Developmental Changes in Trial-to-Trial Variations in Whisker Barrel Responses Studied Using Intrinsic Optical Imaging: Comparison Between Normal and De-Whiskered Rats

Itaru Yazawa,1 Shinichi Sasaki,1 Hiraku Mochida,1 Kohtaro Kamino,3,4 Yoko Momose-Sato,1,2 and Katsushige Sato1

 1Department of Physiology and  2Department of Neurobehavioral Medicine, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Tokyo 113-8519;  3Hayashibara Biochemical Laboratories, Okayama 701-0221; and  4JAIC College of Medical-Care and Welfare Technology, Fukushima 963-8834, Japan


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Yazawa, Itaru, Shinichi Sasaki, Hiraku Mochida, Kohtaro Kamino, Yoko Momose-Sato, and Katsushige Sato. Developmental Changes in Trial-to-Trial Variations in Whisker Barrel Responses Studied Using Intrinsic Optical Imaging: Comparison Between Normal and De-Whiskered Rats. J. Neurophysiol. 86: 392-401, 2001. We used an intrinsic optical imaging technique to examine postnatal developmental changes in the rat barrel response to a single whisker movement. We compared the optical response patterns between control and de-whiskered rats, from which whiskers were removed except for the D1 whisker just after birth. Barrel responses were evoked by D1-whisker movement stimulation, and the intrinsic optical signals were detected from the somatosensory cortex through the dura mater. In the control rats, the area of the barrel response decreased gradually as postnatal development proceeded from 2 to 7 wk, until reaching the adult pattern. On the other hand, in the de-whiskered rats, the barrel response area did not change during development and showed a larger size than in the control rats. We also compared the trial-to-trial variations in the barrel responses between the two groups. In the control rats, trial-to-trial variations in the optical responses were observed under the same conditions of whisker stimulation, and the extent of the variations decreased with postnatal development up to 7 wk. In the de-whiskered rats, trial-to-trial variations were also observed, but the extent was larger and unchanged during development. In both groups, the positions of the response area were the same with respect to the bregma. These results suggest that the decrease in the area and variations in the optical responses are caused by interactions of the corresponding whisker barrel with neighboring barrels and that these interactions are necessary for the developmental stabilization of the intracortical horizontal connections, which are widespread and have high plasticity in early postnatal periods.


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The posteromedial barrel subfield (PMBSF) of the rodent somatosensory cortex is an interesting model for studying cortical functional organization because each whisker is represented in a one-to-one fashion by a barrel, a discrete aggregate of neurons in layer IV of the contralateral PMBSF. The barrel was first described in the mouse somatosensory cortex by Woolsey and Van der Loos (1970), and many important findings have been demonstrated using anatomical and electrophysiological techniques (for reviews, see Moore et al. 1999; White and Tracy 1995).

The optical imaging technique for intrinsic signals has been developed and used to monitor neuronal activities in the cerebral cortex of in vivo preparations (Chapman et al. 1996; Gödecke and Bonhoeffer 1996; Grinvald et al. 1986). The optical method has proven to be a useful technique for monitoring neural responses to whisker stimulation and offers advantages for studying the functional organization of sensory representation in the barrel cortex (Blood et al. 1995; Dowling et al. 1996; Masino et al. 1993; Peterson and Goldreich 1994; Polley et al. 1999). Recent investigations have indicated that there are at least three components in the intrinsic optical signals (Bonhoeffer and Grinvald 1995; Frostig et al. 1990; Malonek and Grinvald 1996). The first component originates from activity-dependent changes in the oxygen saturation level of hemoglobin. The second component originates from changes in blood volume that are probably due to dilation of venules in an area containing electrically active neurons. The third component arises from light-scattering changes that accompany cortical activation caused by ion and water movement, expansion and contraction of extracellular spaces, capillary expansion, or neurotransmitter release (Momose-Sato et al. 1998; Obaid et al. 1989; Salzberg et al. 1985; Sato et al. 1997; also for a review, see Cohen 1973).

In our previous study (Tanaka et al. 2000), we reported that trial-to-trial variations are often observed in optical responses to the same whisker deflection stimulus and that such variability across trials cannot be controlled. Is there any physiological importance in the trial-to-trial variations? Does the variability reflect some features of the functional organization of the whisker-barrel somatosensory system? To find some clues for these questions, we analyzed developmental changes in the intrinsic optical signals evoked by repeated mechanical deflections of a single D1-whisker and compared the response patterns between control and de-whiskered rats.

Preliminary results have appeared in abstract form (Tanaka et al. 1999; Yazawa et al. 2000).


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Subjects

Male Wistar rats were divided into two groups according to whether or not they underwent sensory deprivation at birth (postnatal day 0: P0). Group I (control rats) involved the study of normal whisker-barrel development, and 35 rats [20-350 g, 2- to 7-wk postnatal and adult (older than 9 wk)] were used to detect D1 barrel responses. Group II (de-whiskered rats) involved the study of changes in whisker barrel development in de-whiskered conditions, and 15 rats (85-160 g, 5- to 7-wk postnatal ages) underwent sensory deprivation as described in the next section. All the rats in both groups were housed in cages in 12 h light/12 h dark cycles with food and water ad libitum.

Sensory deprivation

At birth, Group II rats had all whiskers plucked except the D1 whisker on the right side of the snout. The plucking was done by carefully placing steady tension on the base of the whisker with fine forceps under cooling anesthesia. All regrowing whiskers were replucked until the experimental days. The experimental protocol is summarized in Fig. 1.



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Fig. 1. Schematic drawings of the experimental protocol. The de-whiskered rats had all whiskers except the D1 whisker plucked at birth and had all regrowing whiskers replucked until the experimental days. The control rats had all whiskers except the D1 whisker cutoff just before experiments.

Anesthesia and surgical procedure

All rats were anesthetized initially with urethan (initial dose: 1.4 g/kg ip). The heart rate and respiratory rate were continuously monitored, and body temperature was maintained at 37°C with a heating blanket (KN-474, Natsume Seisaku-sho, Tokyo). Tail pinch and eyeblink reflexes were assessed, and additional supplements of urethan were administered as needed to ensure a satisfactory areflexive state.

After reaching a surgical level of anesthesia, each animal was prepared for surgery. Each rat was mounted in a stereotaxic instrument using blunt ear bars (SR-50, Narishige Scientific Instrument Laboratory, Tokyo). A plastic chamber was affixed to the surface of the skull with dental cement, and the skull overlying the left somatosensory area was removed using a dental drill. The chamber was filled with silicon oil (KE106, Shin-etsu Chemical, Tokyo).

Whisker stimulation

For both groups, all the whiskers except the D1 whisker were cutoff just before the experiments. The right D1 whisker was deflected with a modified galvanometer. The stimulus, consisting of 10 pulses, was delivered at 5 Hz for 2 s with an interstimulus interval of 20 s. Each pulse was a 10-mm deflection of the D1 whisker in a rostral-to-caudal fashion at a position of 15 mm from the root of the whisker, corresponding to an angle of 1.5°. These stimulation conditions produced the maximal optical responses in the rat somatosensory cortex, as described earlier (Tanaka et al. 2000).

Intrinsic optical imaging

The left somatosensory cortex was illuminated using a fiber-optic system driven by a stable DC power supply (Model 66184, Oriel, Darmstadt, Germany). A light was passed through interference filters of different wavelengths. The filter used for visualizing the surface of the cortex, and its vascular pattern had a transmission maximum at 540 ± 30 nm, and the filter used for intrinsic imaging had a passband at 605 ± 5 nm (Asahi Spectra, Tokyo). The depth of focus of the "macroscope" (Ratzlaff and Grinvald 1991) was set to 500 µm under the cortical surface by moving the focal plane of the tandem lens along the z axis (dorsal-ventral). The maximal responses were obtained from this depth of focus for all ages (Tanaka et al. 2000). Intrinsic imaging was performed using a differential video acquisition system, IMAGER 2001 (Optical Imaging, Germantown, NY). During each trial, eight optical images were collected over 5.0 s and stored on a computer with data-acquisition software, VDAQ (Optical Imaging). The whisker was first stimulated for 2 s at the same time as data acquisition. The optical response peaked around 1.4 s after the onset of whisker stimulation, and we used the maximal response image for data analysis (Tanaka et al. 2000). Optical reflectance images were represented by the fractional change (Delta R/R) to correct for uneven illumination using a data-analyzing software program, TVMix (Optical Imaging).

Quantitative measurement of the D1-whisker barrel responses

To quantify the area of the barrel responses, we followed a modified version of the normalized threshold analysis of Chen-Bee and Frostig (1996) and Chen-Bee et al. (1996), using data analyzing software programs, TVMix and Transform (Fortner Research LLC, Sterling, VA). A brief summary, including the modifications, is given in the next paragraph.

The software first located the local peak within the barrel response area. It then calculated the difference between the local peak and the median, and this was normalized to 100% (normalized difference). The normalization is considered to account for any general changes in activity unrelated to whisker stimulation (e.g., potential changes in overall cortical excitability due to fluctuations in the depth of anesthesia) (Chen-Bee et al. 1996). To calculate the barrel response area, the ratio values were processed with a Gaussian filter (a 4 × 4 Gaussian mask) to remove high-frequency noise, and we defined it as the cortical area surrounded by the curve at half of the normalized difference. The noise outside the activated barrel area was not considered to affect the signal (also see Chen-Bee et al. 1996).


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Postnatal changes in D1-whisker responses in control rats

Figure 2 illustrates typical examples of intrinsic optical images obtained from 5- and 7-wk postnatal rats. Optical images were obtained with 10 trials of repeated movements of the D1 whisker on the right side and 5 trials (trials 1, 3, 5, 7, and 9) are presented for each animal. Stimulation which provided the maximal response area (whisker deflections of 1.5° at 5 Hz for 2 s) was applied. The mean values of peak amplitudes of the stimulus-related intrinsic optical signals were 6.8 × 10-4 for 5-wk postnatal rats and 6.2 × 10-4 for 7-wk postnatal rats, and those of noise were 1.5 × 10-4 for 5-wk postnatal rats and 1.6 × 10-4 for 7-wk postnatal rats. Similar values were obtained for other stages (e.g., 6.3 × 10-4 vs. 1.5 × 10-4 for 2-wk postnatal rats and 6.5 × 10-4 vs. 1.4 × 10-4 for adult rats).



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Fig. 2. Intrinsic optical images of D1-barrel responses in 5- and 7-wk postnatal control rats. Optical images were obtained from 10 trials, which were conducted with the same repeated movements of the D1 whisker on the right side, and 5 trials (trials 1, 3, 5, 7, and 9) are presented for each animal.

The images shown in Fig. 2 clearly show that there were variations in the optical response area between trials in each animal. To compare the trial-to-trial variations in the optical responses between different ages, we traced the D1-barrel response area of each trial at half of the normalized difference (see METHODS). The results are summarized in Fig. 3. In this figure, 10 traces from 10 trials are superimposed for each animal. Examples obtained from three animals are presented for 2- to 7-wk postnatal and adult rats. This figure indicates the following characteristics of the D1-whisker barrel responses. The extent of the optical response was largest in the 2-wk rats, and this decreased during postnatal development. And in each animal, there were differences in the extent of the optical response between trials (trial-to-trial variations). The variations were most marked in the 2-wk rats, and they decreased during postnatal development.



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Fig. 3. Traces of the intrinsic optical response obtained from 2- to 7-wk postnatal and adult rats. Examples obtained from 3 animals are presented for each developmental stage. Ten traces are superimposed for each animal. One colored line corresponds to 1 trial.

To quantitatively evaluate the spatial extent of the optical response, we measured the area (S) of the traces. Figure 4 shows the developmental changes in the optical response area and its trial-to-trial variations. In this figure, one symbol corresponds to one trial, and the data obtained from five animals are pooled for each developmental stage. In Table 1, the mean values and standard deviations of the response areas are summarized. The area was largest in the 2-wk animals and gradually decreased as age increased from 2 to 7 wk. In addition, the trial-to-trial variations in the optical response area (SD in Table 1) also decreased during postnatal development. There was no significant difference between 7-wk and adult rats (P > 0.05).



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Fig. 4. Trial-to-trial variations in the optical response area in control rats. open circle , 1 trial. The data obtained from 5 animals are pooled for each developmental stage.


                              
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Table 1. Developmental changes in the area of the optical response (S) in control rats

To examine the characteristics of the optical response areas in more detail, we defined two areas, viz., the "core" and "periphery," as shown in Fig. 5, inset. The core was the area that was responsive to every whisker stimulation trial, and the periphery was the area that was variable between trials. We measured the areas of the core (Sc) and periphery (Sp), and examined their developmental changes (Fig. 5). During postnatal periods, the Sp decreased dramatically (Fig. 5A), while the Sc changed only slightly (Fig. 5B). No significant difference in the Sc between ages was found (P > 0.05). These results show that the developmental changes in the optical response area and its trial-to-trial variations are accompanied by a decrease in the periphery and that the core is relatively stable during development.



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Fig. 5. Developmental changes in the area of the "core" (Sc) and "periphery" (Sp). Inset: definition of the core and periphery. The core is the area that was always responsive to every whisker stimulation, and the periphery is the area that was variable between trials. The Sc in 2-wk postnatal rats was significantly different from those in all other postnatal rats (P < 0.05). No significant difference in the Sc between ages was found (P > 0.05).

Postnatal changes in D1-whisker responses in de-whiskered rats

Next, we studied optical responses in de-whiskered rats and examined whether the developmental process was the same as that observed in control rats. Figure 6 illustrates typical examples of intrinsic optical images obtained from 5- and 7-wk postnatal de-whiskered rats. The de-whiskered rats had all whiskers except the D1 whisker plucked at birth and had all regrowing whiskers replucked until the experimental days. Optical images were obtained with 10 trials of movements of the D1 whisker on the right side, and all the trials are shown for each animal. The same stimuli were applied as to the control rats, thus providing the maximal response area. There was no difference in the time course of optical signals between control and de-whiskered rats (data not shown). These images show that there were also variations in the response area between trials in each de-whiskered animal. The peak amplitudes of the stimulus-related intrinsic optical signals and the noise were almost the same as those in control rats. For example, the mean values of peak amplitudes of the stimulus-related intrinsic optical signals were 7.4 × 10-4 for 5-wk de-whiskered rats and 7.1 × 10-4 for 7-wk de-whiskered rats, and those of noise were 1.6 × 10-4 for 5-wk de-whiskered rats and 1.4 × 10-4 for 7-wk de-whiskered rats.



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Fig. 6. Intrinsic optical images of D1-barrel responses in 5- and 7-wk postnatal de-whiskered rats. Optical images were obtained from 10 trials, and all the trials are shown for each animal.

In Fig. 7, 10 traces from 10 trials are superimposed for each de-whiskered rat. Examples obtained from three animals are presented for the 5- to 7-wk postnatal stages. The following barrel response characteristics can be concluded from this figure: the extent of the optical responses did not to decrease during 5- to 7-wk postnatal development and the trial-to-trial variations in the optical responses were large and showed no decrease during 5- to 7-wk postnatal development. These characteristics are more clearly seen in quantitative representations of the optical response area shown in Fig. 8 and Table 2.



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Fig. 7. Traces of the intrinsic optical response obtained from 5- to 7-wk postnatal rats. Examples obtained from 3 animals are presented for each developmental stage. Ten traces are superimposed for each animal. One colored line corresponds to 1 trial.



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Fig. 8. Trial-to-trial variations in the optical response area in de-whiskered rats. triangle , 1 trial. The data obtained from 5 animals are pooled for each developmental stage.


                              
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Table 2. Developmental changes in the area of the optical response (S) in de-whiskered rats

In Fig. 9, we compared the optical response area (S) between control and de-whiskered rats (, 2- and 5- to 7-wk control rats; , 5- to 7-wk de-whiskered rats). In the control rats, the optical response area and its variations (SD) decreased as development proceeded from 2 to 7 wk as described in the previous section. In the de-whiskered rats, there were no significant changes in the optical response area up to 7 wk (P > 0.05). A marked difference in the size of the optical response area between control and de-whiskered rats at each stage was found (P < 0.05).



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Fig. 9. Comparison of the optical response area between control and de-whiskered rats. , 2- and 5- to 7-wk, control rats; , 5- to 7-wk de-whiskered rats.

We again defined two areas, viz., the core and periphery, as we did for control rats (Fig. 10). In the de-whiskered rats, the Sp slightly decreased with development, but the degree was much smaller than that in control rats (Fig. 10A). The Sc was almost constant during these periods (Fig. 10B).



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Fig. 10. Developmental changes in the area of the core (Sc) and periphery (Sp) in de-whiskered rats. , 2-wk control rats; , 5- to 7-wk de-whiskered rats.

Figure 11 illustrates the locations of the core and periphery in control (A) and de-whiskered (B) rats. The thinner traces show the locations of the core and the thicker ones the locations of the periphery. The data were obtained from three different rats. Although the extent of the periphery was different between control and de-whiskered rats, the locations were almost the same with regard to the bregma. This result suggests that the location of the functional D1-whisker barrel is not rearranged by sensory deprivation.



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Fig. 11. Locations of the core and periphery in control (A) and de-whiskered (B) rats. The thinner traces show the locations of the core and the thicker ones the locations of the periphery. The data were obtained from 3 different animals. The horizontal and vertical lines represent the distance from the bregma.


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In this report, we presented postnatal changes in the intrinsic optical response and its trial-to-trial variations with single whisker movements in the D1-barrel cortex and compared developmental profiles between control and de-whiskered rats. In the control rats, the area of the D1-barrel optical response decreased as development proceeded from 2 to 7 wk, and the extent of the trial-to-trial variations also decreased gradually. On the other hand, in the de-whiskered rats, the area of the optical response and the extent of the trial-to-trial variations were larger than those in the control rats, and did not change during the 5- to 7-wk postnatal periods.

Intrinsic optical response area

In this study, we measured optical response areas in control and de-whiskered rats. Before we have conclusions, we have to consider the following two possibilities about amplitudes of intrinsic optical responses. In the first, the signals in response to whisker movements are large compared with the noise in the measurement (due to technical factors or due to spontaneous activity). In this situation, the noise will not affect the measurement of area. In the second situation, the signals are not so large compared with the noise (signal-to-noise ratio approx 2). In this situation, the noise will affect the area measurement, and it will cause the area measurement to change from trial to trial. In the present experiment, the signal-to-noise ratio was about 4~5, and the values of the signal amplitude and noise were almost the same between young and adult rats and between control and de-whiskered rats. Therefore although there is a possibility that the noise affects the area measurement, the condition seems to be the same between each experiment. Thus we conclude that the developmental change in the optical response area and trial-to-trial variations is not due to an artifact of the area measurement but reflects some physiological changes in whisker barrel responses.

What does the optical response reflect in the whisker-barrel system? In the intrinsic optical imaging, single whisker stimulation defines an area of cortical activity that is larger than the layer IV barrel stained by cytochrome oxidase (Dowling et al. 1996). This means that the optical response area induced by whisker stimulation contains a response area beyond the whisker barrel column, suggesting that the spread of the optical signal is accompanied by an extensive network of horizontal connections for each barrel in cortical layers I-III (Aroniadou and Keller 1993; Bernado et al. 1990; Cauller and Connors 1994; Dodt and Zieglgänsberger 1994). It is also possible that the large activated area is partially due to neurons that receive inputs from more than one whisker. In our experiments, the spatial extent of the D1-whisker barrel optical response in adult control rats was estimated to be 1.07 ± 0.24 mm2, which was larger than the anatomically defined D1-whisker barrel (0.14 mm2) (Chen-Bee and Frostig 1996). Our findings are consistent with previous reports using intrinsic optical imaging (Masino et al. 1993; Peterson and Goldreich 1994) and voltage-sensitive dye optical recording (Grinvald 1985; Orbach et al. 1985). Electrophysiological studies have also demonstrated that single whisker stimulation evokes an excitatory response beyond the boundary of the corresponding barrel (Armstrong-James and Fox 1987) via intracortical horizontal connections (Armstrong-James et al. 1991; Fox 1994).

Postnatal changes in the D1-whisker response area and its variability

As shown in Fig. 4 and Table 1, the spatial extent of the optical response and its trial-to-trial variations evoked by D1-whisker stimulation decreased during postnatal development. They were largest in the 2-wk rats and decreased during 2-7 wk. There was no difference in the size and pattern of the optical responses between 7 wk and adult rats, suggesting that postnatal changes in the D1-whisker optical response are completed by the 7-wk postnatal age. This result is in contrast to a previous study that showed that the barrel cortex in the rat matures much earlier: with a Nissl staining method, Rice (1985) reported that the rat PMBSF barrels are formed as ring-like structures at the end of the second week of postpartum and that they acquire an adult, cell-filled form in the fifth week of life. This discrepancy in the results may be due to the fact that the D1-whisker optical response contains neural activities originating from horizontal connections outside the D1-whisker barrel. Hoeflinger et al. (1995) showed that injections of anterograde and retrograde tracers into a whisker barrel in adult rats labeled widespread horizontal connections within the upper cortical layers extending to a diameter of about 2 mm. This value corresponds to the optical response area recorded in 2- to 3-wk postnatal rats. In conclusion, we suggest that the intracortical horizontal connections from the whisker barrel are morphologically formed and functioning by the 2- to 3-wk postnatal age and that during the subsequent postnatal period, functional modification occurs, and the horizontal connections become gradually masked; this results in a reduction in the optical response area.

The optical responses induced by D1-whisker stimulation showed trial-to-trial variations, and these decreased gradually during postnatal development. To quantitatively evaluate the characteristics of the trial-to-trial variations, we defined two areas, the core and periphery. The size of the core was larger than the size of the anatomically defined D1-whisker barrel (0.14 mm2) (Chen-Bee and Frostig 1996). Therefore it seems likely that the core contains both the D1-whisker barrel and some of the horizontal connections that are activated by every whisker movement and that the periphery corresponds to the horizontal connections that are activated variably between trials.

In control rats, the area of the periphery decreased with development, while that of the core was nearly constant. These results suggest that the neural connections/responsiveness in the core are functionally established at least by 2-wk postnatal and that those in the periphery are unstable in the early postnatal stages, maturing by 7 wk. These results also suggest that the developmental decrease in the optical whisker-response area results from a reduction in the periphery, viz., a reduction in the variably responsive area.

Significance of inter-barrel interactions in normal barrel development

What is the physiological meaning of the change in the optical response pattern? To find out some answers, we compared the extent of the trial-to-trial variations between control and de-whiskered rats. In the rodent somatosensory cortex, it has been shown that sensory deprivation in neonates causes rearrangement of somatotopic representation (Hand 1982; for a review, see Kossut 1992). As shown in Figs. 8 and 9, the optical response area and the extent of trial-to-trial variations to D1-whisker stimulation in de-whiskered rats were larger than those in the control rats and showed no decrease during the 5- to 7-wk postnatal period. Moreover, as shown in Fig. 10, the core and periphery did not change significantly during development. These results show that developmental changes in the optical response area, and the trial-to-trial variations, were blocked by whisker deprivation and suggest that the decrease in the area and variations are caused by the interaction of the corresponding whisker barrel with neighboring barrels. Furthermore, we speculate that, in early postnatal ages, intracortical horizontal connections are widespread and have high plasticity, and that indispensable intracortical connections become tight, while, others are masked by trial-and-error in sensory experience. Indeed, Lendvai et al. (2000) showed the experience-dependent plasticity of dendritic spines in the developing rat barrel cortex using a time-lapse two-photon microscopy technique.


    ACKNOWLEDGMENTS

We thank U. Storz for critically reading the manuscript. We are grateful to Dr. Amiram Grinvald for kind help in constructing the optical recording apparatus and to the reviewer for the helpful comments. We also thank S. Beresford and K. Nakano (MD Anderson Cancer Center) for useful suggestions.

This research was supported by grants from the Ministry of Education-Science-Culture of Japan [Priority Areas (C) Advanced Brain Science Project] and research funds from Japan ALS Foundation, Brain Science Foundation, Toyota Foundation, Asahi Glass Foundation, Konica Imaging Science Foundation, and Nakatani Electric Measuring Technology Association.


    FOOTNOTES

Address for reprint requests: K. Sato, Dept. of Physiology, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: katsushige.phy2{at}tmd.ac.jp).

Received 13 October 2000; accepted in final form 8 February 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society