal unit (CSU-21; Yokogawa), which enabled us to obtain better time resolution of the observation. It takes only 100 ms to obtain one high-quality confocal image from fluo-4-loaded brain slices. We performed several set-up examinations in order to determine whether our method could be used to evaluate the phenomenon of neuronal domains. First, we observed spontaneous neuronal domains using the experimental set up. As shown in Figure 6a
, a calcium wave spreads from central neurons to surrounding cells over a 500 ms period. This result is quite similar to a domain evoked with a temperature drop (Yuste et al., 1995
). Next, we injected IP3 into a single cell. Calcium waves that are very similar to spontaneous neuronal domains were observed (Fig. 6b
). The properties of calcium waves induced by IP3 microinjection are similar to those of spontaneously occurring neuronal domains in terms of appearance, propagation and propagation velocity.
|
The Microinjection of IP3 into a Single Neuron from a GluR2-/- Brain Slice
We assessed whether or not the microinjection of IP3 would produce neuronal domains in the developing neocortex of GluR2-/- neonates. The frequency of IP3-driven neuronal domains in GluR
2-/- cortices was compared to that measured in wild-type cortices (Table 1
). In most trials, neuronal domains were induced in brain slices taken from several wild-type (GluR
2+/+) mice. Representative observations from wild-type brain slices are shown in Figure 7a
. Neuronal domains were not induced in the majority cases of brain slices from GluR
2-/- mice (Table 1
). Instead, only IP3-injected cells showed strong [Ca2+]i elevation (Fig. 7b
). The frequencies of IP3-driven neuronal domains in wild-type and GluR
2-/- mice were thus 91 and 20%, respectively. It is conceivable that during cortical development NMDA receptors would deliver the signal which activates functional cell-coupling via gap-junction channels.
|
|
Connexin Expression in Developing Neocortex of GluR2-/- Mice
There is a possibility that the protein responsible for gap junction, connexin (Cx), might be downregulated in GluR2-/- mice. Sixteen putative Cxs have been described from rodents to date, of which Cxs 26, 32, 36, and 43 are the major isoforms expressed in the developing and adult brain (Nadarajah et al., 1997
). Among them, Cx26 expression is highest prenatally and during the first 3 weeks of postnatal life, suggesting that it may take part in the establishment of neuronal coupling in the developing cortex (Bittman et al., 2002
). We performed an immunohistochemical study on Cx26 expression in developing neocortex of wild-type and GluR
2-/- mice. As shown in Figure 8
, in both types of mice Cx26 was widely expressed in developing cortical plate and no significant differences were found. This suggests that NMDAR does not regulate the expression of Cx26 at developing neocortex.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results have clearly shown that NMDA receptors are involved in the establishment of cell-to-cell interactions based on gap-junction channels, rather than potentiating intracellular calcium elevation driven by IP3. The frequency of occurrence of spontaneous neuronal domains was significantly reduced in GluR2-/- mouse sections, even though the elevation of [Ca2+]i in single cells was frequently observed in brain slices from GluR
2-/- mice (Fig. 5
). In addition, very few neuronal domains were observed following IP3 injection into single neuronal cells in GluR
2-/- mice brain slices, even though this injection resulted in the elevation of [Ca2+]i in the injected cell itself.
Kander and Katz (Kander and Katz, 1998) proposed that the propagation of calcium elevation in the neuronal domain is mediated by the passage of IP3 via gap-junction channels. Based on this scenario, the elevation of intracellular IP3 driven by G-protein activation occurs to the same degree in the GluR2-/- mouse neocortex as in the wild-type mouse, but the propagation of IP3 into the surrounding neurons via gap junctions is virtually absent. Taken together, the decreased frequency of appearance of neuronal domains (10% of that seen in wild-type sections) observed in GluR
2-/- mice suggests that the putative signal driven by the opening of the NMDA receptor channels plays a critical role in the regulation of the generation or the activation of gap-junction channels. Further studies need to be conducted to assess whether NMDA receptor would directly regulate the opening of gap-junction channels between immature neurons. Neurobiotin injection must be a first choice; therefore we have injected neurobiotin along with IP3 into developing cortical neurons from wild-type mice. Unfortunately, we have not been able to detect any dye coupling after the regular staining protocol.
Expression of gap-junction channels in the cortical plate of the mammalian cerebral neocortex has been confirmed in several studies (Peinado et al., 1993a,b
; Nadarajah et al., 1997
; Sohl et al., 1998
). Because gap-junction channels allow the passage of compounds of <1 kDa, as well as of ions, cells coupled via gap-junction channels are able to exchange cations such as sodium and potassium, or second messenger molecules such as calcium and IP3. These physiological events result in the electrical (Gutnick and Prince, 1981
; Deans et al., 2001
) or metabolic (Saez et al., 1989
; Boitano et al., 1992
) synchronization of the coupled cells.
With the above studies in mind, we attempted to analyze the expression of gap-junction channels in the developing neocortex of the GluR2-/- mouse in comparison to the wild-type mouse. Gap-junction channels contain Cx molecules (White and Paul, 1999
; Rörig and Feller, 2000
). In the developing neocortex, the expression of Cx26, Cx32, Cx36 and Cx43, has been reported (Nadarajah et al., 1997
; Naus and Bani-Yaghoub, 1998
; Sohl et al., 1998
; Bittman et al., 2002
). The neuron-specific gap-junction protein Cx36 was recently found and its sequential change of expression was investigated (Sohl et al., 1998
). The expression of Cx36 increases to a maximum up to 7 days postnatally and decreases thereafter (Al-Ubaidi et al., 2000
; Belluardo et al., 2000
; Bittman et al., 2002
). Cx26 is expressed in neuronal progenitor cells in the developing neocortex (Bittman and LoTurco, 1999
). As for Cx32, Cx36 and Cx43, the analysis is not possible at this stage because of the lack of availability of specific antibodies. We carried out an immunohistochemical study on Cx26 expression in the neocortex of wild-type and GluR
2-/- mice, but no significant differences were found (Fig. 8
). It is known that several other factors, such as acidification (pH), [Ca2+]i and protein-phosphorylation regulate the opening of gap-junction channels (Rörig and Feller, 2000
). The putative signal driven by NMDA receptor channels may induce the opening of gap-junction channels rather than promote the upregulation of gene expression of these gap-junction channels.
Our current study may indicate that the generation of neuronal domains could be separated into two parts. First comes the establishment of cell-to-cell interactions via gap-junction channels, presumably promoted by the signal driven by the opening of NMDA receptor channels. Then comes the execution of coordinated neural activity initiated by the activation of Gq protein as previously proposed (Kandler and Katz, 1998). Our study demonstrates an unexpected function of NMDA receptors, which act as inducers of the establishment of functional gap-junction channels in the developing cerebral cortex of the mammalian brain.
![]() |
Notes |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Address correspondence to Tatsuhiro Hisatsune, Department of Integrated Biosciences, The University of Tokyo, Bioscience Bldg 402, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. Email: hisatsune{at}k.u-tokyo.ac.jp.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Belluardo N, Mudo G, Trovato-Salinaro A, Le Gurun S, Charollais A, Serre-Beinier V, Amato G, Haefliger JA, Meda P, Condorelli DF (2000) Expression of connexin36 in the adult and developing rat brain. Brain Res 865:121138.[CrossRef][ISI][Medline]
Bittman K, Lo Turco J (1999) Differential regulation of connexin 26 and 43 in murine neocortical precursors. Cereb Cortex 9:188195.
Bittman K, Becker D, Cicirata F, Parnavelas J (2002) Connexin expression in homotypic and heterotypic cell coupling in the developing cerebral cortex. J Comp Neurol 443:201212.[CrossRef][ISI][Medline]
Boitano S, Dirksen ER, Sanderson MJ (1992) Intercellular propagation of calcium waves mediated by inositol trisphosphate. Science 258:292295.[ISI][Medline]
Chiaia N, Fish S, Bauer WR, Bennett-Clarke C, Rhoades RW (1992) Postnatal blockade of cortical activity by tetrodotoxin does not disrupt the formation of vibrissa-related patterns in the rats somatosensory cortex. Dev Brain Res 66:244250.[ISI][Medline]
Deans MR, Gibson JR, Sellitto C, Connors BW, Paul DL (2001) Synchronous activity of inhibitory networks in neocortex requires electrical synapses containing connexin36. Neuron 31:477485.[ISI][Medline]
Des Rosiers MH, Sakurada O, Jehle J, Shinohara M, Kennedy C, Sokoloff L (1978) Functional plasticity in the immature striate cortex of the monkey shown by the [14C]deoxyglucose method. Science 200: 447449.[ISI][Medline]
Flint AC, Liu X, Kriegstein AR (1998) Nonsynaptic glycine receptor activation during early neocortical development. Neuron 20:4353.[ISI][Medline]
Gutnick MJ, Prince DA (1981) Dye coupling and possible electrotonic coupling in the guinea pig neocortical slice. Science 211:6770.[ISI][Medline]
Henderson T, Woolsey T, Jacquin M (1992) Infraorbital nerve blockage from the birth does not disrupt central trigeminal pattern formation in the rat. Dev Brain Res 66:146152.[ISI][Medline]
Iwasato T, Datwani A, Wolf AM, Nishiyama H, Taguchi Y, Tonegawa S, Knopfel T, Erzurumlu RS, Itohara S (2000) Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature 406:726731.[CrossRef][ISI][Medline]
Kandler K, Katz LC (1998) Coordination of neuronal activity in developing visual cortex by gap junction-mediated biochemical communication. J Neurosci 18:14191427.
Kutsuwada T, Sakimura K, Manabe T, Takayama C, Katakura N, Kushiya E, Natsume R, Watanabe M, Inoue Y, Yagi T, Aizawa S, Arakawa M, Takahashi T, Nakamura Y, Mori H, Mishina M (1996) Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice. Neuron 16:333344.[ISI][Medline]
Lo Turco JJ, Blanton MG, Kriegstein AR (1991) Initial expression and endogenous activation of NMDA channels in early neocortical development. J Neurosci 11:792799.[Abstract]
Luhmann HJ, Prince DA (1991) Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 65:247263.
Mayer ML, Westbrook GL, Guthrie PB (1984) Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309:261263.[ISI][Medline]
Meister M, Wong R, Baylor D, Shatz C (1991) Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 252:939943.[ISI][Medline]
Miyakawa N, Uchino S, Yamashita T, Okada H, Nakamura T, Kaminogawa S, Miyamono Y, Hisatsune T (2002) A glycine receptor antagonist, strychnine, blocked NMDA receptor activation in the neonatal mouse neocortex. Neuroreport 13:16671673.[CrossRef][ISI][Medline]
Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529540.[ISI][Medline]
Mori H, Mishina M (1995) Structure and function of the NMDA receptor channel. Neuropharmacology 34:12191237.[CrossRef][ISI][Medline]
Nadarajah B, Jones AM, Evans WH, Parnavelas JG (1997) Differential expression of connexins during neocortical development and neuronal circuit formation. J Neurosci 17:30963111.
Naus CC, Bani-Yaghoub M (1998) Gap junctional communication in the developing central nervous system. Cell Biol Int 22:751763.[CrossRef][ISI][Medline]
Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307:462465.[ISI][Medline]
OLeary D, Ruff N, Dyck R (1994) Development, critical period plasticity, and adult reorganizations of mammalian somatosensory systems. Curr Biol 4:535544.
Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16:64146423.
Peinado A, Yuste R, Katz LC (1993a) Gap junctional communication and the development of local circuits in neocortex. Cereb Cortex 3:488498.[Abstract]
Peinado A, Yuste R, Katz LC (1993b) Extensive dye coupling between rat neocortical neurons during the period of circuit formation. Neuron 10:103114.[ISI][Medline]
Rabacchi S, Bailly Y, Delhaye-Bouchaud N, Mariani J (1992) Involvement of the N-methyl-D-aspartate (NMDA) receptor in synapse elimination during cerebellar development. Science 256:18231825.[ISI][Medline]
Rakic P (1976) Prenatal genesis of connections subserving ocular dominance in the rhesus monkey. Nature 261:467471.[ISI][Medline]
Rörig B, Feller MB (2000) Neurotransmitters and gap junctions in developing neural circuits. Brain Res Brain Res Rev 32:86114.[ISI][Medline]
Saez J, Connor J, Spray D, Bennett M (1989) Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions. Proc Natl Acad Sci USA 86:27082712.[Abstract]
Shatz C, Stryker MP (1988) Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents. Science 242:8789.[ISI][Medline]
Simon DK, Prusky GT, OLeary DD, Constantine-Paton M (1992) N-methyl-D-aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci USA 89:1059310597.[Abstract]
Sohl G, Degen J, Teubner B, Willecke K (1998) The murine gap junction gene connexin 36 is highly expressed in mouse retina and regulated during brain development. FEBS Lett 428:2731.[CrossRef][ISI][Medline]
Wainai T, Takeuchi T, Seo N, Mishina M (2001) Regulation of acute nociceptive responses by the NMDA receptor GluR2 subunit. Neuroreport 12:31693172.[CrossRef][ISI][Medline]
Watanabe M, Inoue Y, Sakimura K, Mishina M (1992) Developmental changes in distribution of NMDA receptor channel subunit mRNAs. Neuroreport 3:11381140.[ISI][Medline]
Weliky M, Katz LC (1997) Disruption of orientation tuning in visual cortex by artificially correlated neuronal activity. Nature 386: 680685.[CrossRef][ISI][Medline]
White TW, Paul DL (1999) Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61:283310.[CrossRef][ISI][Medline]
Wiesel TN, Hubel DH (1965) Comparison of the effects of unilateral and bilateral eye closure on cortical unit responses in kittens. J Neurophysiol 28:10291040.
Woolsey T, Wann J (1976) Areal changes in mouse cortical barrels following vibrissal damage at different postnatal ages. J Comp Neurol 170:5366.[ISI][Medline]
Yamada K, Hisatsune T, Uchino S, Nakamura T, Kudo Y, Kaminogawa S (1999) NMDA receptor mediated Ca2+ responses in neurons differentiated from p53-/- immortalized murine neural stem cells. Neurosci Lett 264:165167.[CrossRef][ISI][Medline]
Yuste R, Sur M (1999) Development and plasticity of the cerebral cortex: from molecules to maps. J Neurobiol 41:16.[CrossRef][ISI][Medline]
Yuste R, Peinado A, Katz LC (1992) Neuronal domains in developing neocortex. Science 257:665669.[Medline]
Yuste R, Nelson DA, Rubin WW, Katz LC (1995) Neuronal domains in developing neocortex: mechanisms of coactivation. Neuron 14:717.[ISI][Medline]