Role of pre- and postsynaptic activity in thalamocortical axon branching

Abstract

Axonal branching is thought to be regulated not only by genetically defined programs but also by neural activity in the developing nervous system. Here we investigated the role of pre- and postsynaptic activity in axon branching in the thalamocortical (TC) projection using organotypic coculture preparations of the thalamus and cortex. Individual TC axons were labeled with enhanced yellow fluorescent protein by transfection into thalamic neurons. To manipulate firing activity, a vector encoding an inward rectifying potassium channel (Kir2.1) was introduced into either thalamic or cortical cells. Firing activity was monitored with multielectrode dishes during culturing. We found that axon branching was markedly suppressed in Kir2.1-overexpressing thalamic cells, in which neural activity was silenced. Similar suppression of TC axon branching was also found when cortical cell activity was reduced by expressing Kir2.1. These results indicate that both pre- and postsynaptic activity is required for TC axon branching during development.

During development, axons navigate to their target regions and form elaborate branches when they make synaptic connections with multiple target cells. It has been demonstrated that axon guidance to the target region is regulated by attractive and repulsive molecular cues that are expressed in particular spatiotemporal patterns (1). Similar molecular mechanisms are thought to influence axon branching (2). In addition, neural activity such as firing and synaptic activity can also affect branch formation (3–5). An intriguing and unanswered problem is how neural activity regulates axonal branching.

The thalamocortical (TC) projection in the mammalian cortex is a well-characterized system in which to investigate activity-dependent axon branching. In the developing sensory cortices, TC axons form elaborate terminal arbors, whose size and complexity are altered by neural activity. In the primary visual cortex of higher mammals such as cats, ferrets, and monkeys, TC axons serving left and right eyes are segregated into eye-specific stripes (6). This segregation is established during development (7), but is disrupted by blockade of retinal activity (8, 9). Regarding individual axon arbors, it is known that after monocular deprivation, TC axons serving the deprived and nondeprived eyes shrink and expand their arbors, respectively (10, 11). A recent study in which monocular deprivation was combined with silencing cortical activity has further suggested that correlations between pre- and postsynaptic activity play a dominant role in segregation of axon arbors (12). In accordance with this view, molecular machinery in pre- and postsynaptic sites has also been shown to affect arbor formation of TC axons in the somatosensory cortex (13–15). However, the relative role of pre- and postsynaptic activity in TC axon branching remains unclear.

To address this issue, we investigated TC axon branching in cocultures of the thalamus and cortex by manipulating the firing activity of thalamic (presynaptic) and cortical (postsynaptic) cells. To do this, neural activity of either thalamic or cortical cells was selectively silenced by means of overexpression of Kir2.1, an inward rectifying potassium channel (16, 17). Our findings suggest that TC axon branching is inhibited by reducing the activity at either of these locations, suggesting that both pre- and postsynaptic activity is required for the development of TC axon branching.

Results

TC axon branching was studied in cocultures of the thalamus and cortex by introducing a plasmid encoding enhanced yellow fluorescent protein (EYFP) into thalamic cells. Spontaneous firing activity was monitored with multielectrode dishes (MED) during culturing (Fig. 1A). As previously shown (18), spontaneous firing (mostly field potential-like activity) developed during the second week in vitro in both thalamic and cortical cells (1.34 ± 0.45 Hz in the cortical explant, n = 4; 0.96 ± 0.32 Hz in the thalamic explant, n = 4; Fig. 1 C and H and Table 1). Furthermore, cortical activity was highly correlated with thalamic activity (Fig. 1C and Fig. S1). Most of the negative potentials measured in the cortex were synchronized with thalamic potentials (85.7 ± 8.5%, n = 4). In the presence of such spontaneous activity, most EYFP-labeled axons formed elaborate branches (the number of branch points, 13.2 ± 2.5, n = 17) in cortical explants after 2 weeks in vitro (Table 1; see also Figs. 2A and 3A). In contrast, TC axons formed few branches (2.2 ± 0.6, n = 17) in cocultures where all firing activity was blocked by tetrodotoxin (TTX) application during the second week in vitro (Table 1 and Fig. S2) (18).

Fig. 1.

Suppression of spontaneous firing in Kir2.1-overexpressing thalamic cells in vitro. (A) Cocultures of thalamic and cortical slices on the MED after 2 weeks in culture. (B) Kir2.1 was expressed together with EYFP in thalamic neurons in a coculture. (C) The upper and lower traces show extracellular recording from cortical and thalamic explants, respectively, in a normal coculture preparation. In C, 85% of cortical cell firing is synchronized with thalamic cell firing with a delay of ≈4 ms (estimated by cross-correlation analysis; see Materials and Methods). (D–F) Immunohistochemistry of Kir2.1-overexpressing thalamic cells. EYFP-labeled cells (D) were immunostained with anti-FLAG antibody (E). (F) A merged image. (G) Perforated-patch recording from intact (Upper) and transfected (Lower) thalamic cells. (H) Quantitative analysis of spontaneous firing activities of cortical, thalamic, and Kir2.1-overexpressing thalamic cells. The numbers in parentheses represent the number of cultures tested. CTX, cortex; Th, thalamus; KirTh, Kir2.1-overexpressing thalamus. (Scale bar: A and B, 500 μm; D–F, 20 μm.)

Table 1.

Branching aspects of TC axons and firing activity of thalamic and cortical cells

	Th→CTX	KirTh→CTX	mKirTh→CTX	Th→KirCTX	Th→KirCTX (tri)	Th→CTX (tri)	Th→CTX (TTX)
	(na = 17, nc = 7)	(na = 14, nc = 7)	(na = 9, nc = 6)	(na = 12, nc = 5)	(na = 12, nc = 4)	(na = 7, nc = 3)	(na = 17, nc = 9)
Branch points	13.2 ± 2.5	2.9 ± 0.7*	12.2 ± 3.4	5.3 ± 1.4†	5.8 ± 1.6†	11.9 ± 3.9	2.2 ± 0.6*
Total length, mm	2.14 ± 0.22	1.59 ± 0.21	2.84 ± 0.57	1.77 ± 0.24	1.55 ± 0.16	2.48 ± 0.65	1.22 ± 0.11†
Density	5.4 ± 0.7	2.1 ± 0.3*	4.0 ± 0.5	3.1 ± 0.4†	3.7 ± 0.7	4.7 ± 0.8	2.4 ± 0.3*
Tip length, μm	61.8 ± 7.0	268 ± 54*	121 ± 19	141 ± 39	109 ± 27	119 ± 21	244 ± 52*
	(nc = 4)	(nc = 2)		(nc = 4)	(nc = 5)	(nc = 5)	(nc = 2)
Th activity, Hz	0.96 ± 0.32	1.03 ± 0.20	N.D.	0.27 ± 0.09	0.81 ± 0.11	0.81 ± 0.11	0.0 ± 0.0
		0.017 ± 0.01‡
CTX activity, Hz	1.34 ± 0.45	1.21 ± 0.10	N.D.	0.17 ± 0.04	0.61 ± 0.14	1.40 ± 0.30	0.0 ± 0.0

Each value represents the mean and SEM. For branching, cross-comparison analysis shows no significant difference between KirTh→CTX, Th→KirCTX, Th→KirCTX (tri), and Th→CTX (TTX). Likewise, there is no significant difference between Th→CTX, mKirTh→CTX and Th→CTX (tri). CTX, cortex; Th, thalamus; KirTh, Kir2.1-overexpressing thalamus; mKirTh, mutant Kir2.1-overexpressing thalamus; KirCTX, Kir2.1-overexpressing cortex; (tri), the triple culture; (TTX), TTX treatment; n_a, the number of axons sampled; n_c, the number of cultures.

*P < 0.01.

^†P < 0.05 (Dunnett's test).

^‡This value was obtained from Kir2.1-expressing thalamic cells (n = 3) by whole-cell patch recording.

Fig. 2.

Control and Kir2.1-overexpressing thalamic axons in coculture preparations. Thalamic cells were electroporated with eyfp (A) or with eyfp + kir2.1 (B) after 1-2 days in culture. Labeled TC axons were observed in the cocultured cortical explants after 14 days in culture. (C) TC axons transfected with eyfp and kir2.1were observed after 9 days in culture. Contrast is reversed. (Scale bar: A, 100 μm.) (A–C) Interrupted lines indicate the pial surface of cortical explant. Arrows indicate the presumed layer 4 boundaries.

Fig. 3.

Decreased axonal branching in Kir2.1-overexpressing thalamic neurons. (A) TC axon branching in normal cocultures of the thalamus and cortex after 2 weeks in culture. (B) Axon branching of Kir2.1-overexpressing thalamic neurons in the cortex. Arrows indicate the presumed layer 4 boundaries. CTX, cortex; Th, thalamus; KirTh, Kir2.1-overexpressing thalamus.

We then studied the influence of pre- and postsynaptic activity on TC axon branching by specifically reducing neural activity in either thalamic or cortical cells. First, axon branching was examined in Kir2.1-overexpressing thalamic cells. To observe axonal extension, the eyfp vector was cotransfected with a kir2.1 expression vector into thalamic cells (Fig. 1B). Immunohistochemistry with an antibody against a peptide tag indicated that more than 95% of EYFP-labeled cells expressed Kir2.1 protein (Fig. 1 D–F). In most cases, up to 20 labeled cells (less than 0.2% of the total number of thalamic cells) were sparsely distributed in the thalamic explant (Fig. 1B). Whole-cell perforated-patch recordings showed that spontaneous action potential firing was dramatically diminished in Kir2.1-overexpressing thalamic cells (0.017 ± 0.01 Hz, n = 3; Fig. 1 G and H and Table 1), with hyperpolarized membrane potentials (Fig. S3A). In addition, membrane resistance was substantially decreased (Fig. S3A), which may be attributable to an increase in conductance caused by overexpression of the potassium channel. However, excitatory postsynaptic responses were evoked readily in Kir2.1-overexpressing thalamic cells when recurrent corticothalamic fibers were stimulated electrically (Fig. S3B). The morphological features of these transfected cells were also similar to those of intact thalamic cells (Fig. S4 A, B, E, and F). Thus, it is unlikely that the basic aspects of cellular morphology and the appearance of corticothalamic synaptic transmission are altered by Kir2.1 overexpression. Furthermore, overall firing activity in cortical and thalamic explants was not affected by introducing kir2.1 into a small number of thalamic cells (Fig. S3C and Table 1).

Under these conditions, TC axon branching was investigated after 2 weeks in culture. Kir2.1-overexpressing TC axons extended into the cortical explant with little branching (Fig. 2 A and B). The number of branch points was roughly one-fourth in Kir2.1-overexpressing cells (2.9 ± 0.7, n = 14), compared with control (Fig. 3 A and B and Table 1). The branch density (the number of branch tips per the total branch length) was also much decreased (Table 1). Conversely, branch tip length was significantly increased by Kir2.1 overexpression (Table 1). In contrast, these parameters including branch points (12.2 ± 3.4, n = 9) were unchanged in cells that expressed a mutant Kir2.1 lacking rectification (Table 1 and Fig. S5). Thus, TC axon branching was markedly reduced by silencing thalamic cell activity.

We also observed Kir2.1-expressing TC axons at earlier developmental stages. They grew extensively in the cortical explant, and some of them traveled with growth cones (Fig. 2C). This aspect was similar to that for normal TC axons in vitro (18, 19), which indicates that axonal growth is not influenced by Kir2.1 overexpression. It is also unlikely that overproduced branches are later eliminated.

Branch formation was further studied in cocultures where cortical cell activity was suppressed. As TC axons form connections with multiple target cells in vitro as well as in vivo, we attempted to overexpress Kir2.1 in numerous cortical cells. To achieve this, kir2.1 and dsred were cotransfected into the cells that are destined to become layer 4, using in utero electroporation. After birth, cortical slices containing DsRed-positive cells were dissected and cocultured with a thalamic slice (Fig. 4A). To label TC axons, the eyfp vector was electroporated to thalamic neurons at 1 day in vitro, as described.

Fig. 4.

Spontaneous activity of thalamic and cortical cells in cocultures or triple cultures containing Kir2.1-overexpressing cortex. (A) Coculture of the thalamus with the cortical slice where Kir2.1 plus DsRed were overexpressed before culturing by in utero electroporation. Thalamic and cortical explants were placed on an MED (Top). The same cultures were observed by fluorescence microscopy (Bottom) after 2 weeks in culture. (B) Similar to A, with the exception that a thalamic explant was sandwiched with between a Kir2.1-overexpressing and an untransfected cortical explant on the MED. (C) The upper two traces show the extracellular recording from cocultures of the thalamic explant and Kir2.1-overexpressing cortical explant. The lower three traces correspond to the extracellular potentials from the triple culture. (D) Relative firing activity (normalized by the average firing frequency of normal thalamic or cortical activity) in thalamic and Kir2.1-overexpressing explants in the cocultures (Left). Similarly, the relative activities of thalamic explants, Kir2.1-overexpressing cortical explants and additional cortical explants are shown for the triple culture (Right). The numbers in brackets represent the numbers of samples tested. (Scale bar: A and B, 1 mm.)

After 2 weeks in vitro, more than a thousand DsRed-labeled cells (1,000–2,000 cells, roughly 4–8% of the total number of cortical cells) were primarily found in the upper cortical layers (Fig. 4A and Fig. S4H). Kir2.1-overexpressing cortical cells had slightly hyperpolarized membrane potentials and smaller membrane resistances, as was the case for thalamic cells (Fig. S3A; see also Fig. S5). Postsynaptic responses evoked by thalamic stimulation were also examined in Kir2.1-expressing cortical cells. The amplitude of excitatory postsynaptic currents was 3× smaller than in naïve cortical explants (Fig. 5; see also Fig. S3D), but was not different from that in nonexpressing cells in kir2.1-transfected cortical explants (Fig. 5). This suggests that basic synaptic properties are affected by the silencing effect of kir2.1 rather than by transfection itself. The cellular morphology was not obviously different from that of normal cortical cells (Fig. S4 C, D, E, and G), although the possibility cannot be ruled out that Kir2.1 expression affects fine morphology (20).

Fig. 5.

Postsynaptic responses in kir2.1-transfected cortical explants. Postsynaptic currents were recorded in control (A) and Kir2.1-expressing (B) cells in the same kir2.1-transfected cortical explants by stimulating the thalamic explant. The current was also recorded in cortical cells (naïve control) in normal cocultures (C). The peak amplitudes of EPSCs were plotted against stimulus intensity (D). All electrophysiological experiments were performed after 13–15 days in culture. *P < 0.05 (Dunnett's test).

We found that TC axon branching was markedly decreased in cocultures of kir2.1-transfected cortex with thalamus (branch points, 5.3 ± 1.4, n = 12; Table 1). The frequency of spontaneous activity was decreased considerably in kir2.1-transfected cortical explants (0.17 ± 0.04 Hz, n = 4; Fig. 4 C and D and Table 1). The silenced cells, which were densely distributed in the upper layers (10–20% of upper-layer cells; Fig. S4 H and I), are thought to impair the emergence or spreading of field potential-like firing activity by suppressing synchronous firing, which may be generated by local circuits consisting of subsets of cortical neurons (21–23). As a consequence, firing activity was also suppressed in thalamic explants (0.27 ± 0.09 Hz, n = 4; Fig. 4 C and D and Table 1). This may be attributable to a significant reduction in excitatory transmission from the cortex to the thalamus, as spontaneous firing originated from the cortical explant more frequently (Fig. S1) (24).

To make thalamic cells active, another cortical slice was added to the coculture (Fig. 4B). Consequently, firing activity in the thalamus was restored substantially in the triple cultures (0.81 ± 0.11 Hz in the thalamus of the triple culture, n = 5; 0.96 ± 0.32 Hz in the thalamus of the normal coculture, n = 4; Fig. 4 C and D and Table 1). In contrast, spontaneous firing remained low in Kir2.1-transfected cortical explants (0.61 ± 0.14 Hz, n = 5), compared with firing activity in normal cocultured cortex (1.34 ± 0.45 Hz, n = 4) and the additional cortex in the triple cultures (1.40 ± 0.30 Hz, n = 5; Fig. 4 C and D and Table 1). In the triple cultures, TC axons did not form extensive branches in the Kir2.1-transfected cortex (Fig. 6A). Quantitative analysis confirmed that the number of branch points was significantly smaller (5.8 ± 1.6, n = 12) for TC axons invading the Kir2.1-trasnsfected cortex than control, although the total branch length and the density were not statistically different (Table 1). In contrast, TC axons invading the intact cortical explant in the triple culture formed branches in a fashion (11.9 ± 3.9, n = 7) similar to that in control (Fig. 6B and Table 1). Thus, axonal branching was decreased in the more silent cortex, which indicates that cortical cell activity is also required for TC axon branching.

Fig. 6.

TC axon branching in Kir2.1-overexpressing and intact cortical explants in the triple cultures. TC axon branching was reduced in Kir2.1-overexpressing cortical explants (A) but not in intact cortical explants (B). Th, thalamus; CTX (tri), intact cortex in the triple culture; KirCTX (tri), Kir2.1-overexpressing cortex in the triple culture. All TC axons were observed after 2 weeks in culture. Arrows indicate the presumed layer 4 boundaries.

Discussion

The present findings show that TC axon branching is impaired in coculture preparations, where either thalamic or cortical cell activity is silenced by Kir2.1 overexpression. Together with the finding that axon branching is reduced in the presence of TTX (Fig. S2) (18), it is likely that both pre- and postsynaptic activity is required for normal TC axon branching (Fig. 7; see also Table 1).

Fig. 7.

Relation between the number of branch points and firing activity. Firing activities of thalamic and cortical explants are shown in X and Y axes, respectively. The number of branch points in each culture type is shown on the z axis. CTX, cortex; Th, thalamus; KirTh, Kir2.1-overexpressing thalamus; KirCTX, Kir2.1-overexpressing cortex; (tri), triple culture; (TTX), TTX treatment.

The finding that branch elaboration is promoted only by coactivation of pre- and postsynaptic cells may not fit the case of axon arbor remodeling in eye-specific projections of the visual cortex. Indeed, elaborate axon arbors are observed in the cat visual cortex when both pre- and postsynaptic cells are silent (12, 25). This is also true in the remodeling of axon arbors in the retinotectal projection (26, 27). In contrast, TC axons did not form elaborate branches in TTX-treated cocultures and in cocultures of the thalamus and Kir2.1-expressing cortex, in which both pre- and postsynaptic activity was suppressed. This is consistent with the features of geniculocortical axons observed at the early developmental stages in TTX-infused visual cortex (28). Therefore, the mechanism that underlies initial arbor formation seems to be different from those regulating plastic changes or maintenance in later developmental stages.

Previous studies using neuronal silencing with Kir2.1 have suggested that presynaptic activity plays a dominant role in axon branching (27, 29, 30). In particular, competitive interactions between a retinal ganglion cell and adjacent cells have been suggested to be crucial in retinotectal axon branching (27). However, it is unlikely that the same mechanism acts on branch formation of TC axons. When kir2.1 was introduced into a larger number of thalamic cells, the labeled axons still formed few branches in the cortical explant (Fig. S6). In accordance with this, recent evidence suggests that competition among presynaptic axons may be less important than homosynaptic mechanisms for the elaboration of eye-specific geniculocortical axon arbors (12).

A characteristic aspect of our cultures was that thalamic and cortical cell firing was highly synchronized (Fig. 1C and Fig. S1). Our previous study using the same coculture preparations showed that synchronized firing activity between thalamic and cortical cells develops during the second week in vitro, when axonal branches are added in great numbers (18). Thus, precise synchrony rather than the overall amounts of pre- and postsynaptic activity for a given period may play an important role in the promotion of axon branching (18, 31–34), although our investigations did not distinguish between these two possibilities. The NMDA receptor, a coincidence detector of pre- and postsynaptic activity, is likely to be involved in this process (13, 35, 36). Indeed, a large NMDA receptor-mediated component was generated in cortical cells by thalamic stimulation (Fig. S7), as found in the developing sensory cortices (37–39). In addition, the fact that the AMPA/NMDA ratio was lower in the silenced cortical explants than in naïve ones implies that the normal development of the AMPA component might also be related to branch formation (Fig. S7).

There are several possible mechanisms to account for how coactivation of pre- and postsynaptic elements might underlie the axonal morphological changes. A plausible mechanism is that a retrograde signal may induce branches in growing axons. The retrograde substance could be released from postsynaptic cells when both pre- and postsynaptic elements are coactivated. Neurotrophins are candidate molecules for this process (40), as they promote thalamic axon growth in vitro (41). Similarly, the expression of branch-promoting and inhibiting molecules (2, 42) synthesized by postsynaptic cortical cells may be regulated by electrical activity. Activity-dependent expression of receptor molecules for these ligands may also be crucial. For example, Eph expression in motor axons is altered by their firing activity (43). It has also been shown that activity blockade inhibits axon responsiveness to ephrin-As and leads to the disruption of topographic mapping in the retinotectal projection (44). Finally, regulation of cytoplasmic signaling such as that driven by NMDA receptor activation may also be specifically involved in activity-dependent processes (45, 46).

In summary, TC axon branching was promoted only when both thalamic and cortical cells were coactivated in vitro. This suggests that coactivation of pre- and postsynaptic elements not only strengthens synaptic plasticity but also induces structural changes in cortical circuits during development.

Materials and Methods

A schematic timeline of the present experiments, including culture preparations, gene transfer, electrophysiological recording, and anatomical observation, is provided in Fig. S8.

Organotypic Slice Culture.

All experiments were performed according to the guidelines established by the animal welfare committees of Osaka University. Cocultures of the cortex with the thalamus were prepared as described previously (47). In brief, the dorsal thalamic region was dissected from E15 rat embryos (Sprague-Dawley), and cortical slices were dissected from sensory cortices of postnatal day (P)1 or P2 rats. The thalamic and cortical slices were plated on a membrane filter (Millicell-CM PICMORG50; Millipore), which was coated with rat tail collagen. To monitor neural activity, cortical and thalamic explants were plated on MEDs (Alpha MED Sciences) (18). The culture medium consisted of a 1:1 mixture of DMEM and Ham's F-12 (Invitrogen) with several supplements (47). These cultures were maintained at 37 °C in an environment of humidified 95% air and 5% CO₂.

Preparation of Plasmids and Gene Delivery into Cultured Cells.

The coding region of eyfp was cloned into a pCAGGS vector (48). Rat kir2.1 cDNA (GenBank accession no. NM017296, Ensemble ENSRNOG00000004720) was obtained by RT-PCR with primers (forward; 5′-TTCTAAAGCAGAAACACTGG-3′, reverse; 5′-CATCAGACTGTGTAGCGA -3′) and P2 rat brain cDNA. The product was further processed by PCR (forward primer; 5′-GCTCGAGGAAGCATGGGCAGTGTGCGT-3′, reverse primer; 5′-GAATTCCTACTTGTCGTCATCGTCTTTGTAGTCTACTATCTCCGATTCTCGCCT-3′) to add FLAG tag (underlined) to the carboxyl terminus. The PCR product was inserted into the pCAGGS vector. To generate nonconducting kir2.1 mutant, the pore-region amino acid motif GYG was mutated to AAA (16). Cotransfection of pCAGGS-eyfp and pCAGGS-kir2.1 (or pCAGGS-kir2.1 mutant) was performed for organotypic coculture preparations to examine the detail of axon morphology.

Electroporation with glass microelectrodes was performed after 1–2 days in culture to introduce these vectors into thalamic cells (18, 49). In brief, the plasmid solution of pCAGGS-eyfp (1–2 μg/μL) or a mixture of pCAGGS-eyfp (1–2 μg/μL) + pCAGGS-kir2.1 (4–5 μg/μL) was pressure ejected onto the surface of the explants with a glass micropipette, and electrical pulses (10 trains of 200 square pulses of 1 ms duration, 200 Hz, 500 μA) were applied with another glass microelectrode.

In Utero Electroporation.

In utero electroporation was performed to overexpress Kir2.1 in the cortical cells that are destined to become layer 4 cells (50, 51, 52). Pregnant rats at E16 were deeply anesthetized with Nembutal. The abdomen was surgically opened without opening the uterus itself. A mixture of pCAGGS-dsred (2 μg/μL) and pCAGGS-kir2.1 (2 μg/μL) was injected into one cerebral vesicle. Platinum electrodes were positioned beside the uterus, and square pulses (30 V; 50 ms) were delivered five times with electroporator (CUY20; NepaGene). After electroporation, embryos were allowed to develop until birth.

Confocal Imaging and Quantitative Analysis.

EYFP-labeled TC axons were observed by confocal microscopy (MRC-600; Bio-Rad) (18), with an argon laser and a filter set (excitation, 488 nm; emission long-pass filter, 515 nm). DsRed-positive cells were observed with another filter set (excitation, 514 nm; emission long-pass filter, 565 nm). EYFP-labeled axons were easily distinguished with this filter set even when DsRed-labeled cells were present in cortical explants. Images were collected with 10× and 40× objective lenses (768 × 512 pixels for 1,097 × 731 μm or 274 × 183 μm) at 1- to 5-μm steps to obtain the entire axon arbors (2–20 optical sections). Individually distinguishable axons were drawn using National Institutes of Health image custom-made macros (a generous gift from Edward Ruthazer, Montreal Neurological Institute, McGill University, Montréal, Canada). Small processes (<5 μm) were excluded from analysis. Quantitative analysis, including the number of branch points, axonal tip length, total branch length, and branch density (the number of branches/total branch length), were performed. Statistical evaluation was performed by means of one-way ANOVA followed by Dunnett's post hoc test.

Analysis of Cellular Morphology and Cell Counts.

To study cellular morphology and the number of labeled cells, images were taken with a 10× or 20× objective lens. To determine the total number of cells in thalamic and cortical slices, cultures were cut into 20-μm sections, followed by Nissl staining. Then, the ratio of the number of labeled cells to the total number of cells was estimated for each cortical or thalamic explant. Indeed, the percentage was estimated to be 3–6%. The ratio of Kir2.1-expressing cortical cells was also evaluated by dissociating cortical slices, which had been transfected with dsred + kir2.1 by in utero electroporation. The cortical slices were dissected at P1, and were incubated in Ca²⁺ Mg²⁺-free Hanks’ solution containing trypsin (0.25%). After dissociation, the number of labeled cells and the total number of dissociated cells were counted. The percentage was estimated to be 5–10% by this method. Finally, the ratio of Kir2.1-expressing cells was evaluated as the average of the values obtained by the both methods.

Immunohistochemistry.

Cultured slices were fixed with 4% paraformaldehyde followed by extensive washes with PBS. The slices were incubated overnight at 4 °C with anti-FLAG mAb (1:1,000, ANTI-FLAG M2; Sigma). The fluorescence signal was visualized by donkey anti-mouse IgG-Cy3 (1:200; Sigma).

Recording of Spontaneous Activity.

To examine spontaneous activities of cortical and thalamic cells, extracellular recording was performed on MED (interpolar distance, 0.3 mm; input impedance with the preamplifier >1 MΩ), as described previously (18). In brief, extracellular voltages were recorded at several locations for more than 5 min every day. Negative potentials with amplitudes above a set threshold (1.5× the maximal amplitude of the baseline noise) were counted as spikes with Axograph software (Axon Instruments). Firing activity of each explant was represented as an average of the firing rates during 11–14 days in culture. The synchronism between spontaneous thalamic and cortical activities was defined as the percentage of thalamic firing that time-overlapped with cortical firing during the observation time (5 min). Cross-correlation analysis was performed to examine the time difference between thalamic and cortical potentials, using Axograph software.

To measure spontaneous activity from individual Kir2.1-overexpressing cells in the cocultures, whole-cell perforated-patch recordings were performed under current-clamp mode using an upright microscope (BX51WI; Olympus). The pipette solution consisted of (in mM) 140 D-gluconate potassium salt, 10 NaOH, 10 Hepes, 8 MgCl₂; pH was adjusted to 7.35 with HCl; 0.005 volume dimethyl sulfoxide solution of amphotericin B (0.2 mg/mL) was added before recordings. The recording chamber (culture dish) was perfused at a rate of 1–2 mL/min with a saline whose composition was (mM) 123 NaCl, 4.2 KCl, 10 Hepes, 10 D-glucose, 2 CaCl₂, and 1 MgCl₂ (pH 7.35).

Recording of Synaptic Currents.

To obtain synaptic currents from thalamic and cortical cells, whole-cell recordings were performed. All experiments were carried out at 32 °C. Resistances of patch pipettes were 2–3 MΩ when filled with an intracellular solution composed of (in mM) 60 CsCl, 10 Cs D-gluconate, 20 TEA-Cl, 20 BAPTA, 4 MgCl₂, 4 ATP, and 30 Hepes (pH 7.3, adjusted with CsOH). The pipette access resistance was compensated by 70%. The composition of the standard bathing solution was (in mM) 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled with 95% O₂ and 5% CO₂. Picrotoxin (100 μM) was always added to block inhibitory synaptic transmission. Electrical stimulation (duration, 0.1 ms; amplitude, 0–50 μA) to the cortical and thalamic explants was applied with bipolar tungsten electrodes (interpolar distance, 0.5 mm).