SEARCH

SEARCH BY CITATION

Keywords:

  • enriched environment;
  • stereology;
  • myelinated fiber;
  • corpus callosum;
  • aged rat

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

In this study, the effect of enriched environment (EE) on the spatial learning of aged rats was examined, and then the effects of EE on the aged corpus callosum (CC) were investigated by means of the modern stereological methods. We found that EE significantly improved the spatial learning of aged rats. The CC volume, the total volume of the myelinated fibers and total volume of the myelin sheaths in the CC, the total length of the myelinated fibers in the CC of enriched rats were significantly increased when compared to standard rats. The increase of the myelinated fibers in enriched rat CC might provide one of the structural bases for the enrichment-related improvement of the spatial learning. This study provided, to the best of our knowledge, the first evidence of environmental enrichment-induced increases of the CC and the myelinated fibers in the CC of aged rats. Anat Rec, , 2012. © 2012 Wiley Periodicals Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

It has been reported that in rodents, complex environment improved performance on cognition tests (Hymovitch, 1952), and reduced the learning and memory decline typically associated with aging (Kempermann et al., 1998; van Praag et al, 2000; Frick and Fernandez, 2003). Moreover, the protection effects of complex environment on the age-related cognitive decline of murine were correlated well with the increases in neurogenesis, synaptic density, and neurotrophic factors (Mora et al. 2007; Segovia et al., 2009). After exposure to an enriched environment (EE), an increase was found in BrdU-labeled neurons in mouse dentate gyrus (Kempermann et al., 1997, 1998, 2002), the oligodendrocyte nucleus density in the rat visual cortex (Szeligo and Leblond, 1977; Sirevaag and Greenough, 1987), the ratio of synapses per neuron in the cortex of male rats (Turner and Greenough, 1985; Briones et al., 2004) and the levels of the presynaptic protein synaptophysin in the hippocampus and neocortex (Frick and Fernandez, 2003; Lambert et al., 2005). Recent studies found the localized increase in the structural integrity and coherence of white matter tracts in the healthy adult human white matter with 6-week juggling training (Scholz et al., 2009) and in the long-term trained adult Baduk players (Lee et al., 2010).

The corpus callosum (CC) plays an important role in interhemispheric integration and communication as the main interhemispheric fiber tract (Biegon et al., 1994; Schlaug et al., 1995; Bloom and Hynd, 2005; Glickstein and Berlucchi, 2008). Evidence from previous studies suggested that the CC could alter with experience or training. Using MRI technique, Sánchez et al. (1998) found the mean mid-sagittal area of the CC was significantly increased in enriched rhesus monkeys from the age of 2–12 months. In humans, the mid-sagittal area of the anterior CC of adult musicians with a long-term musical training was increased when compared to controls (Schlaug et al., 1995) and amount of piano practicing in childhood and early adulthood correlated with CC fiber tract as observed with diffusion tensor imaging (DTI) (Bengtsson et al., 2005). Similarly, area and fiber tract of the human anterior CC was found modifiable by experience from adulthood into old age (Lövdén et al., 2010). However, the histological investigations about the effects of EE on the CC are still insufficient and highly controversial. Juraska and Kopcik (1988) reported that the CC of rats remained sensitive to extrinsic influences well beyond the early postnatal period. In contrast, Markham et al. (2009) concluded that the myelination in rat CC was not readily sensitive to the manipulation of housing environment during adulthood. To the best of our knowledge, there has been no study investigating the effects of EE on the CC and on the myelinated fibers in the CC using the three-dimensional (3D) quantitative techniques, the modern stereological techniques. Therefore, this study investigated the effects of EE on the CC and on the myelinated fibers in the CC of middle-aged female rats using the modern stereological techniques.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Housing Conditions

Twenty-four female Sprague Dawley rats (14-months-old) were divided randomly into two groups, 12 rats lived in an EE (one large cage containing wood shavings, a rearrangeable set of tunnels, various toys and other small constructions, the cage dimension is 120 × 60 × 50 cm3) (Kempermann et al., 1997, 1998) and 12 control rats were reared four per cage in standard laboratory cages (SE, 40 × 30 × 30 cm3) containing only wood shavings. The toys and constructions were changed once a week at the time of cage cleaning (Kempermann et al., 1997, 1998). The rats were housed for 4 months in a temperature-controlled room, with a constant 12:12 h light/dark cycle. Animal care and treatment were conducted in accordance with NIH guidelines (NIH Publications No. 80-23).

Behavioral Testing

Morris water maze test used a circular tank with a diameter of 200 cm and a height of 80–90 cm, which was filled with water (24 ± 2°C). The water was made opaque with nontoxic paint. The tank was divided into four quadrants. The four start positions were located at the intersections of the quadrants. A platform with a diameter of 10 cm was hidden 2 cm below the water level. Rats were first placed on the platform for 15 sec and then placed into water. Each rat received four trials per day for four consecutive days. The start position was changed on every trial. The swim time to reach platform was recorded in each trial. If a rat failed to reach the platform in 3 min, it was taken out and placed on the platform for 15 sec. On day 5, the time to reach the visible-platform was measured (Morris, 1984; Brandeis et al., 1989).

Tissue Preparation

Five rats from each group were anaesthetized using 4% chloral hydrate intraperitoneally. Then, they were perfusion-fixed with 2% paraformaldehyde and 2.5% gluraraldehyde, the cerebrum was taken out and divided into two hemispheres along the mid-sagittal plane. Each hemisphere was then coronally cut into serial 1-mm thick parallel slabs, starting randomly at the rostral pole. On average, 12 (CV = 0.14) slabs were obtained from each hemisphere.

Estimation of the Total CC Volume

A transparent counting grid with an area of 0.39 mm2 associated with each point was placed randomly on the caudal surface of each slab under the anatomy microscopy, the total number of points hitting the CC was counted, and the total volume of CC, V (cc), was calculated according to the Cavalieri's principle (Tang et al., 1997, 2003).

Estimation of the Volume Density and Total Volume of Myelinated Fibers in CC

The right or left hemisphere was sampled randomly. Every third slab was sampled systematically from the slabs of the sampled hemisphere, the first one being sampled from the first three slabs randomly. A plastic sheet with equidistant points was placed randomly on the caudal surface of the sampled slabs. The tissue blocks were obtained where the points in the sheet hit the CC. The tissue blocks were then fixed in 4% glutaraldehyde at 4°C, rinsed in 0.1 M phosphate buffered saline (pH 7.2) three times followed by osmicated in 1% 0.1 M phosphate buffered osmium tetroxide at 4°C. The blocks were gradually dehydrated, then infiltrated with epoxy resin 618 (Chen Guang Chemical Industry, Sichuan, China). The tissue was pre-embedded in 5-mm spheres with Epon. After being solidified, the spheres were rotated randomly before re-embedded. This operation process was called isector (Nyengaard and Gundersen, 1992), which ensured to obtain isotropic, uniform and random sections so that each tissue sample had a uniformly random orientation before being cut (Tang and Nyengaard, 1997). The tissue blocks were then re-embedded in epon resin.

One section with the thickness of 60 nm was obtained from the center of each epon block using an ultramicrotome. The ultrathin sections were then viewed in a TEM (Hitachi-7500, made by Hitachi, Japan). From each section, four fields of vision were randomly photographed at a magnification of 6,000×. A transparent counting grid with total points of 360 was randomly placed on the photographs. The points hitting the myelinated fibers, ∑P (mf), the points hitting the myelin sheaths, ∑P (ms), and the points hitting the CC, ∑P (cc), were counted, respectively. The volume density and total volume of the myelinated fibers in CC, the volume density, and total volume of myeline sheaths in CC were estimated as previously described (Tang et al., 1997, 2003).

Estimation of the Length Density and Total Length of Myelinated Fibers in CC

The unbiased counting frame (Gundersen, 1977) was superimposed randomly on the randomly captured photographs. Myelinated fiber profiles inside the counting frame or touching the top and right lines (inclusion lines) were included for counting and myelinated fiber profiles touching the left line and bottom line and the extensions of the right line and left line (exclusion lines) were excluded for counting. The length density and total length of the myelinated fibers in CC were estimated as previously described (Tang et al., 1997, 2003).

Estimation of Mean Diameter of Myelinated Fibers in CC

The diameter of myelinated fibers sampled by the unbiased counting frame was estimated by measuring the longest profile diameter perpendicular to its longest axis (Tang et al., 1997, 2003). The measurements were obtained from the outer limit of the myelin sheath.

Estimation of Tissue Shrinkage

One tissue block was randomly taken from each CC. The dimensions of these pieces of blocks were measured carefully before being processed, and the tissue cross sectional area of each block was calculated. After being processed, the dimensions of these pieces of tissue blocks were measured carefully again, and the area of the tissue sections was computed again. The measurements were compared to see if significant shrinkage had occurred. The amount of shrinkage was estimated as previously described (Tang et al., 1997, 2003).

Statistics

All statistical analyses were performed with SPSS 16.0. Morris water maze data between the two groups from day 1 to 4 were analyzed using repeated measures analysis of variance (ANOVA). The outcome of day 5 was analyzed with an one way ANOVA. Unpaired, two-tailed Student's t test was used to determine whether the stereological data in the entire CC were significantly different between standard environment (SE) group and EE group. Coefficient of error and observed interbrain coefficient of variation were estimated as before (Gundersen et al., 1999).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Morris Water Maze Tests

For hidden-platform test, the mean escape latency of EE group from day 1 to 4 was significantly shortened when compared to SE group (P = 0.001, Fig. 1). There was no significant difference in the visible-platform test between EE group and SE group (P = 0.069, Fig. 1).

thumbnail image

Figure 1. Effects of EE on spatial learning capacity. Escape latency in EE group from day 1 to 4 is significantly shortened when compared to SE group (P = 0.001). For visible-platform task on day 5, there is no significant difference between EE rats and SE rats (P = 0.069). Each point represents the mean ± SEM (standard error of the mean) for each group during one test session.

Download figure to PowerPoint

Stereological Estimations

The average areal shrinkage induced by tissue processing was 8.0%. As the tissue shrinkage was not statistically significant (P = 0.054), the final stereological estimates were not corrected for the tissue-processing induced shrinkage. Stereological measurements of the CC and Stereological measurements of the myelinated nerve fibers in the CC were presented in Table 1.

Table 1. Stereological estimates of the corpus callosum and the myelinated fibers in the corpus callosum of middle-aged female rats
AnimalV(corpus callosum) (mm3)VV (mf)V (mf) (mm3)LV (mf) (km/mm3)L (mf) (km)D (mf) (μm)
  1. SE: standard environment, EE: enriched environment, Mean: mean value of each variable, SD: standard deviation, OCV: observed coefficient of variance, CE: coefficient of error.

SE
 Mean68.90.641.10.568.80.7
 SD7.10.18.70.120.70.04
 OCV (%)10.313.821.120.230.15
 CE (%)4.66.29.49.013.42.3
EE
 Mean92.70.766.10.7125.60.63
 SD16.20.112.70.136.50.02
 OCV (%)17.47.519.216.329.13.9
 CE (%)7.83.48.67.3131.8

The mean volume of the CC in EE rats was significantly increased by 34.5% (P = 0.017) when compared to that in SE rats (Fig. 2). The myelinated fiber volume and myelin sheath volume in the CC of EE rats were significantly increased by 60.8% (P = 0.007) and 57.7% (P = 0.008), respectively, when compared with those of SE rats (Fig. 3). The total length of myelinated fibers in the CC of EE rats was significantly increased by 82.5% when compared to that of SE rats (P = 0.016, Fig. 4A). In contrast, the mean diameter of myelinated fibers in the CC of EE rats was significantly decreased by 9.3% when compared to that of SE rats (P = 0.045, Fig. 4B).The absolute distributions of the size-category myelinated fiber length in the CC of EE rats and SE rats were shown in Fig. 4C, which indicated that the enrichment-induced changes of the myelinated nerve fibers in the CC of EE rats were mainly due to the marked increases of the myelinated nerve fibers with diameter less than 1 μm, especially the fibers with diameter of 0.4–0.6 μm (Fig. 4C).

thumbnail image

Figure 2. The comparison of the mean volume of the CC between EE group and SE group. * indicates the mean volume of the CC in EE rats is significantly increased (P = 0.017) when compared to that in SE rats.

Download figure to PowerPoint

thumbnail image

Figure 3. The comparisons of the mean myelinated fiber volume and mean myelin sheath volume in the CC between EE group and SE group. ** indicates both the myelinated fiber volume (P = 0.007) and myelin sheath volume (P = 0.008) in the CC of EE rats are significantly increased when compared with those of SE rats.

Download figure to PowerPoint

thumbnail image

Figure 4. The comparisons of the total length of the myelinated fibers (A), the mean diameter of the myelinated fibers (B), the absolute distributions of the size-category myelinated fiber length (C) in the CC between EE group and SE group. * indicates the total length (P = 0.016) and mean diameter (P = 0.045) of myelinated fibers in the CC of EE rats are significantly changed when compared to those of SE rats.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

In previous studies, the effects of experience and EE on the size of the CC were investigated by means of MRI techniques (Schlaug et al., 1995; Sánchez et al., 1998) and DTI methods (Bengtsson et al., 2005; Scholz et al., 2009; Lee et al., 2010; Lövdén et al., 2010). Although neuroimaging techniques such as MRI and DTI provided opportunities for whole-brain studies in living subjects, the measures derived from magnetic resonance imaging were indirect and their interpretation was complex (Scholz et al., 2009). Therefore, studies using cellular and biochemical techniques were required to determine the biological basis of the observed changes (Scholz et al., 2009). In previous histological studies, the number of myelinated axon profiles in the mid-sagittal sections of splenium was counted (Juraska and Kopcik, 1988; Markham et al., 2009). According to what they described, they estimated the length density of the myelinated axons in the splenium of complex rats and isolated housing rats. However, biological conclusions based on density were very difficult to interpret because it would never be known if changes in density were due to an alteration of total quantity and/or an alteration in the reference volume (Braendgaard and Gundersen, 1986). As they did not estimate the total volume of the splenium part of the CC in both complex rats and isolated housing rats, they could not obtain the total length of the myelinated axons in the splenium part of the CC in both groups. Furthermore, in previous studies, they had to define the boundary of certain portion of the CC. It was very difficult to keep the definition identity for each animal. In addition, previous studies were based on the assumption that the sections were cut perpendicular to the nerve fibers of the CC. The assumption was hard to be true. Therefore, they could not make firm conclusions on the changes of the myelinated axons in the splenium part of the CC between two groups (Juraska and Kopcik, 1988; Markham et al., 2009). In this study, we quantitatively investigated the total volume of the CC, the total volume of the myelinated nerve fibers, and total volume of the myelin sheaths and the total length of the myelinated fibers in the CC using 3D unbiased stereological methods and electron microscopical techniques. Therefore, our results are the estimates of total quantities of the CC and myelinated nerve fibers in the CC and can be interpreted unambiguously.

In this study, we found that the spatial learning of the enriched rats was significantly improved, which was in accordance with the reports by other groups (Kempermann et al., 1998; Nilsson et al., 1999; Frick and Fernandez, 2003; Leggio et al., 2005). By means of MRI techniques, Sánchez et al (1998) found the corrected volume of prefrontal white matter and the corrected volume of parietal white matter of social environmental macaque infants were significantly larger than those of nursery animals. Using DTI methods, Lee et al (2010) found that the cluster volume of white matter with increased fractional anisotropy values was increased in long-term trained Baduk players when compared to the controls. The mid-sagittal area of CC and mid-sagittal area of subregions of CC of social environmental macaque infants (Sánchez et al., 1998), adult musicians (Schlaug et al., 1995) and enriched rats (Juraska and Kopcik, 1988; Markham et al., 2009) were found significantly increased when compared to those of controls. Despite the cluster volume and the mid-sagittal area of CC were not equal to the total volume of the CC, they partly reflected the size of the CC. In this study, we found that EE induced increase in the total volume of the CC in middle-aged female rats. Larger CC area should bring a performance advantage for difficult and demanding tasks and allow for optimal integration of cortical activity (Pandya and Seltzer, 1986; Yazgan et al., 1995). Previous studies demonstrated that more symmetrically organized brains had larger callosum (DeLacoste-Utamsing and Holloway, 1982; Witelson, 1985; O'Kusky, 1988; Witelson, 1989; Steinmetz, 1992), and increase in callosal size was generally considered to be a morphological base of interhemispheric connectivity and of hemispheric (a)symmetry (Schlaug et al., 1995). The enrichment-induced structural changes of CC might potentially be involved in neural plasticity, such as learning and/or memory consolidation (Fields, 2008). Therefore, we speculated that the enrichment-induced changes in the CC volume might be one of the structural bases for the enrichment-induced improvement of spatial learning. However, the exact functional consequence of the enrichment-induced structural changes in the CC volume of the middle-aged female rats needs to be further investigated.

In previous histological studies, only the density of nerve fibers in the CC has been investigated from 2D microscopic images (Juraska and Kopcik, 1988; Markham et al., 2009). This study used new stereological methods to investigate the effects of EE on the myelinated fibers in the CC of middle-aged female rats. We found that the total volume of the myelinated fibers and the total volume of the myelin sheaths in CC were all significantly increased in enriched rats when compared to standard rats. The enrichment-induced significant increase of the total volume of the myelinated fibers and myelin sheaths might partly account for the enrichment-induced increase of the CC volume. Furthermore, the current results demonstrated that EE increased the total length of myelinated fibers in the aged CC, especially the myelinated fibers with smaller diameters. The significant prolongation of myelinated nerve fibers with smaller diameters suggested that the thinner, later myelinating fibers be susceptible to environmental enrichment, as the thinner, later myelinating fibers were susceptible to the age-related changes (Tang et al., 1997; Marner et al., 2003; for review, see Bartzokis, 2004). Our research team found enrichment-induced new oligodendrocytes in aged CC (personal communication). Therefore, we speculated that the enrichment-induced increase in the thin myelinated nerve fibers in aged CC might indicate the enrichment-induced remyelination in the CC. The increased length of the myelinated fibers with small diameters in the enriched rat CC might lead to our present result, a reduction in the mean diameter of the myelinated fibers in the CC after environmental stimulation. However, the exact mechanism that EE leads to a reduction of the mean diameter of the myelinated fibers in the CC needs to be further studied. Neuroanatomical tracing studies indicated that the thin fibers (less than 1 μm) concentrated in the CC seemed to connect prefrontal and higher-order processing areas of the temporal and parietal lobes (Pandya and Seltzer, 1986). The fibers with smaller diameters were thought to be important in maintaining the balance between excitation and inhibition in cerebral hemispheres (Yazgan et al., 1995; for review, see Bloom and Hynd, 2005). The delay produced by interhemispheric transfer through smaller diameter fibers might permit cascade processing, where computation in separate hemispheric modules can be time-staggered (Pandya and Seltzer., 1986). Moreover, the efficiency of myelination in CC related closely to the cortical cognition (Peters and Sethares, 2002; Bloom and Hynd, 2005). Changes in these properties might underlie behavioral improvements by altering conduction velocity and synchronization of nervous signals (Fields, 2008). Therefore, we speculated that the enrichment-induced increases in the total volume and the total length of the myelinated nerve fiber, especially the thin myelinated nerve fibers, in aged CC might be related to the enrichment-induced improvement of spatial learning. However, the relationship between the enrichment-induced increases of the myelinated nerve fiber in the CC and the functional consequence of this structural plasticity need to be further investigated in the future.

In conclusion, our results further confirmed that EE significantly enhanced the spatial learning of middle-aged female rats. The current study for the first time provided cellular evidence to demonstrate the enrichment-induced plasticity of the CC of the middle-aged female rats. The enrichment-induced increase of the CC volume was mostly due to enrichment-induced increase of the myelinated nerve fibers in the CC. The enrichment-induced change in the CC might be one of the structural bases for the enrichment-induced improvement of spatial learning ability.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED