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Neonatal whisker trimming causes long-lasting changes in structure and function of the somatosensory system
Experimental Neurology 219 (2009) 524–532
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Neonatal whisker trimming causes long-lasting changes in structure and function of the somatosensory system Li-Jen Lee a,b,⁎, Wan-Jung Chen a, Ya-Wen Chuang a, Yu-Chun Wang a a b
Department of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 10 April 2009 Revised 10 July 2009 Accepted 11 July 2009 Available online 18 July 2009 Keywords: Sensory deprivation Tactile Barrel Dendritic spine Explorative behavior Social interaction
a b s t r a c t The significance of very early experience in the maturation of whisker-to-barrel system comes primarily from neonatal whisker or infraorbital nerve lesion studies conducted prior to the formation of cortical barrels. However, the surgical procedures damage the sensory pathway; it is difficult to examine the consequence after the recovery of sensory deprivation. To address this issue, we performed a neonatal whisker-cut (WC) paradigm and examined their behavioral performance during P30 to P35. With fully regrown whiskers, the rats that had whisker cut from the date of birth (P0) to postnatal day (P) 3 (WC 0–3) exhibited shorter crossable distance in the gap-crossing test. However, the rats had whisker cut at P3 only (WC 3) behaved normally in this test, suggesting the critical period for the development of whisker-specific tactile function is P0–P3, agreed with previous findings demonstrated by lesion methods. In the WC 0–3 rats, the cortical areas in the layer IV somatosensory region in relation to the trimmed whiskers were enlarged and the spiny stellate neurons within had larger dendritic span and greater spine density. Furthermore, more long and multiplehead spines were found in these rats. With abnormal structure and function in the somatosensory system, the WC 0–3 rats showed higher explorative activity and more frequent social interactions. Our results have demonstrated that the early tactile deprivation, similar to early visual deprivation, perturbed the developmental program of the brain and affected later behaviors in various aspects. © 2009 Elsevier Inc. All rights reserved.
Introduction Early sensory experience plays a critical role in the structural and functional maturation of the sensory systems (Grubb and Thompson, 2004; Hensch, 2004; Fox and Wong, 2005). For example, whisker deprivation in the neonatal rodents affects dendritic morphology and receptive field of neurons in the somatosensory cortex (Feldman and Brecht, 2005; Alvarez and Sabatini, 2007) and certain whisker-specific behavioral performance later in life (Symons and Tees, 1990; Carvell and Simons, 1996; Shishelova, 2006). The beauty of the whiskerspecific sensory system is not only its functional significance but also its structural characteristics. The whisker-specific neural modules are topographically organized in the somatosensory cortex, known as “barrels” (Woolsey and Van der Loos, 1970). Layer IV spiny stellate neurons in the barrel cortex receive sensory inputs from the patches of thalamocortical afferent and relay information to other cortical layers within the cortical column (Lübke et al., 2000). The thalamocortical afferent develops the typical barrel pattern during the first few postnatal days (Erzurumlu and Jhaveri, 1991, Rebsam et al., 2002, Lee ⁎ Corresponding author. Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No 1, Ren-Ai Rd, Section 1, Taipei 100, Taiwan. Fax: +886 2 23915292. E-mail address: [email protected] (L.-J. Lee). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.07.012
et al., 2005a). LTP-like synaptic plasticity in the thalamocortical synapses is also evident during this period of time (Crair and Malenka, 1996; Isaac et al., 1997). These findings suggest that the early postnatal period consists a critical time window for the structural and functional maturation of the somatosensory system (Fox, 2002; Inan and Crair, 2007). Anatomical evidences supporting the significance of early sensory experience for the development of the whisker-to-barrel system come primarily from lesion studies (Kaas et al., 1983; Fox, 2002). Cauterization of whisker follicles or lesion of infraorbital nerve causes significant morphological alterations in barrel patterns and dendritic structures, especially when surgeries are conducted before the formation of cortical barrels (~ P4) (Van der Loos and Woolsey, 1973; Killackey and Belford, 1979; Steffen and Van der Loos, 1980; Wong-Riley and Welt, 1980; Harris and Woolsey, 1981; Killackey and Shinder, 1981; Schlaggar et al., 1993; Catalano et al., 1995). However, the effects of surgical procedures are not merely sensory deprivation (Calia et al., 1998) and most importantly, the sensory pathway is damaged, it is difficult to address the function of whisker afterward. Whiskers regrow rapidly after trimming. Despite of less morphological alterations, significant changes in cellular and behavioral aspects are observed in rodents following neonatal whisker removal (Simons and Land, 1987; Symons and Tees, 1990; Fox, 1992; Carvell and Simons, 1996; Vees et al., 1998; Keller and Carlson, 1999; Lendvai
L.-J. Lee et al. / Experimental Neurology 219 (2009) 524–532
et al., 2000; Stern et al., 2001; Erchova et al., 2003; Sadaka et al., 2003; Schierloh et al., 2004; Shoykhet et al., 2005; Jiao et al., 2006; Shishelova, 2006; Lee et al., 2007; McRae et al., 2007; Cheetham et al., 2008). Regrettably, in most of the whisker trimming experiments, complete whisker regrowth are not permitted, making the functional examination of the trimmed whisker difficult. There are relatively fewer studies in which the trimmed whiskers are allowed to regrow fully before subsequent assessment (Simons and Land, 1987; Carvell and Simons, 1996; Shoykhet et al., 2005; Zuo et al., 2005; Lee et al., 2007; McRae et al., 2007). Yet, in these experiments, the manipulated whiskers are kept short for weeks before regrowth, which is way beyond the proposed critical period time window (Shoykhet et al., 2005). Up to date, little is known about the impact of brief (days) early-life whisker removal, corresponding to the postnatal critical time window, on the structure and function of the somatosensory system. To address the influence of early sensory deprivation on brain development, we performed neonatal whisker trimming, a noninvasive manipulation compared with previous cauterization of whisker follicles or lesion of infraorbital nerve. All the whiskers were bilaterally trimmed to avoid the uneven sensory inputs in the partial whisker trimming paradigms (Vees et al., 1998). Furthermore, the whiskers were trimmed from the date of birth (P0) to postnatal (P) day 3, a relatively short-term treatment, compared with previous long-term whisker trimming protocol. This period also matches the postnatal critical time window before the barrel formation (Hensch, 2004). Most importantly, the trimmed whiskers were allowed to fully regrow. The length-related influences in whisker-specific tactile function can be excluded. Besides, the whisker functions in the daily activities of rodents, such as decision making, exploration of environment and social interactions can be examined. Our results demonstrated that short-term sensory deprivation during early postnatal period perturbs neuronal development in the somatosensory cortex and, surprisingly, affects later behaviors in various aspects. Materials and methods Subjects All animal handling was in accordance with a protocol approved by National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. Newborn (postnatal day 0, P0) male Wistar rat pups from six different litters were used in this study. Rat pups in the same litter were randomly divided into whisker removal and control groups. For the whisker removal group, all whiskers, including supraorbital whiskers, were carefully trimmed bilaterally twice a day from the date of birth (P0) to postnatal day (P) 3 (WC 0–3) or at P3 only (WC 3) with a sterilized curved eye scissor. For the control group, rat pups were also taken from their dam and handled the same way as the whisker-cut rats except the trimming of the whiskers. Due to the short manipulation time (about 10 s for each animal), no anesthesia was used during the whisker trimming. All rat pups were kept with their dam until weaned after three weeks of age. The animals were housed in the Laboratory Animal Center of National Taiwan University College of Medicine under 12-hour light/dark cycle with free access to food and water. Adolescent rats (P30–P35) were used for behavioral examinations. After behavioral tests, all rats were sacrificed and the brains were processed for cytochrome oxidase histochemistry or Golgi–Cox impregnation. Behavioral tests Adolescent rats (P30–P35) of control group (CON, n = 23) and whisker-cut group (WC 0–3, n = 21; WC 3, n = 15) were used in behavioral tests. The sequence of behavioral tests was fixed: open field
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test, gap-crossing test and followed by social interaction test. In each test, the behaviors of rats were examined only once. The minimum time interval between tests was 24 h. Gap-crossing test Whisker-specific tactile function was evaluated by a gap-crossing test. The test apparatus was modified from our previous version (Lee, 2009). In brief, the custom-made apparatus (78 cm long × 29.5 cm wide × 29.5 cm high) had an 8 cm high floor which was composed of two platforms (39 cm long) with a gap in between (Figs. 1A and B). The fixed platform was covered with a wood ceiling as the dark side, and the movable platform was left uncovered as the bright side. These were the most important features of our device. A rat placed in the bright side of the testing apparatus would scuttle to the dark side by animal nature without training. To reduce explorative activity, a set of transparent plastic wall was used to form a no return pathway in the bright side. The gap-crossing procedure was conducted in a series of increasing gap distance, centimeter by centimeter. Two attempts were allowed for a rat in a given distance and the cut-off time was 120 s. If the rat was not able to cross the gap within 120 s, twice, the distance was recorded as the uncrossable distance, and the maximum crossable distance was thus determined. Open field To assess the explorative activity, individual rats were placed in an open field arena (40 cm × 40 cm). Animals were habituated to the testing environment for 30 min before the test. Once placed in the center of the open field, animals were videotaped for 10 min. The travelled distance and the number of rearing were examined. The arena was cleaned and wiped after each test. Social interaction test The test arena (43 cm long × 22 cm wide × 21 cm high) contains clean wood shavings and was novel to all experimental subjects. One naive and one test rats (either CON or WC 0–3), from different litters, unfamiliar with each other, were brought to the test arena simultaneously. During the 10-minute test session, the behaviors of the animals were recorded with a video camera. After each test, the arena was cleaned and wiped. The social behaviors of the test rats were analyzed by experienced observer without knowing of the
Fig. 1. Gap-crossing test. Lateral (A) and top (B) views of the custom-made testing apparatus are illustrated. The maximum crossable gap distance of adolescent rats (P30–P35, n = 13) was between 6 cm and 9 cm. This distance was significantly reduced in the rats of same age that their whiskers were cut from P0 to P3 (WC 0–3, n = 16) but not in the rats that had whiskers cut at P3 only (WC 3, n = 15) (C). Results are mean ± SEM, ⁎⁎⁎p b 0.001.
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condition of any animal. The following social activities were counted. (1) Investigative behaviors: sniffing any part of the body of the other rat. (2) Contact behaviors: crawling over or burrowing under the other rat and social grooming. (3) Following behaviors: pouncing, following or chasing the other rat which is at a distance greater than half of the body length. The total time of all social interactions was accumulated. In the present experiments, animals did not exhibit serious attacks or threats; therefore aggressive behavior was not counted. Histology
Dendritic spines The number and shape of spines were examined under an Olympus light microscope with a 100× lens using Golgi–Cox impregnated layer IV spiny stellate neurons. The spine density was determined from the 1st to 4th dendritic orders. Statistical analysis Data were expressed as mean ± SEM. Statistical analyses were performed between different groups using two-tailed unpaired student's t-test or univariate analysis of variance. Asterisks were used to indicate significant differences (⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001).
After behavioral tests, rats were sacrificed by overdose of Avertin and transcardially perfused with phosphate-buffered saline followed by fixative (4% paraformaldehyde in phosphate buffer, PB, pH 7.4). After perfusion, the brains were taken. Each hemisphere was separated and flattened between glass slides in the same fixative.
Results
Cytochrome oxidase histochemistry Paraformaldehyde-fixed rat brains were subjected to cytochrome oxidase (CO) histochemistry, a typical method for revealing somatosensory-related structures in the brain (Wong-Riley and Welt, 1980). Sixty μm-thick tangential sections were taken with a vibratome (Warner instruments, Hamden CT, USA). Sections were then transferred to a reaction solution containing (mg/ml): 0.5 cytochrome C, 0.5 diaminobenzidine and 40 sucrose in PB at room temperature until a brownish color was reached.
Neonatal bilateral whisker-cut rats survived well and developed normally in their body length and body weight. In the meantime, the trimmed whiskers regrew rapidly. There are five rows of major whiskers (A–E) on the face of rat and the whiskers are numbered in a temporal-to-nasal order. By the time of behavioral examination (P30), most of the major whiskers examined (whiskers 1, 2 and 3 of each row) had achieved the normal length (Table 1) implying that there was no physical damage in the hair follicles as well as the nerve terminals within in the whisker-cut rat pups (Li et al., 1995).
Golgi–Cox impregnation Dendritic structures were revealed by Golgi–Cox method as previously described (Lee, 2009). In brief, flattened cortices were placed in an impregnation solution (mixture of solution A: 1.0 g potassium dichromate and 1.0 g mercuric chloride in 85 ml distilled water with solution B: 0.8 g potassium chromate and 0.5 g sodium tungstate in 20 ml distilled water) at room temperature for 8–10 days. After impregnation, specimens were cut tangentially at thickness of 100 μm with a vibratome at low speed. Collected sections were then reacted with ammonium hydroxide followed by diluted (1:5) rapid fixer solution (Ilford, Marly, Switzerland). All sections were then counterstained with cresyl violet, dehydrated through series of alcohols, and mounted with Permount (Fisher Scientific, Pittsburgh, PA, USA).
Whisker-specific tactile function is impaired in early sensory-deprived rats
Morphometric analysis Somatosensory barrel patterns Barrel patterns were revealed in layer IV of the somatosensory cortex by CO histochemistry. The areas of the whole flattened cortex and large barrels (A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4) in the posteromedial barrel subfield were measured with ImageJ program (1.36, NIH, Bethesda, MD, USA). Brightness and contrast were adjusted to achieve the sharpest boundaries of individual barrels. The septal regions between barrels were not included in the area measurement. Dendrites Golgi–Cox impregnated neurons were examined under an Olympus light microscope (Tokyo, Japan) with a 40× lens and imaged by a CCD camera (Jenoptic, Jena, Germany). Layer IV spiny stellate cells were selected for reconstruction and morphological examinations by the following criteria: 1) in the barrel wall; 2) spiny appearance; 3) orientated dendrites and 4) complete dendritic arbor. The area occupied by the dendrites is defined as dendritic span which was estimated as the area circled by the dendritic tips (Lee et al., 2005b). The dendritic parameters such as dendritic span and total dendritic length were measured from the two-dimensional display by ImageJ program. The number of primary dendrites, branching points, and terminal endings were counted manually. The concentric sphere method of Sholl (1953) was used to analyze dendritic complexity.
Recovery of neonatal whisker trimming
Rodents are tactile animals and very reliant on the function of whiskers in their daily activities (Brecht, 2007; Petersen, 2007; Diamond et al., 2008). We first asked if the whisker-dependent behaviors were altered after the recovery of early-life sensory deprivation. To examine the whisker-dependent tactile function, a basic gap-crossing test was used (Fig. 1). This type of test required no prior training of the animals. Once a rat was placed in the bright side of the testing apparatus, it would move to the dark side by animal nature. Rats moved rapidly to the dark side while crossing the short gap distance. However, when the gap distance was widened up to 4 or 5 cm, roughly the length of the rats' head, rats extended their head, used their whiskers to explore the surroundings and spent time by the edge. Generally, the maximum crossable gap distance of normal adolescent male (CON) rats was between 6 cm and 9 cm (7.08 ± 0.79 cm, n = 13). However, the maximum crossable distance of the WC 0–3 (whisker cut during P0 to P3) rats was reduced remarkably (4.94 ± 1.34 cm, n = 16) compared with controls (p b 0.001, Fig. 1C). The results demonstrated that the early-life sensory experience is required for the proper performance in the gap-crossing test. Furthermore, to test if there is a critical period in this behavioral task, we also bilaterally trimmed all the whiskers of rat pups at P3 only Table 1 Recovery of neonatally trimmed whiskers. Whisker 1 CON Row A Row B Row C Row D Row E
Whisker 2 WC 0–3
CON
Whisker 3 WC 0–3
CON
38.5 ± 1.72 36.4 ± 1.27 32.7 ± 4.24 28.7 ± 1.38 21.3 ± 3.06 40.7 ± 1.06 38.7 ± 1.5 36.6 ± 4.41 33.1 ± 1.35⁎ 25.6 ± 1.9 42.1 ± 1.73 40.1 ± 1.07 38.3 ± 1.42 34.4 ± 1.72⁎ 26.5 ± 2.37 43.5 ± 1.84 41.9 ± 1.21 37.3 ± 1.89 34.4 ± 1.27 27.5 ± 2.64 38.3 ± 3.02 34.7 ± 1.38 28.7 ± 1.89 27.9 ± 1.46 18.6 ± 1.65
WC 0–3 20.9 ± 1.07 23.7 ± 0.49 24.7 ± 0.76 26.6 ± 1.51 17.6 ± 1.51
Major whiskers were cut bilaterally in neonatal rats during P0 to P3 and allowed to regrow afterward. By the time of P30, most of the major whiskers examined had achieved normal length (in mm). Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 8) and neonatal whisker-cut (WC 0–3, n = 6) rats. ⁎ p b 0.05.
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(WC 3). Surprisingly, the very early (P0–P3) whisker cut-induced behavioral change in gap-crossing test did not occur in WC 3 rats (n = 15). The critical period, P0 to P3, determined by whisker-specific gap-crossing test matched well with the developmental time window for the maturation of whisker-to-barrel system as previously demonstrated by lesion studies (Van der Loos and Woolsey, 1973; Killackey and Belford, 1979; Schlaggar et al., 1993). Changes of cortical barrel patterns in the whisker-cut rats Whisker-specific tactile function is defective in the WC 0–3 rats, we next examined if there is an anatomical basis for this functional impairment. The cortical barrel pattern was the key structure to check. The barrels in the layer IV somatosensory cortex were revealed by cytochrome oxidase histochemistry in both control and whisker-cut rats (Figs. 2A and B). We first measured the overall area of each flattened cortex. No significant difference was found between the two (CON and WC 0–3) groups (122.1 ± 2.72 mm2 in CON, n = 10; 117.2 ± 4.53 mm2 in WC 0–3, n = 9; p = 0.63). Apparently, no difference in the overall pattern between the two groups was observed at first sight. However, the size of individual barrels corresponding to the trimmed whiskers was somewhat altered by early sensory deprivation. For all the barrels examined, the area of barrels in the WC 0–3 rats was larger than the normal ones. In some barrels, the difference achieved statistic significance (e.g. A2, A3, A4, C1, C2, D1, D3, E1 and E3 barrels, in Fig. 2C). When we combined the areas of all the measured barrels, there was a 14.5% enlargement in the WC 0–3 rats, compared with CON rats.
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Dendritic arbors are affected by early whisker trimming The subtle changes in the cortical barrels suggest cortical mechanism for the tactile functional impairment in the WC 0–3 rats. We then examined dendritic structures of the cortical neurons in the barrel field corresponding to the major whiskers. The layer IV spiny stellate neurons were chosen for morphological analyses. These cells were located in the barrel wall and orientated most of their dendrites toward the barrel center. Golgi–Cox impregnated neurons in the barrel field were selected for reconstruction according to their location, orientation and spiny appearance (Fig. 3A). The neurons from the WC 0–3 rats (n = 46 neurons from 6 animals) and normal rats (n = 59 neurons from 8 animals) had comparable soma size, number of branching nodes, terminal tips and total dendritic length; whereas neurons in WC 0–3 rats had more primary dendrites, less dendritic orders but greater dendritic span (Table 2). The enlarged dendritic span matched up well with the greater barrel areas (Fig. 2C) and enlarged receptive field in the neonatal whisker-deprived rats (Simons and Land, 1987). The complexity of dendrites was estimated by Sholl analysis. The numbers of intersections were comparable between the two groups except for the more distal regions (beyond 100 μm, Fig. 3B). The number of branching nodes and terminal tips was counted in relation to the distance from the soma center. The quantity and distribution of branching nodes in the two groups were similar (Table 2, Fig. 3C) whereas the spatial distribution of the terminal tips varied between the two groups. There were less terminals in the proximal regions yet more tips beyond 100 μm from the soma center in the neurons of WC 0–3 rats (Fig. 3D). These results implied the fault of dendritic pruning in these rats. Dendritic spines The morphology and number of dendritic spine are sensitive to neural activity (Alvarez and Sabatini, 2007). Sensory deprivation or pharmacological blockade of synaptic transmission alters the stability of spines (Zuo et al., 2005). We therefore examined the effects of bilateral early whisker trimming on the shape and density of dendritic spines of layer IV spiny stellate neurons (Fig. 4). The morphology of dendritic spines was affected. There were more branched (multiple heads) spines in the WC 0–3 rats (93 in 598 spines examined), which rarely occurred (38 in 585 spines) in the control ones (Fig. 4A). This distorted spine phenotype correlated with deafferentation-induced spine immaturity (Fiala et al., 2002). Furthermore, the density of total spines was also found to be changed in the WC 0–3 rats. The spine density was significantly (p b 0.05) increased in the 2nd (1.62± 0.06/μm, versus 1.30 ± 0.08/μm of controls), 3rd (1.62 ± 0.06/μm, as compared with controls, 1.39± 0.07/μm) and 4th (1.66 ± 0.06/μm, as compared with controls, 1.42± 0.08/μm) dendritic orders (Fig. 4B). Taken together, in the WC 0– 3 rats, dendritic arborization and spine density in the barrel cortex were changed. These alterations in the dendritic structures of layer IV spiny stellate neurons in these rats may serve as the anatomical basis for the whisker-specific tactile function impairments. Altered explorative activity in the early-life whisker-deprived rats
Fig. 2. Barrel patterns in the layer IV somatosensory cortex. The barrels in the posteromedial barrel subfield corresponding to the major whiskers were revealed by cytochrome oxidase histochemistry. Schematic outlines of individual barrels were drawn accordingly (A and B). The areas of large barrels (A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4) were measured (C). Bar is 1 mm. Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 10) and neonatal whisker-cut (WC 0–3, n = 9) rats (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
With abnormal somatic sensation, the WC 0–3 rats may exhibit abnormal explorative behavior. To test this possibility, individual rats were placed in an open field, and their locomotor activity was taperecorded and analyzed. Compared with normal rats, the total travelled distance was significantly increased in the whisker-cut rats (675.50 ± 119.11 cm in CON, n = 6; 1103.12 ± 118.34 cm in WC 0–3, n = 6, p b 0.01, Fig. 5A). The number of rearing was also increased in WC 0–3 rats (39.83 ± 3.57) compared with controls (24.20 ± 3.97, p b 0.05, Fig. 5B). Together, these results indicated that the early-life (P0–P3) whisker-deprived rats exhibited increased explorative activity in a novel open field.
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Fig. 3. Layer IV spiny stellate cells in the rat barrel cortex. Golgi–Cox impregnated layer IV spiny stellate cells from both groups were reconstructed. In this illustration, all dendritic spines are omitted (A). Dendritic complexity was estimated by Sholl analysis (B). The number of branching nodes (C) and terminal tips (D) were counted in relation to the distance from the soma center. Bar is 100 μm. Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 59 neurons) and neonatal whisker-cut (WC 0–3, n = 46 neurons) groups (⁎p b 0.05, ⁎⁎⁎p b 0.001).
Early-life sensory deprivation affects later social interactions We next examined the effect of early-life (P0–P3) whisker deprivation on social behaviors. Investigative, contact and following behaviors as well as the total social interaction time of male adolescent rats were observed for 10 min (Fig. 6). Six normal (CON) and 7 neonatal whisker-cut (WC 0–3) rats were used in this experiment. The occurrences of investigative and contact behaviors as well as the total social interaction time were significantly increased in the neonatal sensory-deprived rats (for investigative behaviors, 23.17 ± 3.68 in CON, 35.67 ± 3.72 in WC 0–3, p b 0.05, Fig. 6A; for contact behaviors, 9.33 ± 3.68 in CON, 20.83 ± 2.72 in WC 0–3, p b 0.05, Fig. 6B; for total social interaction time, 69.83 ± 8.16 s in CON, 130.03 ± 17.63 s in WC 0–3, p b 0.05, Fig. 6D). However, such increased frequency was not observed in the following behaviors (2.83 ± 1.22 in CON, 2.51 ± 1.12 in WC 0–3, p = 0.84, Fig. 6C). When the observation time was divided into two 5-minute episodes, further Table 2 Dendritic parameters of layer IV spiny stellate cells in the barrel cortex. Parameters
CON (n = 59 neurons)
WC 0–3 (n = 46 neurons)
Primary dendrites Branching nodes Terminal tips Highest order Total dendritic length (μm) Dendritic span (μm2)
3.15 ± 0.13 12.81 ± 0.42 17.80 ± 0.52 5.76 ± 0.16 839.88 ± 26.23 9327.45 ± 367.89
3.59 ± 0.16⁎ 12.72 ± 0.49 17.48 ± 0.59 5.20 ± 0.17⁎ 910.07 ± 41.8 13543.80 ± 823.51⁎⁎⁎
The morphological features of neurons were measured from the two-dimensional display. Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON) and neonatal whisker-cut (WC 0–3) groups. ⁎ p b 0.05. ⁎⁎⁎ p b 0.001.
information was revealed. During the first 5-minute period, all behaviors examined and the total social interaction time were comparable between control and whisker-cut rats. However, during the second 5-minute period, sensory-deprived rats exhibited significantly higher frequency in the investigative (6.83 ± 1.08 in control, 15.02 ± 1.56, p b 0.01, Fig. 6A) and contact (3.67 ± 1.69 in control, 8.52 ± 0.99, p b 0.05, Fig. 6B) behaviors as well as in the total social interaction time (19.17 ± 4.36 s in CON, 70.01 ± 9.27 s in WC 0–3, p b 0.001, Fig. 6D). Discussion The present study has demonstrated the first time the consequences after the recovery of early short-term whisker deprivation. Our results also confirm the critical time window for the functional development of the whisker-to-barrel system. A temporary sensory deprivation during this early postnatal period (P0–P3) disturbs the normal development of the sensory system both in structure and in function. With impaired perceptual faculty, the early-life sensorydeprived animals exhibit abnormal explorative activity and social behaviors. Structural alterations after early-life whisker deprivation It has been argued that the structural plasticity in layer IV somatosensory cortex can only be demonstrated by lesion strategies during early development (Kaas et al., 1983; Fox, 2002). However, surgical procedures damage the integrity of the sensory system and may also induce side effects (Calia et al., 1998). Since non-invasive manipulation (e.g. whisker trimming) causes little or no macroscopic changes (Kaas et al., 1983), the robust morphological alterations in the
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Fig. 4. Layer IV spiny stellate cells in the rat barrel cortex. The dendritic spines of layer IV spiny stellate cells in the somatosensory cortex were revealed by Golgi–Cox impregnation method (A). Note that the numerous branched or multiple-head spines (arrowheads) appeared in the whisker-cut rats. Spine density was determined at each dendritic order (1st to 4th). The density of total spine was significantly increased in whisker-cut (WC 0–3) rats compared to controls, especially in the 2nd, 3rd and 4th dendritic orders (B). Bar is 10 μm in (A). Results are mean ± SEM (⁎p b 0.05, ⁎⁎p b 0.01).
barrel cortex following early surgical lesions might be the mixture of sensory deprivation and other effects. Here, we have confirmed that even minor manipulation such as whisker trimming during early postnatal period can also cause subtle yet significant morphological alterations in the barrel field. The early short-term sensory deprivation produces enlarged cortical barrels and increased dendritic spans corresponding to the manipulated whiskers. Moreover, the spine density and morphology are also sensitive to the early sensory deprivation. Our findings are consistent with the previous electrophysiological recordings. Layer IV cortical neurons in the barrel cortex exhibit larger excitatory receptive field and higher neural activity after the recovery of whisker removal began at birth (Simons and Land, 1987; Shoykhet et al., 2005; Lee et al., 2007). It is known that during neural development, considerable amount of dendritic processes and spines are overproduced and then later eliminated (Warren and Jones, 1997; Innocenti and Price, 2005). This pruning program takes place during the first postnatal weeks and is neural activity-dependent (Segal and Andersen, 2000; Cline and Haas, 2008). An exciting study using two-photon microscopy technique has shown that the elimination of dendritic spine in the layer V pyramidal neurons of barrel cortex is greatly reduced by whisker trimming (Zuo et al., 2005). Similar results can be reproduced by chronic blockade of NMDA receptors, the authors has concluded that glutamatergic neurotransmission plays a critical role in this process. In cortexspecific NMDA receptor deficient mice, the excitatory neurotransmission is greatly reduced (Iwasato et al., 2000), the dendritic arbor and spine density are also altered (Datwani et al., 2002). In these mice, the dendrites of layer IV spiny stellate neurons have longer length and greater spine density than the wildtype controls. These results show the fault of dendritic pruning in NMDA receptor deficient mice and again support the neural activity-dependent sculpture of dendritic arbors (Segal and Andersen, 2000; Lee et al., 2005b; Cline and Haas, 2008). In the present study, with deficient sensory input, the layer IV
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spiny stellate neurons in sensory-deprived rats may receive fewer amount of or less organized neural activity insufficient to activate NMDA receptor (Liu, 2003). As such, the spiny stellate neurons in sensory-deprived rats, like in the NMDA receptor mutant mice, exhibit impaired pruning of dendritic arbors and spines. Since the pruning process occurs during a limited time window, the longer dendritic length, greater dendritic span and higher spine density are left in the whisker-deprived rats even though the whiskers have fully regrown. In the present study, we have demonstrated that the morphology and density of dendritic spine are also sensitive to early-life temporal sensory deprivation. The increased spine density and altered spine shape reflect the shortness of neural activity and spine immaturity that frequently appeared in deafferentation (Fiala et al., 2002) and mental retardation (Kaufmann and Moser, 2000). In fact, the failure of dendritic pruning is observed in an animal model of Fragile X mental retardation syndrome (Comery et al., 1997). The cortical neurons of Fragile X mental retardation patients have distorted spine features. These spines are longer, thinner, tortuous and sometimes multiple swellings are present (Kaufmann and Moser, 2000; Fiala et al., 2002). The increased spine density and the appearance of multiple-head spines in WC 0–3 rats resemble the dendritic phenotype of Fragile X syndrome, again supporting neural activity-dependent mechanism in the morphogenesis of dendritic structures. On the other hand, the Fragile X mental retardation protein (FMRP), absent in Fragile X patients, is normally located in the synapse and is important for the integrity of synaptic structure (Weiler et al., 1997). The expression of FMRP is up-regulated in the barrel field after whisker stimuli (Todd et al., 2003). Hence, it is important to examine the expression of FMRP, as well as other synaptic proteins, in the barrel cortex of whiskerdeprived animals. Several synaptic proteins including NMDA receptor, Dynamin I, and Synaptotagmin I, are found to be changed in the cortical neurons of visual deprivation animal models (Murphy et al., 2004; Cnops et al., 2008). In the visual system, early visual disruption often results in later poor visual acuity known as amblyopia. Dark rearing and monocular deprivation are commonly used protocols for visual deprivation (Fagiolini et al., 1994). Monocular deprivation causes robust functional and structural changes along the visual pathway by creating a mixture condition of altered visual stimulation and binocular competition (Murphy et al., 2004). We therefore suggest that the dark rearing paradigm for visual deprivation is relatively close to the bilateral whisking trimming protocol in the present study. In dear-reared rats, larger receptive field and lower visual acuity are found in the cortical level (Fagiolini et al., 1994). Layer IV stellate neurons in the visual cortex of dark-reared rats lost their polarity resulting from uneven growth of dendrites (Borges and Berry, 1978). Our findings of enlarged
Fig. 5. Explorative activity in the open field. Locomotor activities of adolescent rats were examined in an open field for 10 min. The total travelled distance (A) and number of rearing (B) were analyzed. Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 6) and neonatal whisker-cut (WC 0–3, n = 6) rats (⁎p b 0.05).
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Fig. 6. Altered social behaviors in the early-life sensory-deprived rats. Effects of sensory deprivation on social interactions were evaluated by the investigative (A), contact (B), following (C) behaviors and overall social activities (D) of male adolescent rats during a 10-minute observation period. Counts of a particular behavior or total time of all behaviors were presented as in the first 5 min (0–5:00), in the succeeding 5 min (5:00–10:00) and in the whole 10 min (total). Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 6) and neonatal whisker-cut (WC 0–3, n = 7) rats (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
barrels and prolonged dendrites in the somatosensory cortex of whisker-trimmed rats are in accordance with the results produced by visual deprivation. Despite the fundamental differences between the visual and somatosensory systems, they share some common developmental procedures that require proper sensory experiences for their maturation (Grubb and Thompson, 2004; Fox and Wong, 2005). Behavioral performances after the recovery of early-life whisker deprivation Since the dendritic spines of layer IV spiny stellate neurons are the first receiving sites of thalamic inputs in the sensory cortex, altered dendritic structures imply functional deficits. With tortuous dendritic spines, the sensory-deprived animals may suffer poor synaptic transmission and integration. Consequently, the animals are less perceptive to the environmental stimuli, which in turn affects the decision making (Celikel and Sakmann, 2007). Indeed, these rats with fully regrown whiskers exhibit shorter crossable gap distance, confirming that the function of their whisker-dependent sensory system is impaired. The present study is the first experiment, to our knowledge, that demonstrates functional deficit after the recovery of short-term (few days) early-life whisker deprivation. Furthermore, by using whisker-specific gap-crossing test, we have also defined the critical time window for the functional maturation of the whisker-tobarrel system. However, we cannot exclude deficits, if any, in the WC 3 rats, insensitive to the gap-crossing test (Shoykhet et al., 2005). Neither can we extract the emotional constituent in this test. Nevertheless, the results still clearly demonstrate that the early-life sensory experience is required for the proper performance in the gapcrossing test. A number of whisker-related behavioral changes are observed in rats which have their whiskers removed from birth but did not recover (Symons and Tees, 1990; Shishelova, 2006). However, without full regrowth, it is difficult to address the temporal importance of the manipulation. Simons and Carvell have beautifully presented whiskerdependent behavioral changes in rats after recovery from 45-day whisker trimming which began at birth. The behavioral abnormality they demonstrated is task-sensitive. Early sensory experience is essential for conducting behavioral task that requires two or more
whiskers, implying that the ability of integrating inputs is established based on normal sensory experience (Carvell and Simons, 1996). Based on their findings and ours, we conclude that the concept of the external three-dimensional dynamic world is built upon the internal sensory map which is shaped by sensory experience during early development. With altered somatic sensation, neonatal whisker-cut rats later exhibit increased explorative behaviors. Similar findings are observed in children with inadequate sensory stimuli particularly during their early childhood (Lewis, 1978; Lin et al., 2005). It has been pointed out that these children are hyperactive and have poor discrimination. The increased physical activity and reduced discrimination together produce more random or meaningless activities. However, the excess activities do little to help extract information from the environment. As such, the favorable opportunities for learning may be reduced (Lewis, 1978). In fact, the sensoryprocessing deficit is one of the major causes for learning difficulties (Goswami, 2006). Experiments are underway to examine whether the capacity of spatial learning is affected in the early-life sensorydeprived animals. Besides altered locomotor activity, abnormal social behaviors in WC 0–3 rats are also observed. These rats behave normally during the first 5-minute section; however, they exhibit significantly higher frequency in investigative and contact behaviors during the second 5minute section. These aberrant behaviors also validate the hyperactive and poor-in-discrimination nature of early-life sensory-deprived animals. The increased social interactions found in the sensorydeprived rats here are also similar to the early-life social-deprived animals (Varlinskaya and Spear, 2008). It is important to examine if the early whisker-deprived rats are, like socially isolated animals, prone to develop emotional problems (Ibi et al., 2008). Conclusion Small initial difference in the sensory-processing systems can lead to significant problems later in life (Beddington et al., 2008). With minor and brief manipulation, we have demonstrated significant structural and, more importantly, functional abnormalities in the somatosensory system. The sequelae of early-life sensory deprivation may be far more serious than estimated.
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Neonatal whisker trimming causes long-lasting changes in structure and function of the somatosensory system Li-Jen Lee a,b,⁎, Wan-Jung Chen a, Ya-Wen Chuang a, Yu-Chun Wang a a b
Department of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 10 April 2009 Revised 10 July 2009 Accepted 11 July 2009 Available online 18 July 2009 Keywords: Sensory deprivation Tactile Barrel Dendritic spine Explorative behavior Social interaction
a b s t r a c t The significance of very early experience in the maturation of whisker-to-barrel system comes primarily from neonatal whisker or infraorbital nerve lesion studies conducted prior to the formation of cortical barrels. However, the surgical procedures damage the sensory pathway; it is difficult to examine the consequence after the recovery of sensory deprivation. To address this issue, we performed a neonatal whisker-cut (WC) paradigm and examined their behavioral performance during P30 to P35. With fully regrown whiskers, the rats that had whisker cut from the date of birth (P0) to postnatal day (P) 3 (WC 0–3) exhibited shorter crossable distance in the gap-crossing test. However, the rats had whisker cut at P3 only (WC 3) behaved normally in this test, suggesting the critical period for the development of whisker-specific tactile function is P0–P3, agreed with previous findings demonstrated by lesion methods. In the WC 0–3 rats, the cortical areas in the layer IV somatosensory region in relation to the trimmed whiskers were enlarged and the spiny stellate neurons within had larger dendritic span and greater spine density. Furthermore, more long and multiplehead spines were found in these rats. With abnormal structure and function in the somatosensory system, the WC 0–3 rats showed higher explorative activity and more frequent social interactions. Our results have demonstrated that the early tactile deprivation, similar to early visual deprivation, perturbed the developmental program of the brain and affected later behaviors in various aspects. © 2009 Elsevier Inc. All rights reserved.
Introduction Early sensory experience plays a critical role in the structural and functional maturation of the sensory systems (Grubb and Thompson, 2004; Hensch, 2004; Fox and Wong, 2005). For example, whisker deprivation in the neonatal rodents affects dendritic morphology and receptive field of neurons in the somatosensory cortex (Feldman and Brecht, 2005; Alvarez and Sabatini, 2007) and certain whisker-specific behavioral performance later in life (Symons and Tees, 1990; Carvell and Simons, 1996; Shishelova, 2006). The beauty of the whiskerspecific sensory system is not only its functional significance but also its structural characteristics. The whisker-specific neural modules are topographically organized in the somatosensory cortex, known as “barrels” (Woolsey and Van der Loos, 1970). Layer IV spiny stellate neurons in the barrel cortex receive sensory inputs from the patches of thalamocortical afferent and relay information to other cortical layers within the cortical column (Lübke et al., 2000). The thalamocortical afferent develops the typical barrel pattern during the first few postnatal days (Erzurumlu and Jhaveri, 1991, Rebsam et al., 2002, Lee ⁎ Corresponding author. Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No 1, Ren-Ai Rd, Section 1, Taipei 100, Taiwan. Fax: +886 2 23915292. E-mail address: [email protected] (L.-J. Lee). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.07.012
et al., 2005a). LTP-like synaptic plasticity in the thalamocortical synapses is also evident during this period of time (Crair and Malenka, 1996; Isaac et al., 1997). These findings suggest that the early postnatal period consists a critical time window for the structural and functional maturation of the somatosensory system (Fox, 2002; Inan and Crair, 2007). Anatomical evidences supporting the significance of early sensory experience for the development of the whisker-to-barrel system come primarily from lesion studies (Kaas et al., 1983; Fox, 2002). Cauterization of whisker follicles or lesion of infraorbital nerve causes significant morphological alterations in barrel patterns and dendritic structures, especially when surgeries are conducted before the formation of cortical barrels (~ P4) (Van der Loos and Woolsey, 1973; Killackey and Belford, 1979; Steffen and Van der Loos, 1980; Wong-Riley and Welt, 1980; Harris and Woolsey, 1981; Killackey and Shinder, 1981; Schlaggar et al., 1993; Catalano et al., 1995). However, the effects of surgical procedures are not merely sensory deprivation (Calia et al., 1998) and most importantly, the sensory pathway is damaged, it is difficult to address the function of whisker afterward. Whiskers regrow rapidly after trimming. Despite of less morphological alterations, significant changes in cellular and behavioral aspects are observed in rodents following neonatal whisker removal (Simons and Land, 1987; Symons and Tees, 1990; Fox, 1992; Carvell and Simons, 1996; Vees et al., 1998; Keller and Carlson, 1999; Lendvai
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et al., 2000; Stern et al., 2001; Erchova et al., 2003; Sadaka et al., 2003; Schierloh et al., 2004; Shoykhet et al., 2005; Jiao et al., 2006; Shishelova, 2006; Lee et al., 2007; McRae et al., 2007; Cheetham et al., 2008). Regrettably, in most of the whisker trimming experiments, complete whisker regrowth are not permitted, making the functional examination of the trimmed whisker difficult. There are relatively fewer studies in which the trimmed whiskers are allowed to regrow fully before subsequent assessment (Simons and Land, 1987; Carvell and Simons, 1996; Shoykhet et al., 2005; Zuo et al., 2005; Lee et al., 2007; McRae et al., 2007). Yet, in these experiments, the manipulated whiskers are kept short for weeks before regrowth, which is way beyond the proposed critical period time window (Shoykhet et al., 2005). Up to date, little is known about the impact of brief (days) early-life whisker removal, corresponding to the postnatal critical time window, on the structure and function of the somatosensory system. To address the influence of early sensory deprivation on brain development, we performed neonatal whisker trimming, a noninvasive manipulation compared with previous cauterization of whisker follicles or lesion of infraorbital nerve. All the whiskers were bilaterally trimmed to avoid the uneven sensory inputs in the partial whisker trimming paradigms (Vees et al., 1998). Furthermore, the whiskers were trimmed from the date of birth (P0) to postnatal (P) day 3, a relatively short-term treatment, compared with previous long-term whisker trimming protocol. This period also matches the postnatal critical time window before the barrel formation (Hensch, 2004). Most importantly, the trimmed whiskers were allowed to fully regrow. The length-related influences in whisker-specific tactile function can be excluded. Besides, the whisker functions in the daily activities of rodents, such as decision making, exploration of environment and social interactions can be examined. Our results demonstrated that short-term sensory deprivation during early postnatal period perturbs neuronal development in the somatosensory cortex and, surprisingly, affects later behaviors in various aspects. Materials and methods Subjects All animal handling was in accordance with a protocol approved by National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. Newborn (postnatal day 0, P0) male Wistar rat pups from six different litters were used in this study. Rat pups in the same litter were randomly divided into whisker removal and control groups. For the whisker removal group, all whiskers, including supraorbital whiskers, were carefully trimmed bilaterally twice a day from the date of birth (P0) to postnatal day (P) 3 (WC 0–3) or at P3 only (WC 3) with a sterilized curved eye scissor. For the control group, rat pups were also taken from their dam and handled the same way as the whisker-cut rats except the trimming of the whiskers. Due to the short manipulation time (about 10 s for each animal), no anesthesia was used during the whisker trimming. All rat pups were kept with their dam until weaned after three weeks of age. The animals were housed in the Laboratory Animal Center of National Taiwan University College of Medicine under 12-hour light/dark cycle with free access to food and water. Adolescent rats (P30–P35) were used for behavioral examinations. After behavioral tests, all rats were sacrificed and the brains were processed for cytochrome oxidase histochemistry or Golgi–Cox impregnation. Behavioral tests Adolescent rats (P30–P35) of control group (CON, n = 23) and whisker-cut group (WC 0–3, n = 21; WC 3, n = 15) were used in behavioral tests. The sequence of behavioral tests was fixed: open field
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test, gap-crossing test and followed by social interaction test. In each test, the behaviors of rats were examined only once. The minimum time interval between tests was 24 h. Gap-crossing test Whisker-specific tactile function was evaluated by a gap-crossing test. The test apparatus was modified from our previous version (Lee, 2009). In brief, the custom-made apparatus (78 cm long × 29.5 cm wide × 29.5 cm high) had an 8 cm high floor which was composed of two platforms (39 cm long) with a gap in between (Figs. 1A and B). The fixed platform was covered with a wood ceiling as the dark side, and the movable platform was left uncovered as the bright side. These were the most important features of our device. A rat placed in the bright side of the testing apparatus would scuttle to the dark side by animal nature without training. To reduce explorative activity, a set of transparent plastic wall was used to form a no return pathway in the bright side. The gap-crossing procedure was conducted in a series of increasing gap distance, centimeter by centimeter. Two attempts were allowed for a rat in a given distance and the cut-off time was 120 s. If the rat was not able to cross the gap within 120 s, twice, the distance was recorded as the uncrossable distance, and the maximum crossable distance was thus determined. Open field To assess the explorative activity, individual rats were placed in an open field arena (40 cm × 40 cm). Animals were habituated to the testing environment for 30 min before the test. Once placed in the center of the open field, animals were videotaped for 10 min. The travelled distance and the number of rearing were examined. The arena was cleaned and wiped after each test. Social interaction test The test arena (43 cm long × 22 cm wide × 21 cm high) contains clean wood shavings and was novel to all experimental subjects. One naive and one test rats (either CON or WC 0–3), from different litters, unfamiliar with each other, were brought to the test arena simultaneously. During the 10-minute test session, the behaviors of the animals were recorded with a video camera. After each test, the arena was cleaned and wiped. The social behaviors of the test rats were analyzed by experienced observer without knowing of the
Fig. 1. Gap-crossing test. Lateral (A) and top (B) views of the custom-made testing apparatus are illustrated. The maximum crossable gap distance of adolescent rats (P30–P35, n = 13) was between 6 cm and 9 cm. This distance was significantly reduced in the rats of same age that their whiskers were cut from P0 to P3 (WC 0–3, n = 16) but not in the rats that had whiskers cut at P3 only (WC 3, n = 15) (C). Results are mean ± SEM, ⁎⁎⁎p b 0.001.
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condition of any animal. The following social activities were counted. (1) Investigative behaviors: sniffing any part of the body of the other rat. (2) Contact behaviors: crawling over or burrowing under the other rat and social grooming. (3) Following behaviors: pouncing, following or chasing the other rat which is at a distance greater than half of the body length. The total time of all social interactions was accumulated. In the present experiments, animals did not exhibit serious attacks or threats; therefore aggressive behavior was not counted. Histology
Dendritic spines The number and shape of spines were examined under an Olympus light microscope with a 100× lens using Golgi–Cox impregnated layer IV spiny stellate neurons. The spine density was determined from the 1st to 4th dendritic orders. Statistical analysis Data were expressed as mean ± SEM. Statistical analyses were performed between different groups using two-tailed unpaired student's t-test or univariate analysis of variance. Asterisks were used to indicate significant differences (⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001).
After behavioral tests, rats were sacrificed by overdose of Avertin and transcardially perfused with phosphate-buffered saline followed by fixative (4% paraformaldehyde in phosphate buffer, PB, pH 7.4). After perfusion, the brains were taken. Each hemisphere was separated and flattened between glass slides in the same fixative.
Results
Cytochrome oxidase histochemistry Paraformaldehyde-fixed rat brains were subjected to cytochrome oxidase (CO) histochemistry, a typical method for revealing somatosensory-related structures in the brain (Wong-Riley and Welt, 1980). Sixty μm-thick tangential sections were taken with a vibratome (Warner instruments, Hamden CT, USA). Sections were then transferred to a reaction solution containing (mg/ml): 0.5 cytochrome C, 0.5 diaminobenzidine and 40 sucrose in PB at room temperature until a brownish color was reached.
Neonatal bilateral whisker-cut rats survived well and developed normally in their body length and body weight. In the meantime, the trimmed whiskers regrew rapidly. There are five rows of major whiskers (A–E) on the face of rat and the whiskers are numbered in a temporal-to-nasal order. By the time of behavioral examination (P30), most of the major whiskers examined (whiskers 1, 2 and 3 of each row) had achieved the normal length (Table 1) implying that there was no physical damage in the hair follicles as well as the nerve terminals within in the whisker-cut rat pups (Li et al., 1995).
Golgi–Cox impregnation Dendritic structures were revealed by Golgi–Cox method as previously described (Lee, 2009). In brief, flattened cortices were placed in an impregnation solution (mixture of solution A: 1.0 g potassium dichromate and 1.0 g mercuric chloride in 85 ml distilled water with solution B: 0.8 g potassium chromate and 0.5 g sodium tungstate in 20 ml distilled water) at room temperature for 8–10 days. After impregnation, specimens were cut tangentially at thickness of 100 μm with a vibratome at low speed. Collected sections were then reacted with ammonium hydroxide followed by diluted (1:5) rapid fixer solution (Ilford, Marly, Switzerland). All sections were then counterstained with cresyl violet, dehydrated through series of alcohols, and mounted with Permount (Fisher Scientific, Pittsburgh, PA, USA).
Whisker-specific tactile function is impaired in early sensory-deprived rats
Morphometric analysis Somatosensory barrel patterns Barrel patterns were revealed in layer IV of the somatosensory cortex by CO histochemistry. The areas of the whole flattened cortex and large barrels (A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4) in the posteromedial barrel subfield were measured with ImageJ program (1.36, NIH, Bethesda, MD, USA). Brightness and contrast were adjusted to achieve the sharpest boundaries of individual barrels. The septal regions between barrels were not included in the area measurement. Dendrites Golgi–Cox impregnated neurons were examined under an Olympus light microscope (Tokyo, Japan) with a 40× lens and imaged by a CCD camera (Jenoptic, Jena, Germany). Layer IV spiny stellate cells were selected for reconstruction and morphological examinations by the following criteria: 1) in the barrel wall; 2) spiny appearance; 3) orientated dendrites and 4) complete dendritic arbor. The area occupied by the dendrites is defined as dendritic span which was estimated as the area circled by the dendritic tips (Lee et al., 2005b). The dendritic parameters such as dendritic span and total dendritic length were measured from the two-dimensional display by ImageJ program. The number of primary dendrites, branching points, and terminal endings were counted manually. The concentric sphere method of Sholl (1953) was used to analyze dendritic complexity.
Recovery of neonatal whisker trimming
Rodents are tactile animals and very reliant on the function of whiskers in their daily activities (Brecht, 2007; Petersen, 2007; Diamond et al., 2008). We first asked if the whisker-dependent behaviors were altered after the recovery of early-life sensory deprivation. To examine the whisker-dependent tactile function, a basic gap-crossing test was used (Fig. 1). This type of test required no prior training of the animals. Once a rat was placed in the bright side of the testing apparatus, it would move to the dark side by animal nature. Rats moved rapidly to the dark side while crossing the short gap distance. However, when the gap distance was widened up to 4 or 5 cm, roughly the length of the rats' head, rats extended their head, used their whiskers to explore the surroundings and spent time by the edge. Generally, the maximum crossable gap distance of normal adolescent male (CON) rats was between 6 cm and 9 cm (7.08 ± 0.79 cm, n = 13). However, the maximum crossable distance of the WC 0–3 (whisker cut during P0 to P3) rats was reduced remarkably (4.94 ± 1.34 cm, n = 16) compared with controls (p b 0.001, Fig. 1C). The results demonstrated that the early-life sensory experience is required for the proper performance in the gap-crossing test. Furthermore, to test if there is a critical period in this behavioral task, we also bilaterally trimmed all the whiskers of rat pups at P3 only Table 1 Recovery of neonatally trimmed whiskers. Whisker 1 CON Row A Row B Row C Row D Row E
Whisker 2 WC 0–3
CON
Whisker 3 WC 0–3
CON
38.5 ± 1.72 36.4 ± 1.27 32.7 ± 4.24 28.7 ± 1.38 21.3 ± 3.06 40.7 ± 1.06 38.7 ± 1.5 36.6 ± 4.41 33.1 ± 1.35⁎ 25.6 ± 1.9 42.1 ± 1.73 40.1 ± 1.07 38.3 ± 1.42 34.4 ± 1.72⁎ 26.5 ± 2.37 43.5 ± 1.84 41.9 ± 1.21 37.3 ± 1.89 34.4 ± 1.27 27.5 ± 2.64 38.3 ± 3.02 34.7 ± 1.38 28.7 ± 1.89 27.9 ± 1.46 18.6 ± 1.65
WC 0–3 20.9 ± 1.07 23.7 ± 0.49 24.7 ± 0.76 26.6 ± 1.51 17.6 ± 1.51
Major whiskers were cut bilaterally in neonatal rats during P0 to P3 and allowed to regrow afterward. By the time of P30, most of the major whiskers examined had achieved normal length (in mm). Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 8) and neonatal whisker-cut (WC 0–3, n = 6) rats. ⁎ p b 0.05.
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(WC 3). Surprisingly, the very early (P0–P3) whisker cut-induced behavioral change in gap-crossing test did not occur in WC 3 rats (n = 15). The critical period, P0 to P3, determined by whisker-specific gap-crossing test matched well with the developmental time window for the maturation of whisker-to-barrel system as previously demonstrated by lesion studies (Van der Loos and Woolsey, 1973; Killackey and Belford, 1979; Schlaggar et al., 1993). Changes of cortical barrel patterns in the whisker-cut rats Whisker-specific tactile function is defective in the WC 0–3 rats, we next examined if there is an anatomical basis for this functional impairment. The cortical barrel pattern was the key structure to check. The barrels in the layer IV somatosensory cortex were revealed by cytochrome oxidase histochemistry in both control and whisker-cut rats (Figs. 2A and B). We first measured the overall area of each flattened cortex. No significant difference was found between the two (CON and WC 0–3) groups (122.1 ± 2.72 mm2 in CON, n = 10; 117.2 ± 4.53 mm2 in WC 0–3, n = 9; p = 0.63). Apparently, no difference in the overall pattern between the two groups was observed at first sight. However, the size of individual barrels corresponding to the trimmed whiskers was somewhat altered by early sensory deprivation. For all the barrels examined, the area of barrels in the WC 0–3 rats was larger than the normal ones. In some barrels, the difference achieved statistic significance (e.g. A2, A3, A4, C1, C2, D1, D3, E1 and E3 barrels, in Fig. 2C). When we combined the areas of all the measured barrels, there was a 14.5% enlargement in the WC 0–3 rats, compared with CON rats.
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Dendritic arbors are affected by early whisker trimming The subtle changes in the cortical barrels suggest cortical mechanism for the tactile functional impairment in the WC 0–3 rats. We then examined dendritic structures of the cortical neurons in the barrel field corresponding to the major whiskers. The layer IV spiny stellate neurons were chosen for morphological analyses. These cells were located in the barrel wall and orientated most of their dendrites toward the barrel center. Golgi–Cox impregnated neurons in the barrel field were selected for reconstruction according to their location, orientation and spiny appearance (Fig. 3A). The neurons from the WC 0–3 rats (n = 46 neurons from 6 animals) and normal rats (n = 59 neurons from 8 animals) had comparable soma size, number of branching nodes, terminal tips and total dendritic length; whereas neurons in WC 0–3 rats had more primary dendrites, less dendritic orders but greater dendritic span (Table 2). The enlarged dendritic span matched up well with the greater barrel areas (Fig. 2C) and enlarged receptive field in the neonatal whisker-deprived rats (Simons and Land, 1987). The complexity of dendrites was estimated by Sholl analysis. The numbers of intersections were comparable between the two groups except for the more distal regions (beyond 100 μm, Fig. 3B). The number of branching nodes and terminal tips was counted in relation to the distance from the soma center. The quantity and distribution of branching nodes in the two groups were similar (Table 2, Fig. 3C) whereas the spatial distribution of the terminal tips varied between the two groups. There were less terminals in the proximal regions yet more tips beyond 100 μm from the soma center in the neurons of WC 0–3 rats (Fig. 3D). These results implied the fault of dendritic pruning in these rats. Dendritic spines The morphology and number of dendritic spine are sensitive to neural activity (Alvarez and Sabatini, 2007). Sensory deprivation or pharmacological blockade of synaptic transmission alters the stability of spines (Zuo et al., 2005). We therefore examined the effects of bilateral early whisker trimming on the shape and density of dendritic spines of layer IV spiny stellate neurons (Fig. 4). The morphology of dendritic spines was affected. There were more branched (multiple heads) spines in the WC 0–3 rats (93 in 598 spines examined), which rarely occurred (38 in 585 spines) in the control ones (Fig. 4A). This distorted spine phenotype correlated with deafferentation-induced spine immaturity (Fiala et al., 2002). Furthermore, the density of total spines was also found to be changed in the WC 0–3 rats. The spine density was significantly (p b 0.05) increased in the 2nd (1.62± 0.06/μm, versus 1.30 ± 0.08/μm of controls), 3rd (1.62 ± 0.06/μm, as compared with controls, 1.39± 0.07/μm) and 4th (1.66 ± 0.06/μm, as compared with controls, 1.42± 0.08/μm) dendritic orders (Fig. 4B). Taken together, in the WC 0– 3 rats, dendritic arborization and spine density in the barrel cortex were changed. These alterations in the dendritic structures of layer IV spiny stellate neurons in these rats may serve as the anatomical basis for the whisker-specific tactile function impairments. Altered explorative activity in the early-life whisker-deprived rats
Fig. 2. Barrel patterns in the layer IV somatosensory cortex. The barrels in the posteromedial barrel subfield corresponding to the major whiskers were revealed by cytochrome oxidase histochemistry. Schematic outlines of individual barrels were drawn accordingly (A and B). The areas of large barrels (A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4) were measured (C). Bar is 1 mm. Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 10) and neonatal whisker-cut (WC 0–3, n = 9) rats (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
With abnormal somatic sensation, the WC 0–3 rats may exhibit abnormal explorative behavior. To test this possibility, individual rats were placed in an open field, and their locomotor activity was taperecorded and analyzed. Compared with normal rats, the total travelled distance was significantly increased in the whisker-cut rats (675.50 ± 119.11 cm in CON, n = 6; 1103.12 ± 118.34 cm in WC 0–3, n = 6, p b 0.01, Fig. 5A). The number of rearing was also increased in WC 0–3 rats (39.83 ± 3.57) compared with controls (24.20 ± 3.97, p b 0.05, Fig. 5B). Together, these results indicated that the early-life (P0–P3) whisker-deprived rats exhibited increased explorative activity in a novel open field.
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Fig. 3. Layer IV spiny stellate cells in the rat barrel cortex. Golgi–Cox impregnated layer IV spiny stellate cells from both groups were reconstructed. In this illustration, all dendritic spines are omitted (A). Dendritic complexity was estimated by Sholl analysis (B). The number of branching nodes (C) and terminal tips (D) were counted in relation to the distance from the soma center. Bar is 100 μm. Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 59 neurons) and neonatal whisker-cut (WC 0–3, n = 46 neurons) groups (⁎p b 0.05, ⁎⁎⁎p b 0.001).
Early-life sensory deprivation affects later social interactions We next examined the effect of early-life (P0–P3) whisker deprivation on social behaviors. Investigative, contact and following behaviors as well as the total social interaction time of male adolescent rats were observed for 10 min (Fig. 6). Six normal (CON) and 7 neonatal whisker-cut (WC 0–3) rats were used in this experiment. The occurrences of investigative and contact behaviors as well as the total social interaction time were significantly increased in the neonatal sensory-deprived rats (for investigative behaviors, 23.17 ± 3.68 in CON, 35.67 ± 3.72 in WC 0–3, p b 0.05, Fig. 6A; for contact behaviors, 9.33 ± 3.68 in CON, 20.83 ± 2.72 in WC 0–3, p b 0.05, Fig. 6B; for total social interaction time, 69.83 ± 8.16 s in CON, 130.03 ± 17.63 s in WC 0–3, p b 0.05, Fig. 6D). However, such increased frequency was not observed in the following behaviors (2.83 ± 1.22 in CON, 2.51 ± 1.12 in WC 0–3, p = 0.84, Fig. 6C). When the observation time was divided into two 5-minute episodes, further Table 2 Dendritic parameters of layer IV spiny stellate cells in the barrel cortex. Parameters
CON (n = 59 neurons)
WC 0–3 (n = 46 neurons)
Primary dendrites Branching nodes Terminal tips Highest order Total dendritic length (μm) Dendritic span (μm2)
3.15 ± 0.13 12.81 ± 0.42 17.80 ± 0.52 5.76 ± 0.16 839.88 ± 26.23 9327.45 ± 367.89
3.59 ± 0.16⁎ 12.72 ± 0.49 17.48 ± 0.59 5.20 ± 0.17⁎ 910.07 ± 41.8 13543.80 ± 823.51⁎⁎⁎
The morphological features of neurons were measured from the two-dimensional display. Results are presented as mean ± SEM. Asterisks are used to indicate significant differences between control (CON) and neonatal whisker-cut (WC 0–3) groups. ⁎ p b 0.05. ⁎⁎⁎ p b 0.001.
information was revealed. During the first 5-minute period, all behaviors examined and the total social interaction time were comparable between control and whisker-cut rats. However, during the second 5-minute period, sensory-deprived rats exhibited significantly higher frequency in the investigative (6.83 ± 1.08 in control, 15.02 ± 1.56, p b 0.01, Fig. 6A) and contact (3.67 ± 1.69 in control, 8.52 ± 0.99, p b 0.05, Fig. 6B) behaviors as well as in the total social interaction time (19.17 ± 4.36 s in CON, 70.01 ± 9.27 s in WC 0–3, p b 0.001, Fig. 6D). Discussion The present study has demonstrated the first time the consequences after the recovery of early short-term whisker deprivation. Our results also confirm the critical time window for the functional development of the whisker-to-barrel system. A temporary sensory deprivation during this early postnatal period (P0–P3) disturbs the normal development of the sensory system both in structure and in function. With impaired perceptual faculty, the early-life sensorydeprived animals exhibit abnormal explorative activity and social behaviors. Structural alterations after early-life whisker deprivation It has been argued that the structural plasticity in layer IV somatosensory cortex can only be demonstrated by lesion strategies during early development (Kaas et al., 1983; Fox, 2002). However, surgical procedures damage the integrity of the sensory system and may also induce side effects (Calia et al., 1998). Since non-invasive manipulation (e.g. whisker trimming) causes little or no macroscopic changes (Kaas et al., 1983), the robust morphological alterations in the
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Fig. 4. Layer IV spiny stellate cells in the rat barrel cortex. The dendritic spines of layer IV spiny stellate cells in the somatosensory cortex were revealed by Golgi–Cox impregnation method (A). Note that the numerous branched or multiple-head spines (arrowheads) appeared in the whisker-cut rats. Spine density was determined at each dendritic order (1st to 4th). The density of total spine was significantly increased in whisker-cut (WC 0–3) rats compared to controls, especially in the 2nd, 3rd and 4th dendritic orders (B). Bar is 10 μm in (A). Results are mean ± SEM (⁎p b 0.05, ⁎⁎p b 0.01).
barrel cortex following early surgical lesions might be the mixture of sensory deprivation and other effects. Here, we have confirmed that even minor manipulation such as whisker trimming during early postnatal period can also cause subtle yet significant morphological alterations in the barrel field. The early short-term sensory deprivation produces enlarged cortical barrels and increased dendritic spans corresponding to the manipulated whiskers. Moreover, the spine density and morphology are also sensitive to the early sensory deprivation. Our findings are consistent with the previous electrophysiological recordings. Layer IV cortical neurons in the barrel cortex exhibit larger excitatory receptive field and higher neural activity after the recovery of whisker removal began at birth (Simons and Land, 1987; Shoykhet et al., 2005; Lee et al., 2007). It is known that during neural development, considerable amount of dendritic processes and spines are overproduced and then later eliminated (Warren and Jones, 1997; Innocenti and Price, 2005). This pruning program takes place during the first postnatal weeks and is neural activity-dependent (Segal and Andersen, 2000; Cline and Haas, 2008). An exciting study using two-photon microscopy technique has shown that the elimination of dendritic spine in the layer V pyramidal neurons of barrel cortex is greatly reduced by whisker trimming (Zuo et al., 2005). Similar results can be reproduced by chronic blockade of NMDA receptors, the authors has concluded that glutamatergic neurotransmission plays a critical role in this process. In cortexspecific NMDA receptor deficient mice, the excitatory neurotransmission is greatly reduced (Iwasato et al., 2000), the dendritic arbor and spine density are also altered (Datwani et al., 2002). In these mice, the dendrites of layer IV spiny stellate neurons have longer length and greater spine density than the wildtype controls. These results show the fault of dendritic pruning in NMDA receptor deficient mice and again support the neural activity-dependent sculpture of dendritic arbors (Segal and Andersen, 2000; Lee et al., 2005b; Cline and Haas, 2008). In the present study, with deficient sensory input, the layer IV
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spiny stellate neurons in sensory-deprived rats may receive fewer amount of or less organized neural activity insufficient to activate NMDA receptor (Liu, 2003). As such, the spiny stellate neurons in sensory-deprived rats, like in the NMDA receptor mutant mice, exhibit impaired pruning of dendritic arbors and spines. Since the pruning process occurs during a limited time window, the longer dendritic length, greater dendritic span and higher spine density are left in the whisker-deprived rats even though the whiskers have fully regrown. In the present study, we have demonstrated that the morphology and density of dendritic spine are also sensitive to early-life temporal sensory deprivation. The increased spine density and altered spine shape reflect the shortness of neural activity and spine immaturity that frequently appeared in deafferentation (Fiala et al., 2002) and mental retardation (Kaufmann and Moser, 2000). In fact, the failure of dendritic pruning is observed in an animal model of Fragile X mental retardation syndrome (Comery et al., 1997). The cortical neurons of Fragile X mental retardation patients have distorted spine features. These spines are longer, thinner, tortuous and sometimes multiple swellings are present (Kaufmann and Moser, 2000; Fiala et al., 2002). The increased spine density and the appearance of multiple-head spines in WC 0–3 rats resemble the dendritic phenotype of Fragile X syndrome, again supporting neural activity-dependent mechanism in the morphogenesis of dendritic structures. On the other hand, the Fragile X mental retardation protein (FMRP), absent in Fragile X patients, is normally located in the synapse and is important for the integrity of synaptic structure (Weiler et al., 1997). The expression of FMRP is up-regulated in the barrel field after whisker stimuli (Todd et al., 2003). Hence, it is important to examine the expression of FMRP, as well as other synaptic proteins, in the barrel cortex of whiskerdeprived animals. Several synaptic proteins including NMDA receptor, Dynamin I, and Synaptotagmin I, are found to be changed in the cortical neurons of visual deprivation animal models (Murphy et al., 2004; Cnops et al., 2008). In the visual system, early visual disruption often results in later poor visual acuity known as amblyopia. Dark rearing and monocular deprivation are commonly used protocols for visual deprivation (Fagiolini et al., 1994). Monocular deprivation causes robust functional and structural changes along the visual pathway by creating a mixture condition of altered visual stimulation and binocular competition (Murphy et al., 2004). We therefore suggest that the dark rearing paradigm for visual deprivation is relatively close to the bilateral whisking trimming protocol in the present study. In dear-reared rats, larger receptive field and lower visual acuity are found in the cortical level (Fagiolini et al., 1994). Layer IV stellate neurons in the visual cortex of dark-reared rats lost their polarity resulting from uneven growth of dendrites (Borges and Berry, 1978). Our findings of enlarged
Fig. 5. Explorative activity in the open field. Locomotor activities of adolescent rats were examined in an open field for 10 min. The total travelled distance (A) and number of rearing (B) were analyzed. Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 6) and neonatal whisker-cut (WC 0–3, n = 6) rats (⁎p b 0.05).
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Fig. 6. Altered social behaviors in the early-life sensory-deprived rats. Effects of sensory deprivation on social interactions were evaluated by the investigative (A), contact (B), following (C) behaviors and overall social activities (D) of male adolescent rats during a 10-minute observation period. Counts of a particular behavior or total time of all behaviors were presented as in the first 5 min (0–5:00), in the succeeding 5 min (5:00–10:00) and in the whole 10 min (total). Results are mean ± SEM. Asterisks are used to indicate significant differences between control (CON, n = 6) and neonatal whisker-cut (WC 0–3, n = 7) rats (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
barrels and prolonged dendrites in the somatosensory cortex of whisker-trimmed rats are in accordance with the results produced by visual deprivation. Despite the fundamental differences between the visual and somatosensory systems, they share some common developmental procedures that require proper sensory experiences for their maturation (Grubb and Thompson, 2004; Fox and Wong, 2005). Behavioral performances after the recovery of early-life whisker deprivation Since the dendritic spines of layer IV spiny stellate neurons are the first receiving sites of thalamic inputs in the sensory cortex, altered dendritic structures imply functional deficits. With tortuous dendritic spines, the sensory-deprived animals may suffer poor synaptic transmission and integration. Consequently, the animals are less perceptive to the environmental stimuli, which in turn affects the decision making (Celikel and Sakmann, 2007). Indeed, these rats with fully regrown whiskers exhibit shorter crossable gap distance, confirming that the function of their whisker-dependent sensory system is impaired. The present study is the first experiment, to our knowledge, that demonstrates functional deficit after the recovery of short-term (few days) early-life whisker deprivation. Furthermore, by using whisker-specific gap-crossing test, we have also defined the critical time window for the functional maturation of the whisker-tobarrel system. However, we cannot exclude deficits, if any, in the WC 3 rats, insensitive to the gap-crossing test (Shoykhet et al., 2005). Neither can we extract the emotional constituent in this test. Nevertheless, the results still clearly demonstrate that the early-life sensory experience is required for the proper performance in the gapcrossing test. A number of whisker-related behavioral changes are observed in rats which have their whiskers removed from birth but did not recover (Symons and Tees, 1990; Shishelova, 2006). However, without full regrowth, it is difficult to address the temporal importance of the manipulation. Simons and Carvell have beautifully presented whiskerdependent behavioral changes in rats after recovery from 45-day whisker trimming which began at birth. The behavioral abnormality they demonstrated is task-sensitive. Early sensory experience is essential for conducting behavioral task that requires two or more
whiskers, implying that the ability of integrating inputs is established based on normal sensory experience (Carvell and Simons, 1996). Based on their findings and ours, we conclude that the concept of the external three-dimensional dynamic world is built upon the internal sensory map which is shaped by sensory experience during early development. With altered somatic sensation, neonatal whisker-cut rats later exhibit increased explorative behaviors. Similar findings are observed in children with inadequate sensory stimuli particularly during their early childhood (Lewis, 1978; Lin et al., 2005). It has been pointed out that these children are hyperactive and have poor discrimination. The increased physical activity and reduced discrimination together produce more random or meaningless activities. However, the excess activities do little to help extract information from the environment. As such, the favorable opportunities for learning may be reduced (Lewis, 1978). In fact, the sensoryprocessing deficit is one of the major causes for learning difficulties (Goswami, 2006). Experiments are underway to examine whether the capacity of spatial learning is affected in the early-life sensorydeprived animals. Besides altered locomotor activity, abnormal social behaviors in WC 0–3 rats are also observed. These rats behave normally during the first 5-minute section; however, they exhibit significantly higher frequency in investigative and contact behaviors during the second 5minute section. These aberrant behaviors also validate the hyperactive and poor-in-discrimination nature of early-life sensory-deprived animals. The increased social interactions found in the sensorydeprived rats here are also similar to the early-life social-deprived animals (Varlinskaya and Spear, 2008). It is important to examine if the early whisker-deprived rats are, like socially isolated animals, prone to develop emotional problems (Ibi et al., 2008). Conclusion Small initial difference in the sensory-processing systems can lead to significant problems later in life (Beddington et al., 2008). With minor and brief manipulation, we have demonstrated significant structural and, more importantly, functional abnormalities in the somatosensory system. The sequelae of early-life sensory deprivation may be far more serious than estimated.
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