Brain weight differences associated with induced focal microgyria
© Peiffer et al; licensee BioMed Central Ltd. 2003
Received: 28 January 2003
Accepted: 24 June 2003
Published: 24 June 2003
Disrupting neural migration with bilateral focal freezing necrosis on postnatal day 1 (P1) results in the formation of 4-layered microgyria. This developmental injury triggers a pervasive neural reorganization, which is evident at the electrophysiological, behavioral, and anatomical levels. In this experiment, we investigated changes in brain weight as an index of global disruption of neural systems caused by focal damage to the developing cortical plate.
We found a dramatic reduction in overall brain weight in microgyric subjects. This reduction in brain weight among animals with microgyria is reflected in decreased total brain volume, with a disproportionate decrease in neocortical volume. This effect is so robust that it is seen across varied environments, at variable ages, and across the sexes.
This finding supports previous work suggesting that substantial reorganization of the brain is triggered by the induction of bilateral freezing damage. These results have critical implications for the profound re-organizational effects of relatively small focal injuries early in development to distributed systems throughout the brain, and particularly in the cerebral cortex.
Focal damage to the developing brain can have widespread consequences for structures and regions that project to or receive direct or even indirect projections from the damaged area [1–3]. Focal neocortical malformations induced by freeze injury to the developing cortical plate exemplify this fact. It has been shown that these malformations, resembling human microgyria, are associated with widespread disturbances in neuronal organization[4, 5]. For example, brain slices containing microgyric cortex show increased epileptogenic activity [6–9]. Microgyric anomalies are also associated with connectional alterations [10–12], affecting both thalamic and cortico-cortical connectivity. In addition, changes in neuronal cell size distribution in the medial geniculate nuclei associated with the presence of neocortical malformations, have been demonstrated in both human dyslexic brains and rat brains [14–16].
Along with these structural alterations, damage to the developing brain produces functional changes, suggesting that structural changes are not necessarily maladaptive in all cases. For example, the presence of neocortical malformations in certain strains of inbred mice are correlated with better performance on tasks of spatial reference memory[17, 18]. On the other hand, induced microgyric animals show a variety of behavioral deficits that suggest disturbances to multiple brain systems [15, 19–22]. Rapid auditory processing deficits have been found in males with induced microgyria using both an operant condition paradigm and a reflex modification paradigm [15, 19–21]. In both paradigms, auditory processing deficits were found only on rapidly changing stimuli, and not when these same stimuli were delivered at slower rates. Further, animals with induced microgyria showed poorer performance in discrimination learning and took longer paths to the target in the Morris Water Maze than sham littermates.
The extent of brain-wide changes induced by the relatively restricted freezing lesion that produces microgyria is, however, unknown. Here we present evidence that induced bilateral focal microgyria leads to significant reductions in overall brain weight that are quite robust, being seen at all ages measured (P30 – P118), in both males and females, and are unaffected by acoustic rearing experiences. Moreover, volumetric analysis suggests this reduction largely reflects a disproportionate decrease of neocortical tissue.
The present experiment was comprised of three studies (see Methods for complete details). Study 1 examined the effect of rearing in three different acoustic Environments (enriched, deprived, and control). The effect of Age (P30, P52, P83) was investigated in Study 2, while the effect of Sex was assessed in Study 3.
Body Weight Analysis
Mean (± SEM) Body Weights for All Studies
506.8 ± 22.4
459.9 ± 25.4
449.4 ± 10.6
462.1 ± 8.3
408.9 ± 14.1
364.0 ± 9.3
97.1 ± 4.7
98.2 ± 3.7
230.6 ± 8.2
233.0 ± 11.9
381.0 ± 10.4
382.5 ± 9.5
443.8 ± 9.7
459.3 ± 7.3
238.4 ± 8.1
254.5 ± 5.6
Brain Weight Analyses
Mean (± SEM) Residual Brain Weight Scores Regressed Against Body Weight for All Studies
-0.097 ± 0.035
0.079 ± 0.039
-0.044 ± 0.038
0.105 ± 0.028
-0.092 ± 0.027
0.040 ± 0.033
-0.070 ± 0.025
0.041 + 0.026
-0.068 ± 0.018
0.093 ± 0.015
-0.079 ± 0.023
0.044 ± 0.026
-0.050 ± 0.031
0.030 ± 0.034
-0.058 ± 0.038
0.077 ± 0.041
Brain Volume Analyses
Mean (± SEM) Residual Volume Scores Regressed Against Body Weight.
-51.44 ± 11.26
7.92 ± 12.36
-94.55 ± 21.32
89.35 ± 34.37
45.73 ± 23.36
-15.74 ± 35.59
-13.38 ± 3.68
-14.09 ± 5.28
-31.16 ± 5.46
35.53 ± 10.64
19.51 ± 8.28
0.01 ± 8.41
-38.06 ± 8.83
22.01 ± 12.72
-63.50 ± 17.59
53.82 ± 24.03
26.22 ± 18.01
-15.75 ± 27.60
Cortex: Total Brain
-0.00 ± 0.004
-0.02 ± 0.008
-0.01 ± 0.007
0.02 ± 0.004
0.01 ± 0.008
0.01 ± 0.004
-0.35 ± 1.90
1.79 ± 1.31
-1.75 ± 1.15
An ANOVA using the residuals of neocortical volume as the dependent measure were similar to those of brain volume (Figure 3B). Microgyric subjects had smaller neocortical volumes than shams (F1,31 = 38.00, p < 0.001), irrespective of environmental condition. Environmental effects remained significant (F2,31 = 6.27, p< 0.01), with those subjects in the acoustically Enriched group having smaller neocortical volume residuals than those in the other conditions. There was no interaction between Lesion and Environment (F2,31 < 1, NS).
An ANOVA was performed for non-neocortical volume (computed by total brain volume minus neocortical volume), to assess whether Lesion effects and Environmental effects were specific to cortex, or distributed through non-neocortical areas as well. Analysis showed a main effect of Lesion (F1,31 = 9.62, p < 0.01), and Environment (F2,31 = 5.83, p < 0.01), with no interaction (F2,31 = 2.85, p > 0.05; see Figure 3C). However, relative F values suggested that while Environment main effects were equivalent between cortex and non-cortex, Lesion effects were more pronounced in the neocortex.
To directly assess the issue of a disproportionate loss of neocortical volume in Lesion subjects, we computed the ratio of neocortical to total brain volume, regressed those values against body weight, and analyzed the residuals. An ANOVA revealed a significant effect of Lesion (F1,31 = 25.66, p < 0.001) but not Environment (F2,31 = 2.97, p > 0.05), and no interaction between the two (F2,31 < 1, NS). Thus, microgyric subjects had disproportionately smaller cortices than their sham counterparts (Figure 3D). Environment, conversely, affected the whole brain and had no significant effects on this ratio measure. It is important to note that the enrichment procedure of this study differed significantly from those used previously in that it began prenatally. Future research will be needed to determine what possible effects this additional variable had on subjects' body weight and brain volume.
The current findings show that early focal brain injuries, specifically P1 focal freezing necrosis producing microgyria, lead to reductions in brain weight that are so robust that the main effect of Lesion does not interact with Environment, Age, or Sex. Analysis of histologic brain and neocortical volume confirmed the effects of lesion on brain weight, and suggest that the reduction in volume associated with microgyria is proportionally greater in the cerebral cortex. This finding supports previous work suggesting that substantial reorganization of the brain is triggered by the induction of bilateral freezing damage. Surprisingly, this reorganization was not differentially affected across ages, by an enriched environment, nor by the difference in brain weight between the sexes (e.g.,.Refs. [27, 28]). Further, reorganization (i.e., brain weight and volume reduction due to microgyria) was evident by P30 and remained consistent into adulthood.
Since reductions in brain weight and total brain volume in Microgyric (Lesion) subjects reflect disproportionate loss of neocortical tissue, it is important to address the unlikely possibility that this effect reflects direct tissue loss from lesion induction. This is unlikely because no tissue was removed. Further, though microgyric neocortical tissue showed anomalous organization, there was no demonstrable tissue reduction localized to the malformation itself (see Figure 1).
Unlike Lesion effects, the reduction in brain volume seen in subjects raised in an acoustically Enriched environment was evident throughout neocortical and non-neocortical regions, with no disproportionate effects in cortex. It is possible that this reduction reflects the smaller body size of enriched animals (who were highly active), and which may not have been fully accounted for in the residual measure due to a low n and accordingly low correlation. Further research will be needed to reconcile this smaller brain size with reports of increased brain weight in enriched subjects [24–26]. It is important to note that the environmental enrichment methods employed in this study are fundamentally different from those used in other studies; for example, our auditory environment differences began prenatally and extended until adulthood.
Finally, Kolb and Cioe also found a reduction in brain weight following aspiration lesions of frontal cortex performed on P2. Although aspiration differs from freezing injury, both techniques were applied within the period of neural migration, and have demonstrated little effect of specific location of the lesion on a variety of measures (e.g., behavior, brain weight, thalamic morphology[14, 30]). These factors indicate that subsequent systematic alterations (e.g., in function) may reflect a developmental cascade leading to distal changes, rather than stemming from the focal injury per se.
An animal model of microgyric neuromigrational anomalies has been employed to demonstrate a reduced brain weight in microgyric subjects that is consistent across variable ages, environments, and sex. Importantly, this effect appears to reflect a disproportionate reduction of neocortical volume. Since differences in brain weight have been shown to be proportional to total brain DNA content and thus total CNS cell number[31, 32], and since brain weight has been suggested to be a good surrogate measure for total cell number in mice (as in humans ), we hypothesize that total cell number – particularly in cortex – may be decreased in microgyric subjects.
Alternatively, the decrease in brain weight could reflect changes in neuropil rather than cell numbers. It is important to note that the highly significant Microgyria effects on whole brain, and particularly cerebral cortex, are seen despite a lack of body weight differences between Sham and Lesion groups. Environment effects, conversely, may be accounted for at least partly by significant body weight differences (due to activity differences) that may not fully have been accounted for in residual scores (due to relatively small subject numbers). Collective results have critical implications for the profound re-organizational effects of relatively small focal injuries early in development, specifically to affect distributed systems throughout the brain and in particular the cerebral cortex. Future research will explore these issues.
Wistar rats (N = 96) from three separate study groups born at the University of Connecticut to purchased dams from Charles River Laboratories (Wilmington, MA) were used. All groups received induced focal microgyric lesions on P1 using identical methods (see below), and took part in a battery of auditory perception testing (data not shown). All procedures were approved by the University of Connecticut's Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with adequate measures to minimize pain or discomfort to the animals.
Induction of Microgyric Lesions
On P1 (day of birth = P0), litters were culled to 10 rat pups for focal microgyria induction. Pups were randomly designated to receive bilateral freezing lesions or sham surgery, balancing treatment within litters. Focal microgyric lesions were induced using a modification of the technique employed by Dvorák and associates[33, 34]; and explained in detail elsewhere. Briefly, pups assigned to the lesion condition received hypothermia induced anesthesia followed by a small midline incision over the skull. A cooled (-70°C) 2-mm diameter stainless steel probe was placed on the skull approximately 2 mm lateral of the sagittal suture overlying bregma for 5 sec. Following the initial lesion, an identical lesion was placed in the opposite hemisphere (first lesion side randomly determined) using a second cooled probe. Sham subjects had identical treatment, except the steel probe was maintained at room temperature. Following treatment, the skin was sutured, and subjects were marked with ink footpad injections, warmed, and returned to the dam.
Study Group Composition
Study 1: Environment
Study 2: Development
Study 3: Sex
Age at Time of Brain Analysis
P30 (N = 7L/11s) P52 (N = 8L/10S) P83 (N = 10L/10S)
Standard Housing (N = 7L/7S) White Noise (N = 6L/7S) Enriched (N = 7L/6S)
Male (N = 10L/10S) Female (N = 10L/10S)
Brain Weight Analysis
Subjects were weighed, anesthetized and transcardially perfused (0.9% saline, 10% Phosphate Buffered Formalin). Heads were removed, placed in formalin, and shipped to GDR for anatomical analysis. Brains were removed (spinal cord removed at the caudal extent of the cerebellum) and weighed. The brains were then embedded in celloidin as described previously[14, 15, 35], sliced at 30 μm, and every tenth section stained with cresyl violet and mounted onto glass slides. Microgyric lesions were confirmed and location assessed.
Separate ANOVA's were performed on brain weight residuals for each study group after regressing against body weight (see Tables 1 and 2), using either a linear regression (Studies 1 and 3), or a logarithmic regression (Study 2). The logarithmic regression for Study 2 compensated for the confound between brain weight and age in this study. Each study's specific treatment groups served as Between Subject variables – Study 1: Environment (3 levels: Enriched, White Noise, and Control), Lesion (2: Microgyric Lesion and Sham); Study 2: Age (3: P30, P52, and P83), Lesion (2: Microgyric Lesion and Sham); Study 3: Sex (2: Male and Female), Lesion (2: Microgyric Lesion and Sham).
Brain Volume Analysis
Volumes were estimated using Stereo Investigator (Microbrightfield Corp., USA) interfaced to a Nikon E800 microscope (Nikon Instruments, USA) fitted with a Prior motorized stage (Prior Scientific, USA). The areas of systematic series of sections through the entire region of interest were measured using point counting, and the volumes estimated using Cavalieri's rule. In cases where there were missing sections, volumes were estimated using a parabolic approach. Measures of total brain volume included the entire brain from olfactory bulb to cerebellum. Cerebral cortex was measured from the first section on which it appeared through its caudal extent, with the lateral boundary defined by entorhinal cortex. For estimates of total brain and cerebral cortical volume, we measured every 40th section using a 1 mm2 point counting grid (13–18 sections for total brain volume, 7–13 sections for cerebral cortex). Microgyric volume was measured on every 20th section using a 300 μm2 grid (6–10 sections/subject). The coefficient of error for these measures ranged from 0.01 to 0.10, with means of 0.02 ± 0.004 for brain volume, 0.05 ± 0.011 for neocortical volume, and 0.06 ± 0.02 for microgyria volume.
List of Abbreviations
Primary somatosensory cortex
Secondary somatosensory cortex
Hindlimb area of somatosensory cortex
Forelimb area of somatosensory cortex
Analysis of variance
Research was supported, in part, by the National Institutes of Health, grant HD20806. The authors would like to thank Dr. Victor Denenberg for statistical advice and discussion and Stefany Palmieri, Phyllis Itoka, and Sidhardha Kamaraju for technical assistance.
- Goldman PS, Galkin TW: Prenatal removal of frontal association cortex in the fetal rhesus monkey: anatomical and functional consequences in postnatal life. Brain Res. 1978, 152: 451-485. 10.1016/0006-8993(78)91103-4.PubMedView ArticleGoogle Scholar
- Miller B, Windrem MS, Finlay BL: Thalamic ablations and neocortical development: alterations in thalamic and callosal connectivity. Cereb Cortex. 1991, 1: 241-61.PubMedView ArticleGoogle Scholar
- Schneider GE: Early lesions and abnormal neuronal connections. Trends Neurosci. 1981, 4: 187-192. 10.1016/0166-2236(81)90061-8.View ArticleGoogle Scholar
- Galaburda AM, Sherman GF, Rosen GD, Aboitiz F, Geschwind N: Developmental dyslexia: four consecutive patients with cortical anomalies. Ann Neurol. 1985, 18: 222-33.PubMedView ArticleGoogle Scholar
- Humphreys P, Rosen GD, Press DM, Sherman GF, Galaburda AM: Freezing lesions of the developing rat brain: a model for cerebrocortical microgyria. J Neuropathol Exp Neurol. 1991, 50: 145-60.PubMedView ArticleGoogle Scholar
- Jacobs KM, Gutnick MJ, Prince DA: Hyperexcitability in a model of cortical maldevelopment. Cereb Cortex. 1996, 6: 514-23.PubMedView ArticleGoogle Scholar
- Jacobs KM, Hwang BJ, Prince DA: Focal epileptogenesis in a rat model of polymicrogyria. J Neurophysiol. 1999, 81: 159-73.PubMedGoogle Scholar
- Luhmann HJ, Raabe K: Characterization of neuronal migration disorders in neocortical structures: I. Expression of epileptiform activity in an animal model. Epilepsy Res. 1996, 26: 67-74. 10.1016/S0920-1211(96)00041-1.PubMedView ArticleGoogle Scholar
- Luhmann HJ, Raabe K, Qu M, Zilles K: Characterization of neuronal migration disorders in neocortical structures: extracellular in vitro recordings. Eur J Neurosci. 1998, 10: 3085-94. 10.1046/j.1460-9568.1998.00311.x.PubMedView ArticleGoogle Scholar
- Giannetti S, Gaglini P, Granato A, Di Rocco C: Organization of callosal connections in rats with experimentally induced microgyria. Childs Nerv Syst. 1999, 15: 444-8. 10.1007/s003810050435.PubMedView ArticleGoogle Scholar
- Giannetti S, Gaglini P, Di Rocco F, Di Rocco C, Granato A: Organization of cortico-cortical associative projections in a rat model of microgyria. Neuroreport. 2000, 11: 2185-9.PubMedView ArticleGoogle Scholar
- Rosen GD, Burstein D, Galaburda AM: Changes in efferent and afferent connectivity in rats with induced cerebrocortical microgyria. J Comp Neurol. 2000, 418: 423-40. 10.1002/(SICI)1096-9861(20000320)418:4<423::AID-CNE5>3.3.CO;2-X.PubMedView ArticleGoogle Scholar
- Galaburda AM, Menard MT, Rosen GD: Evidence for aberrant auditory anatomy in developmental dyslexia. Proc Natl Acad Sci U S A. 1994, 91: 8010-3.PubMedPubMed CentralView ArticleGoogle Scholar
- Herman AE, Galaburda AM, Fitch RH, Carter AR, Rosen GD: Cerebral microgyria, thalamic cell size and auditory temporal processing in male and female rats. Cereb Cortex. 1997, 7: 453-64. 10.1093/cercor/7.5.453.PubMedView ArticleGoogle Scholar
- Peiffer AM, Rosen GD, Fitch RH: Rapid auditory processing and MGN morphology in microgyric rats reared in varied acoustic environments. Brain Res Dev Brain Res. 2002, 138: 187-93. 10.1016/S0165-3806(02)00472-8.PubMedView ArticleGoogle Scholar
- Rosen GD, Herman AE, Galaburda AM: Sex differences in the effects of early neocortical injury on neuronal size distribution of the medial geniculate nucleus in the rat are mediated by perinatal gonadal steroids. Cereb Cortex. 1999, 9: 27-34. 10.1093/cercor/9.1.27.PubMedView ArticleGoogle Scholar
- Denenberg VH, Sherman GF, Morrison L, Schrott LM, Waters NS, Rosen GD, Behan PO, Galaburda AM: Behavior, ectopias and immunity in BD/DB reciprocal crosses. Brain Res. 1992, 571: 323-329. 10.1016/0006-8993(92)90671-U.PubMedView ArticleGoogle Scholar
- Denenberg VH, Sherman G, Schrott LM, Waters NS, Boehm GW, Galaburda AM, Mobraaten LE: Effects of embryo transfer and cortical ectopias upon the behavior of BXSB-Yaa and BXSB-Yaa plus mice. Brain Res Dev Brain Res. 1996, 93: 100-108. 10.1016/0165-3806(96)00010-7.PubMedView ArticleGoogle Scholar
- Clark MG, Rosen GD, Tallal P, Fitch RH: Impaired processing of complex auditory stimuli in rats with induced cerebrocortical microgyria: An animal model of developmental language disabilities. J Cogn Neurosci. 2000, 12: 828-39. 10.1162/089892900562435.PubMedView ArticleGoogle Scholar
- Fitch RH, Tallal P, Brown CP, Galaburda AM, Rosen GD: Induced microgyria and auditory temporal processing in rats: a model for language impairment?. Cereb Cortex. 1994, 4: 260-70.PubMedView ArticleGoogle Scholar
- Fitch RH, Brown CP, Tallal P, Rosen GD: Effects of sex and MK-801 on auditory-processing deficits associated with developmental microgyric lesions in rats. Behav Neurosci. 1997, 111: 404-12. 10.1037//0735-7044.111.2.404.PubMedView ArticleGoogle Scholar
- Rosen GD, Waters NS, Galaburda AM, Denenberg VH: Behavioral consequences of neonatal injury of the neocortex. Brain Res. 1995, 681: 177-89. 10.1016/0006-8993(95)00312-E.PubMedView ArticleGoogle Scholar
- Zilles K: The Cortex of the Rat: A Sterotaxic Atlas. Berlin: Springer-Verlag. 1985.Google Scholar
- Rosenzweig MR, Krench D, Bennett EL, Diamond MC: Effects of environmental complexity and training on brain chemistry and anatomy: A replication and extension. J Comp Physiol Psychol. 1962, 55: 429-437.PubMedView ArticleGoogle Scholar
- Bennett EL, Rosenzweig MR, Diamond MC: Rat brain: effects of environmental enrichment on wet and dry weights. Science. 1969, 163: 825-6.PubMedView ArticleGoogle Scholar
- La Torre JC: Effect of differential environmental enrichment on brain weight and on acetylcholinesterase and cholinesterase activities in mice. Exp Neurol. 1968, 22: 493-503.PubMedView ArticleGoogle Scholar
- Falk D, Froese N, Sade DS, Dudek BC: Sex differences in brain/body relationships of Rhesus monkeys and humans. J Hum Evol. 1999, 36: 233-8. 10.1006/jhev.1998.0273.PubMedView ArticleGoogle Scholar
- Pakkenberg B, Gundersen HJ: Neocortical neuron number in humans: effect of sex and age. J Comp Neurol. 1997, 384: 312-20. 10.1002/(SICI)1096-9861(19970728)384:2<312::AID-CNE10>3.3.CO;2-G.PubMedView ArticleGoogle Scholar
- Kolb B, Cioe J: Recovery from early cortical damage in rats, VIII. Earlier may be worse: behavioural dysfunction and abnormal cerebral morphogenesis following perinatal frontal cortical lesions in the rat. Neuropharmacology. 2000, 39: 756-64. 10.1016/S0028-3908(99)00260-9.PubMedView ArticleGoogle Scholar
- Kolb B, Holmes C, Whishaw IQ: Recovery from early cortical lesions in rats. III. Neonatal removal of posterior parietal cortex has greater behavioral and anatomical effects than similar removals in adulthood. Behav Brain Res. 1987, 26: 119-137. 10.1016/0166-4328(87)90161-6.PubMedView ArticleGoogle Scholar
- Zamenhof S, Guthrie D, van Marthens E: Neonatal rats with outstanding values of brain and body parameters. Life Sci. 1976, 18: 1391-6. 10.1016/0024-3205(76)90355-6.PubMedView ArticleGoogle Scholar
- Zamenhof S, van Marthens E, Grauel L: DNA (cell number) in neonatal brain: second generation (F2) alteration by maternal (F0) dietary protein restriction. Science. 1971, 172: 850-1.PubMedView ArticleGoogle Scholar
- Dvorak K, Feit J: Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats-contribution to the problems of morphological development and developmental period of cerebral microgyria. Histological and autoradiographical study. Acta Neuropathol (Berl). 1977, 38: 203-12.View ArticleGoogle Scholar
- Dvorak K, Feit J, Jurankova Z: Experimentally induced focal microgyria and status verrucosus deformis in rats – pathogenesis and interrelation. Histological and autoradiographical study. Acta Neuropathol (Berl). 1978, 44: 121-9.View ArticleGoogle Scholar
- Rosen GD, Press DM, Sherman GF, Galaburda AM: The development of induced cerebrocortical microgyria in the rat. J Neuropathol Exp Neurol. 1992, 51: 601-11.PubMedView ArticleGoogle Scholar
- Rosen GD, Harry JD: Brain volume estimation from serial section measurements: A comparison of methodologies. J Neurosci Methods. 1990, 35: 115-124. 10.1016/0165-0270(90)90101-K.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.