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prefrontal cortex
Zdravko Petanjeka, Miloš Judaša, Goran Šimić a, Mladen Roko Rašina,b,c, Harry B. M. Uylingsd, Pasko Rakicb,c,1,
and Ivica Kostović a
a
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, 10,000 Zagreb, Croatia; bDepartment of Neurobiology and cKavli Institute
for Neuroscience, Yale University, New Haven, CT 06520; and dDepartment of Anatomy and Neuroscience, VU University Medical Center, 1007 MB,
Amsterdam, The Netherlands
Edited* by Jean-Pierre Changeux, Institut Pasteur, Paris Cedex 15, France, and approved June 27, 2011 (received for review March 30, 2011)
The major mechanism for generating diversity of neuronal connec- detectable anatomical phenotype in some human cognitive dis-
tions beyond their genetic determination is the activity-dependent orders such as nonsyndromic mental retardation (16). Finally, this
stabilization and selective elimination of the initially overproduced biomedically and socially important concept (9) is also the subject
synapses [Changeux JP, Danchin A (1976) Nature 264:705–712]. The of continuing dialogue between proponents of selectionism versus
largest number of supranumerary synapses has been recorded in the constructionism (17).
cerebral cortex of human and nonhuman primates. It is generally The end of the critical period of synaptic spine elimination in
accepted that synaptic pruning in the cerebral cortex, including pre- the human cortex basically relies on the pioneering study of
frontal areas, occurs at puberty and is completed during early ado- Huttenlocher and colleagues (2, 4). Thus, it usually is assumed
lescence [Huttenlocher PR, et al. (1979) Brain Res 163:195–205]. In the tacitly that the period of synaptic overproduction in the human
present study we analyzed synaptic spine density on the dendrites cerebral cortex is completed by the end of puberty (18), even
of layer IIIC cortico–cortical and layer V cortico–subcortical projecting though Huttenlocher’s study contains only a single 19-y-old brain
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pyramidal neurons in a large sample of human prefrontal cortices in specimen in the age group between 15 and 32 y. In contrast,
subjects ranging in age from newborn to 91 y. We confirm that more recent studies using electroencephalography (15, 19), PET
dendritic spine density in childhood exceeds adult values by two- (20), and functional MRI (18, 21–26) have suggested that the
to threefold and begins to decrease during puberty. However, we dynamic changes in gray matter density and white matter in-
also obtained evidence that overproduction and developmental tegrity in the human association neocortex extend into the third
remodeling, including substantial elimination of synaptic spines, decade of life (21, 23, 26, 27). These changes, observed by MRI,
continues beyond adolescence and throughout the third decade cannot be explained by a capacious increase in dendritic length,
of life before stabilizing at the adult level. Such an extraordinarily because the dendritic growth in the human neocortex is limited
long phase of developmental reorganization of cortical neuronal mainly to early childhood (28). Therefore, it has been assumed
circuitry has implications for understanding the effect of environ- that functional plasticity probably reflects reorganization of cir-
mental impact on the development of human cognitive and emo- cuitry, including synaptic elimination (29–32), essential for ac-
tional capacities as well as the late onset of human-specific neu- quisition of the highest brain functions in humans, including
ropsychiatric disorders. affective modulation of emotional cues, self-conceptualization,
mentalization, cognitive flexibility, and working memory (33–36).
association cortex | critical period | schizophrenia | synaptogenesis However, the cellular data supporting this assumption have
been missing.
Fig. 1. (A) Representative low-magnification photographs of the rapid Golgi-impregnated layer IIIc and V pyramidal cells in the dorsolateral prefrontal
cortex of a 16-y-old subject. Black arrows indicate basal dendrites, and gray arrows indicate oblique dendrites. (Scale bar: 100 μm.) (B) Neurolucida re-
construction of layer IIIc pyramidal neuron of a 49-y-old subject, illustrating sites selected for counting spines over a 50-μm length of apical distal oblique
dendrites (green), apical proximal oblique dendrites (blue), and basal dendrites (red). (Scale bar: 100 μm.) (C) Representative high-power magnification
images of rapid Golgi-impregnated layer IIIc pyramidal neurons from the dorsolateral prefrontal cortex showing basal dendrites (Left) and distal apical
oblique dendrites (Right) during different stages: an infant 1 mo of age, a 2.5-y-old child, and 16-y-old, 28-y-old, and 49-y-old subjects. (Scale bar: 10 μm.) (D)
Graphs representing number of dendritic spines per 50-μm dendrite segment on basal dendrites after the first bifurcation (red); apical proximal oblique
dendrites originating within 100 μm from the apical main shaft (blue); and apical distal oblique dendrites originating within the second 100-μm segment from
the apical main shaft (green) of layer IIIc (filled symbols) and layer V (open symbols) pyramidal cells in the dorsolateral prefrontal cortex. Squares represent
males; circles represent females. The age in postnatal years is shown on a logarithmic scale. Puberty is marked by a shaded bar. B, birth (fourth postnatal day);
P, puberty. Specification of tissue analyzed is given in Table S1, and the positions of sections on which pyramidal neurons were measured are indicated on the
reconstructed pyramidal neuron shown in B.
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Fig. 2. The DSD, as defined in Fig. 1, plotted at the linear scale to illustrate the dynamics of changes occurring during the 100-y human lifespan. Regression
curves fit the distribution of data from the basal dendrites (A), apical proximal oblique dendrites (B), and apical distal oblique dendrites (C) of pyramidal cells
from layer IIIc and V. In all cases the equation of the curves is a double exponential function in the form: y = a*exp(−bt) + c*exp (−dt) + e, where a, b, c, d, and
e are fixed coefficients, and t is time in years (Table S3).
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(28). In the statistical analysis every subject represents a separate age. The
Tissue Preparation. The parts of the prefrontal cortex examined included the a posteriori Student-Newman-Keuls test for multiple comparisons was ap-
superior and middle frontal gyrus, mainly defined as the frontal granular and plied to determine which subjects were significantly different. P values <0.05
magnopyramidal Brodmann’s area 9 (76). Blocks of tissue (1 cm3) were sec- were considered statistically significant. Statistical analysis with parametric
tioned perpendicular to the long axis of the frontal gyrus, from the right and nonparametric procedures showed comparable results.
hemisphere in the majority of cases. The classical rapid Golgi method was used, DSD was plotted at the linear scale to illustrate the dynamics of changes
as described previously (28, 46). The pia was not removed. After sectioning, the
occurring during the 100-y human lifespan (Fig. 2) and to obtain regression
cortical tissue was immersed immediately in rapid Golgi solution (0.3% os-
curves fitting the distribution of data from the basal, apical proximal oblique,
mium tetroxide and 3% potassium dichromate) and kept in the dark. After 7 d,
and apical distal oblique dendrites of the pyramidal cells from layer IIIc and V.
the dichromate solution was replaced by 1% silver nitrate for 2 d. Then the
In all cases the equation of the curves was a double exponential function in the
tissue was dehydrated and embedded rapidly in 8% celloidin. After embed-
form: y = a*exp(−bt) + c*exp(−dt) + e, where a, b, c, d, and e are fixed coef-
ding, a microtome was used to section the blocks serially into coronal sections
ficients and t is time in years (Table S3).
160–200 μm in thickness. This thickness was chosen as a compromise to have
many dendrites and good microscopic clarity. Nissl-stained sections from ad-
jacent blocks were cut at 30 μm to check and additionally ensure that the ACKNOWLEDGMENTS. We thank J. Arellano for insightful discussions and A.
Bernacchia for help with regression curves in Fig. 2. This work was supported
neurons quantified were taken from Brodmann’s area 9 (37).
by grants from the Ministry of Science, Education, and Sports of the Republic
of Croatia (to Z.P., M.J., I.K., and G.Š.), the Unity Through Knowledge Fund
Quantitative Analysis. The criteria for cell selection for quantitative analysis (to I.K.), the National Institutes of Health, and the Kavli Institute for Neuro-
included clear impregnation of the finest dendrites and dendritic spines science at Yale University (to P.R.).
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