Download Microstructure of the neocortex: Comparative aspects

Journal of Neurocytology 31, 299–316 (2002)
Microstructure of the neocortex: Comparative aspects
J AV I E R D E F E L I P E ∗ , L I D I A A L O N S O - N A N C L A R E S
and JON I. ARELLANO
Instituto Cajal (CSIC), Avenida Dr. Arce, 37, 28002 Madrid, Spain
[email protected]
Received December 16, 2002; accepted January 8, 2003
Abstract
The appearance of the neocortex, its expansion, and its differentiation in mammals, represents one of the principal episodes in
the evolution of the vertebrate brain. One of the fundamental questions in neuroscience is what is special about the neocortex of
humans and how does it differ from that of other species? It is clear that distinct cortical areas show important differences within
both the same and different species, and this has led to some researchers emphasizing the similarities whereas others focus on
the differences. In general, despite of the large number of different elements that contribute to neocortical circuits, it is thought
that neocortical neurons are organized into multiple, small repeating microcircuits, based around pyramidal cells and their
input-output connections. These inputs originate from extrinsic afferent systems, excitatory glutamatergic spiny cells (which
include other pyramidal cells and spiny stellate cells), and inhibitory GABAergic interneurons. The problem is that the neuronal
elements that make up the basic microcircuit are differentiated into subtypes, some of which are lacking or highly modified
in different cortical areas or species. Furthermore, the number of neurons contained in a discrete vertical cylinder of cortical
tissue varies across species. Additionally, it has been shown that the neuropil in different cortical areas of the human, rat and
mouse has a characteristic layer specific synaptology. These variations most likely reflect functional differences in the specific
cortical circuits. The laminar specific similarities between cortical areas and between species, with respect to the percentage,
length and density of excitatory and inhibitory synapses, and to the number of synapses per neuron, might be considered as the
basic cortical building bricks. In turn, the differences probably indicate the evolutionary adaptation of excitatory and inhibitory
circuits to particular functions.
Introduction
The appearance of the neocortex, along with its expansion and differentiation in mammals represents one of
the fundamental events in the evolution of the vertebrate brain. This brain region is where faculties such
as speech and thought are localized, those which distinguish humans from other mammals. Thus, understanding its structure and function is one of the prime
aims in neuroscience. In general, the neocortex of all
species contains a set of elements similar to that of
any other part of the brain. That is, two major types
of neurons (projection cells and interneurons), glia (astrocytes, oligodendrocytes and microglia), nerve fibers
(extrinsic and intrinsic) and blood vessels. Furthermore, the physiological properties, neurotransmitters
and neuroactive peptides, receptors and ion channels,
and other compounds generally expressed by cortical
neurons are not unique to the neocortex, but are found
throughout the brain. Thus, one of the fundamental
questions in neuroscience is, what neural substrates
make a human being human? In other words, what is
∗ To
whom correspondence should be addressed.
0300–4864
C
2003 Kluwer Academic Publishers
special about the neocortex of humans and how does
it differ from that of other species? Santiago Ramón y
Cajal, made some of the most important early contributions to the study of the cortical microstructure in
different species (DeFelipe & Jones, 1988) and he highlighted the need to answer these questions in his book
Recuerdos de mi vida (Cajal, 1917, pp. 345–350):
“At that time, the generally accepted idea that the differences between the brain of [non-human]mammals
(cat, dog, monkey, etc.) and that of man are only
quantitative, seemed to me unlikely and even a little
offensive to human dignity. . . but do not articulate
language, the capability of abstraction, the ability to
create concepts, and, finally, the art of inventing ingenious instruments. . . seem to indicate (even admitting fundamental structural correspondences with
the animals) the existence of original resources, of
something qualitatively new which justifies the psychological nobility of Homo sapiens?. . . ’’.
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In spite of the many comparative studies performed
since the original findings of Cajal, it is somehow
surprising that we still don’t have any real answers to
this fundamental question. In this presentation we shall
mainly deal with some of the structural aspects of the
neocortex that are related to our knowledge of cortical
microorganization.
Fundamental aspects of the intrinsic organization
of the neocortex
The neocortex is commonly described as a 6-layered
structure that is subdivided into cortical areas. These
areas are distinguished by their histological and neurochemical characteristics, their connections (afferent/efferent fiber systems) and their functional properties. There are two major groups of cortical neurons:
spiny neurons and smooth or sparsely spiny neurons. Spiny
neurons include the population of pyramidal and spiny
stellate cells that are characterized by the excitatory
asymmetric synapses they form (glutamatergic). The
majority of cortical neurons are pyramidal cells that
are found in layers II–VI and constitute the bulk of the
projection cells (commonly they are classified according to their projection site). Most spiny stellate cells
are interneurons and they are located in the middle
cortical layers. Smooth or sparsely spiny neurons are interneurons and include a large population of neurons
that form inhibitory symmetric synapses (GABAergic),
as well as a small population that are non-GABAergic.
Smooth interneurons are found in layers I–VI and form
a morphologically and physiologically heterogeneous
group. Furthermore, a number of smooth interneurons
have been shown to contain more than one neuroactive substance, commonly a neuroactive peptide. Different types of interneurons can also be recognized by
their chemical and electrical synaptic connectivity. Additionally, specific morphological types, with particular molecular attributes and connections, have been
shown to present distinct physiological characteristics
(for reviews see Peters & Jones, 1984; Jones & Hendry,
1986; White, 1989; DeFelipe & Fariñas, 1992; DeFelipe,
1993, 2002; Nieuwenhuys, 1994; Kawaguchi & Kubota,
1997; Thomson & Deuchars, 1997; Somogyi et al., 1998;
Galarreta & Hestrin, 2001; Silberberg et al., 2002; Wang
et al., 2002).
In general, cortical neurons are thought to be organized into multiple, small repeating microcircuits.
However, as pointed out by Silberberg et al. (2002),
given the great diversity of anatomical, molecular and
physiological types of neurons, and the intricate connectivity, it is evident that a unique, discrete microcircuit cannot exist. However, in spite of the large number
of different elements that constitute cortical circuits, it is
possible to draw up a common basic microcircuit. The
skeleton of this basic microcircuit is formed by a pyramidal cell and its input-output connections (DeFelipe,
2002). That is excitatory inputs (asymmetrical synapses)
only arrive at the dendritic arbor and originate from
extrinsic afferent systems and spiny cells (which include other pyramidal cells and spiny stellate cells). Inhibitory inputs (symmetrical synapses), which mostly
originate from GABAergic interneurons, terminate on
the dendrites, soma and axon initial segment. These
interneurons are interconnected between themselves
with the exception of chandelier cells, which only form
synapses with the axon initial segment of pyramidal
cells. These microanatomical characteristics have been
found in all cortical areas and species examined so far
and, therefore, they can be considered as fundamental
aspects of cortical organization.
It is clear that cortical areas show important differences within the same and different species. The contention is that, since the times of Cajal, some researchers
have emphasized the similarities whereas others the
differences. For the former group of researchers
the histological differences are essentially fortuitous,
the functional differences of the various cortical areas
are a consequence of the differential connectivity of
their afferent and efferent fiber systems. For the second
group, the morphological differences between cortical
areas are fundamental as are the differences in connectivity. Thus, the working hypothesis posed by Cajal
when he started to examine the human cerebral cortex (Cajal, 1899), can still be taken as a current working
hypothesis.
“. . . if the gray cerebral cortex is an aggregation of organs with diverse functions, each of them must possess a special structure, within a [fundamental] plan
whose general lines are appropriate for the whole
cortex.’’
The basic uniformity of the neocortex
and species-specific variations
COLUMNAR ORGANIZATION
In 1938, Lorente de Nó introduced the important idea of
the “elementary cortical unit of operation.’’ He considered
the cortex as consisting of small cylinders composed
of vertical chains of neurons that crossed all cortical
layers and that had specific afferent fibers as their axis
(Lorente de Nó, 1938):
“. . . all the elements of the cortex are represented in
it, and therefore it may be called an elementary unit,
through which, theoretically, the whole process of the
transmission of impulses from the afferent fibre to the
efferent axon may be accomplished.”
This idea formed the basis of the hypothesis of the
columnar organization of the cerebral cortex that developed later, mainly after the works of Mountcastle,
Hubel and Wiesel (for reviews see Hubel & Wiesel, 1977;
Cortical microstructure
Mountcastle, 1978, 1997; Jones, 2000a; Buxhoeveden &
Casanova, 2002). The columnar organization is mostly
related to the migration of neurons from the ventricular
and subventricular zones into radial columns during
development (Rakic, 1988). This radial migration has
been suggested as a mechanism, known as the radial
unit hypothesis (Rakic, 1988), by which the neocortex
could expand enormously during evolution as a sheet
of cells with a basically uniform thickness rather than
increasing in size as a globe (reviewed in Rakic, 2002;
see also Chenn & Walsh, 2002).
NUMBER AND PROPORTION OF NEURONAL TYPES
WITHIN A CORTICAL COLUMN
In 1980, a highly influential paper by Rockel, Hiorns
and Powell emphasized the basic uniformity of the cortex (Rockel et al., 1980). These authors counted the number of neurons in tissue samples (30 µm wide by 25 µm
thick) across the whole cortex, from the pial surface to
the white matter from different functional areas (motor,
somatic sensory, visual, frontal, parietal and temporal)
of a variety of species (mouse, rat, cat, monkey and
man). They found that the absolute number of neurons
was very similar in all areas and in all species (approximately 110), with the exception of the binocular
part of area 17 of the monkey and human where there
were approximately 2.5 times more neurons. They assessed tissue columns of 30 µm width because, according to the studies of Mountcastle, Hubel and Wiesel,
this was approximately the width of the smallest functional column in the neocortex. Thus, the number of
neurons within this column would represent a quantitative reflection of the anatomical basis of the functional column. Rockel et al. (1980) also pointed out that
the pattern of the intrinsic cortical connections, as revealed by various anatomical methods in the laboratory
of Powell, and by other authors (e.g., Gilbert & Kelly,
1975; Lund et al., 1975; Jones & Wise, 1977; for a review,
see Jones, 1983), was essentially uniform throughout
the neocortex. They also pointed out that the cytoarchitectonic variations between different areas of the same
brain and between the same areas in different species
were correlated with variations in the relative sizes of
the major afferent and efferent fiber pathways of each
area.
Early electron microscope studies identified ultraestructural characteristics that distinguish the somata
of pyramidal and nonpyramidal neurons (Colonnier,
1968; Jones & Powell, 1970). On the basis of these, several authors have reported that the proportions of these
two major neuronal types were the same in visual, motor and somatic sensory cortex of the monkey and in the
visual and motor areas of the rat and cat (Sloper, 1973;
Tömböl, 1974; Sloper et al., 1979; Winfield et al., 1980).
All these features led to Rockel et al. (1980) to conclude
that
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“in mammalian evolution the area of neocortex increases in larger brains but the number of and proportions of neuronal types through the depth remains
constant, except in area 17 of primates. From these
and other findings it is suggested that the intrinsic
structure of the neocortex is basically more uniform
than has been thought and that differences in cytoarchitecture and function reflect differences in connections.’’ (see also Powell, 1981).
However, using other more appropriate quantitative
methods (including the disector method), it has not
been possible to confirm these conclusions in the rat
and cat (Beaulieu & Colonnier, 1989; Beaulieu, 1993;
Skoglund et al., 1996), nor that each cortical unit contains a similar number of neurons in a variety of
other species (Haug, 1987; Stolzenburg et al., 1989; see
Table 1).
SPECIES - SPECIFIC VARIATIONS IN CORTICAL
MICROSTRUCTURE
The majority of the studies of cortical microstructure
have been based on the examination of relatively few
species (mouse, rat, cat, macaque monkey and human).
But, when other less commonly used species are considered, such as, elephants, pigmy shrew, manatees, dolphins, giraffes or apes, it is obvious that their cortical
microanatomy is rather different (e.g., Haug, 1987; Reep
et al., 1989; Stolzenburg, 1989; Glezer et al., 1988, 1993;
Hof et al., 1999, 2000; Preuss & Coleman, 2002). For example, Dexler (1913) described clumps of neuronal cell
bodies in layer VI of the cerebral cortex of dugongs
and he called these aggregations “basal Rindenkerne’’
(basal cortical nuclei). These cellular aggregates were
also been found in the cerebral cortex of manatees, in a
region that Reep et al. (1989) called the “cluster cortex’’
to emphasize this feature. This peculiar cytoarchitecture has not been described in any other species, suggesting that it is a unique trait of the sirenia (Reep et al.,
1989; see also Johnson et al., 1994).
Dramatic differences in the neuron density and
cytoarchitectonic organization can be seen in Nisslstained sections from the dorsal parietal cortical region of the human (somatosensory area 1), the giraffe
(unchacterized cytoarchitectonic area), and the platypus (somatosensory bill area; Fig. 1). For example, in
the human vertical aggregates of neurons can be distinguished that form relatively thin and long minicolumns. The giraffe neocortex is mainly characterized
by a thick layer I, the presence of short and thick minicolumns or clusters (“septa’’) separated by regions with
few neurons (“hollows’’) in layer V and, above all, the
existence of clusters of neurons in layer II. The neocortex of the platypus is characterized by having a high
density of neurons, a dense and thin layer II, and short
minicolumns in layer VI (see also Elston et al., 1999b).
D E F E L I P E , A L O N S O - N A N C L A R E S and A R E L L A N O
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Table 1. Data accumulated when considering all cortical layers.
Total number of synaptic
profiles studied
Range (mean ± s.e.m.) of
asymmetrical and symmetrical
synaptic profiles per 100 µm2
Range (mean ± s.e.m.) of all
synaptic profiles per 100 µm2
Mean cross-sectional lengths of
all synapses (µm ± s.e.m.)
Synaptic density of
asymmetrical and symmetrical
synaptic profiles per 108 /mm3
(mean ± s.e.m.)
Synaptic density of all synapses
per 108 /mm3 (mean ± s.e.m.)
% of asymmetrical synapses
% of symmetrical synapses
No. of neurons/mm3
No. of neurons per column of
30 × 25 µm through the depth
of the cortex
No. of synapses/neuron
Human temporal
cortex (n = 3)
Rat hindlimb
Mouse barrel
somatosensory y cortex (n = 3) cortex (n = 2)
Mouse visual
cortex (n = 1)∗
2195
2523
2054
897
11–18 (14.9 ± 0.7)
9–21 (17.9 ± 2.2)
22–35 (28.2 ± 1.4)
24–38 (29.4 ± 1.8)
20–36 (29.9 ± 0.79) 32–46 (38.9 ± 0.95)
50–75 (63.1 ± 1.96) 40–64 (51.3 ± 2.1)
0.28 ± 0.01
0.29 ± 0.01
0.22 ± 0.01
0.21 ± 0.09
5.42 ± 0.28
6.46 ± 0.26
12.83 ± 0.65
14.46 ± 0.93
10.94 ± 0.34
13.97 ± 0.33
29.31 ± 1.02
25.19 ± 1.16
89
11
24186
48
89
11
54468
75
84
16
120315
109
89
11
29821
18015
21983
–
–
–
∗ Data from mouse visual cortex were not used for statistical comparisons with data from mouse barrel cortex, nor with the rat and human
cortex.
All synapses include asymmetrical, symmetrical and uncharacterized synapses.
However, in spite of the importance of applying comparative morphology to understand the basic organization of the neocortex (e.g., Petrides & Pandya, 1994),
relatively little attention has been paid to these studies.
In conclusion, the notion of a basic uniformity in the
neocortex, with respect to the density and types of neurons per column is not valid for all species. Therefore,
these features are not an essential or general feature of
the neocortex.
SPECIES - SPECIFIC VARIATIONS IN NEURONAL TYPES
Immunocytochemical analyses have demonstrated important differences in the proportions of interneurons
between species. For example, in the rat GABAergic
cells form 15% of the population in all cortical areas whereas in the primate, they reach 20% in the
visual cortex and up to 25% in other cortical areas
(Hendry et al., 1987; Beaulieu et al., 1992; Beaulieu, 1993;
Micheva & Beaulieu, 1995; Gabbott & Bacon, 1996; del
Rĺo & DeFelipe, 1996; Gabbott et al., 1997; Meinecke
& Peters, 1997; but see Ren et al., 1992). Interestingly,
while Beaulieu (1993) found the same proportion of
GABAergic neurons (15%) in the occipital, parietal and
frontal cortex of the rat, the numerical density of neurons in the frontal cortex (34,000/mm3 ) was significantly lower than that in the occipital (52,000/mm3 ) and
parietal (48,000/mm3 ) regions. The fixed proportion of
interneurons, irrespective of the number of neurons, is
in keeping with the idea of the uniformity of cortical
Fig. 1. A, B, and C—Low-power photomicrographs of 100-µm-thick vibratome sections stained with thionin from area 1 of
the primary somatosensory cortex of the human (A), dorsal parietal cortex of the giraffe (unchacterized cytoarchitectonic area;
B), and the bill representation of the primary somatosensory cortex of the platypus C). D–I—Higher magnification images
of the regions boxed in A, B, and C. In the human (A, D), vertical aggregations of neurons forming relatively thin and long
minicolumns can be readily observed (arrow). In the giraffe (B, F, G), layer I is thick and there are clusters of neurons in layer
II (arrows in B). One of these clusters is shown at greater magnification in F. In G the thick minicolumns (arrows) present in
layer V are shown and it should be noted that these are separated by relatively large regions with few neurons (“hollows’’) as
indicated with asterisks. In the platypus (C, E, H, I), the neocortex is characterized by the high density of neurons (E), the thin
and very dense layer II (H, indicated between two lines), and the presence of short and relatively thick minicolumns (arrows)
in layer VI (I). Note the high density of neurons in the platypus compared to the human and giraffe. wm, white matter. Scale
bar in I: 620 µm for A, B, C; 145 µm for D–G; 100 µm for, H, I.
Cortical microstructure
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circuits. However, this does not exclude the possibility
that the proportion of the different types of interneurons might vary in each cortical area, resulting in regional specialization of inhibitory circuits (see DeFelipe
et al., 1999b).
The higher proportion of GABA neurons in the primate cortex raises three intriguing but not mutually exclusive possibilities. Firstly, there could be an increase in
the number of all types of interneurons already present
in non-primate mammals. Alternatively, an increase in
the number of certain types of interneurons may have
occurred. Thirdly, new types of interneurons may have
been added during evolution in the primate cortex. This
latter idea was proposed in the times of Cajal, and he indeed thought that unique types of neurons were present
in the human cortex (DeFelipe & Jones, 1988). However,
it is generally thought that the same types of neurons
are found in all species (e.g., Fairén et al., 1984; Tyler
et al., 1998).
Nevertheless, a number of studies support the theory of evolutionary diversity with respect to the morphology and neurochemical characteristics of both
pyramidal cells and interneurons (e.g., Campbell &
Morrison, 1989; Lewis & Lund, 1990; Glezer et al., 1993;
del Rĺo & DeFelipe, 1997b; Nimchinski et al., 1999; Hof
et al., 2000; Elston et al., 1999a, 2001; Preuss & Coleman,
2002; see also Benavides-Piccione & DeFelipe, 2003).
For example, the morphology of pyramidal cells differs
considerably between different cortical areas within a
given species, and between homologous cortical areas of different species. The basal dendritic arbor of
these cells displays notable differences in the size, number of bifurcations and spine density between various
species of monkeys. Since each dendritic spine is assumed to receive at least one excitatory synapse, these
differences indicate species variations in the cortical circuits established by pyramidal cells (see Elston et al.,
1999a). Another example is a type of projection neuron
that can be distinguished by their spindle-shaped morphology (spindle cells) and that are only found in the
cortex of humans and in great apes (Nimchinski et al.,
1999).
With regards interneurons, a remarkable case of interspecies variation is that of the so-called double bouquet cell, the source of a large number of inhibitory
synapses on dendrites within a very narrow column of
cortical tissue. These interneurons have been shown to
form a widespread and regular microcolumnar structure (DeFelipe et al., 1990; del Rĺo & DeFelipe, 1997a),
which is strikingly similar to that shown by bundles
of myelinated axons (radial fasciculi) that originate
from small vertical aggregates of pyramidal cells (radial minicolumns or pyramidal cell modules). Using
immunocytochemical techniques to label double bouquet cells in tangential sections, a high coincidence of
bundles of myelinated axons and double bouquet cell
axons was found, such that one double bouquet cell is
found per pyramidal cell module (del Rĺo & DeFelipe,
1997a). Thus, double bouquet cells seem to constitute
a key element of the minicolumnar organization of the
cortex (DeFelipe et al., 1990; del Rĺo & DeFelipe, 1997a;
Peters & Sethares, 1997; reviewed in DeFelipe, 1997b,
2002; Jones, 2000a). However, while the morphology
and distribution of double bouquet cells is similar in
the human and macaque neocortex, they are modified
or less numerous in the neocortex of other species (e.g.,
the cat), and may even be absent (e.g., in the mouse
and rat). Thus, differences in the morphology, number or distribution of double bouquet cells may represent fundamental differences in cortical microorganization between primates and other species (DeFelipe,
2002).
Recently, it has been proposed that changes in the distribution of transcription factors in the telencephalon
may generate species-specific programs of GABAergic neurogenesis (Letinic et al., 2002). These authors
showed that two lineages of human cortical GABAergic
neurons exist. One lineage, that expresses the transcription factors Dlx1/2 and Mash1, makes up 65% of the
GABAergic neurons. These cells originate in the ventricular and subventricular zones of the dorsal telencephalon and migrate radially across the intermediate
zone to the cerebral cortex. The other lineage, the remaining 35% of GABAergic neurons, express Dlx1/2
but not Mash1, and originate in the ganglionic eminence of the ventral telencephalon from where they
migrate tangentially across the intermediate zone to
the cerebral cortex. However, in rodents the origin
of GABAergic neurons differs since all, or almost all,
GABAergic neurons originate in the ganglionic eminence and migrate tangentially to the cerebral cortex
(Tan et al., 1998; Anderson et al., 1999; Marin et al., 2001).
Thus, we proposed that the double bouquet cells in
the human neocortex might originate in the ventricular/subventricular zone from the GABAergic lineage
expressing Dlx1/2 and Mash1, and migrate radially
along common pathways as the radial units or ontogenetic columns that give rise to the pyramidal cell
modules (reviewed in Rakic, 2002). This could represent a mechanism acquired during evolution by which
the complementary microcolumnar structures formed
by double bouquet cells (and probably, other types
of interneurons), and pyramidal cell modules, might
be constructed in the primate, but not in the rodent
neocortex.
Synaptology of the neuropil: Morphology
and density of synapses
GENERAL CONSIDERATIONS
Generally, cortical synapses are divided into two morphological categories: asymmetrical (type 1) and symmetrical (type 2) synapses (Gray, 1959; Colonnier,
Cortical microstructure
1968, 1981; Peters, 1987; Peters et al., 1991; Peters &
Palay, 1996). These a most easily distinguished by the
postsynaptic density, which is prominent in asymmetrical synapses and thin in symmetrical synapses. Differences or similarities in the density of synapses have
been reported in various cortical areas and species (see
section “Density of synapses’’). However, the number of specimens studied and the extension of cortex examined is frequently relatively small and in
many studies, the cortical layers have not been examined systematically. Other considerations, such as
the age of the individuals, may also differ considerably between studies, and often this information is
not available. This is particularly important since notable decreases in the density of synapses may occur
during puberty and throughout adulthood, depending on the cortical area, layer and species examined
(reviewed in Rakic et al., 1994; see also Bourgeois et al.,
1994; Poe et al., 2001; DeFelipe et al., 1997, 2002). Furthermore, there is no general consensus for counting
synapses and, therefore, it is often difficult to compare
data obtained in different laboratories (DeFelipe et al.,
1999a).
Here we have reviewed some of these issues and
compared them with data we have obtained regarding
the differences in the cortical circuits of distinct species
and cortical areas. We have studied the morphology
and density of synapses, in relation to their depth in
the cortex (from layer I to layer VI), in the adult mouse
(3 months old), rat (5 months old) and human (normal cortex from surgically resected brain tissue from
three male individuals: 24, 27 and 36 years of age).
Some of this material has been used in previous studies
(DeFelipe et al., 1997, 2002; Marco & DeFelipe, 1997). We
applied the same method to quantify synapses in the
thin neuropil (i.e., avoiding the neuronal and glial somata, blood vessels, large dendrites and myelinated axons) of the visual cortex and the somatosensory cortex
of the mouse (area 17 and barrel cortex, respectively),
the rat hindlimb area, and the anterolateral temporal
cortex (T2–T3) of the human (Fig. 2). Furthermore, the
neuronal density was also calculated in this material
to compare and estimate the number of synapses per
neuron in each cortical layer and species. Table 1 data
from the mouse visual cortex were not used for statistical comparisons, therefore, unless otherwise specified,
the mouse cortex refers to the barrel cortex. For more
details of the methods used, see section “methods” on
page 312.
MORPHOLOGY OF SYNAPSES
Types of synapses
In most electron microscope studies, the percentage
of asymmetrical and symmetrical synapses varied between 80–90% and 20–10%, respectively, in all the cortical areas and species examined (e.g., Blue & Parnavelas,
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1983; Rakic et al., 1986; Zecevic et al., 1989; Schüz &
Palm, 1989; Zecevic & Rakic, 1991; Beaulieu et al., 1992,
1994; Bourgeois et al., 1994; Beaulieu & Colonnier, 1985,
1989; Micheva & Beaulieu, 1996; DeFelipe et al., 1997,
2002; White et al., 1997; Marco & DeFelipe, 1997). Our
data confirmed the remarkable consistency in the percentage of asymmetric and symmetric synapses in the
human cortex (89% and 11%), rat (89% and 11%) and
mouse (84% and 16%) when considering all layers together (Table 2). However, when each layer was analyzed individually, a number of differences were found.
For instance, in all layers the percentage of asymmetrical synapses was generally lower in the mouse than
in the rat and human, except for layers I, II and IIIa of
human and layer I of rat (Table 2). The greatest difference was found in layer IV, where 16% and 14% fewer
asymmetrical synapses were seen in the mouse when
compared to rat and human, respectively. The maximum difference between layers in the rat and human
was only 3%.
A given axon terminal forms an asymmetrical or
symmetrical synapse with either one (single synapse)
or with two or more postsynaptic elements (multiple synapses). The large majority of axon terminals
form single synapses with a postsynaptic element in
the three species examined (human, 99.7%; rat, 99%;
mouse, 97.6%), but notable differences between species
were found in certain layers. For example, the greatest
proportion of multiple synapses were found in layer
IV in each of the three species, the proportions being
0.7% in the human, 1.6% in the rat and 4.5% in the
mouse (8 million, 29 million and 173 million multiple
synapses per mm3 , respectively). While in layer I, no
axon terminals formed multiple synapses in the human cortex, whereas in the rat and mouse there were
0.7% and 0.9%, respectively (12 million and 13 million
multiple synapses per mm3 ).
CROSS - SECTIONAL LENGTH OF SYNAPTIC JUNCTIONS
With few exceptions, the cross-sectional length of
synaptic junctions in all cortical areas and species studied so far varies between 0.20–0.40 µm (e.g., Blue &
Parnavelas, 1983; Beaulieu & Colonnier, 1985, 1989;
Schüz & Palm, 1989; Glezer & Morgane, 1990; Beaulieu
et al., 1992, 1994; Huttenlocher & Dabholkar, 1997;
White et al., 1997; DeFelipe et al., 1997, 2002; Marco &
DeFelipe, 1997). As far as we know, no extensive quantitative electron microscope studies have determined
whether interspecies differences might exist between
the mean cross-sectional lengths of synaptic junctions in
the various cortical layers. Therefore, we have used our
material to compare between the human, rat and mouse
(Fig. 3 and Table 3). The mean cross-sectional lengths of
asymmetrical synapses were significantly shorter in all
layers of the mouse cortex when compared to the human and rat (mean length of all layers: 0.30 µm, 0.30 µm
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D E F E L I P E , A L O N S O - N A N C L A R E S and A R E L L A N O
Fig. 2. Electron micrographs illustrating the thin neuropil of layer IIIa in the human temporal cortex, and of layer II/III of the
mouse barrel cortex. Note the higher density of synapses (some of them indicated by arrows) in the mouse cortex. Scale bar:
0.5 µm.
Cortical microstructure
307
Table 2. Thickness of layers, number of neurons and of synaptic profiles in the human, rat and mouse cerebral cortex.
Number neurons/ mm3
(mean ± s.e.m.)
Layer
Thickness
(µm; mean ± s.e.m.)
I
II
IIIa
IIIb
IV
V
VI
I–VI
235 ± 13.5
295 ± 10.5
405 ± 16.5
370 ± 14.1
285 ± 10.2
552 ± 34.0
480 ± 26.7
2622
8333 ± 1531
45563 ± 3010
20964 ± 2709
15090 ± 1804
46167 ± 4073
23076 ± 1734
16774 ± 1875
24186
I
II–III
IV
Va
Vb
VI
I–VI
123 ± 4.9
457 ± 9.4
152 ± 7.0
209 ± 10.2
321 ± 8.8
565 ± 11.5
1827
3472 ± 1273
61670 ± 3996
90965 ± 5911
44868 ± 3450
35536 ± 3068
64286 ± 4520
54483
I
II–III
IV∗
V
VI
I–VI
69 ± 3.7
235 ± 9.9
208 ± 4.6
248 ± 6.1
451 ± 14.1
1210
18229 ± 2915
137645 ± 6410
181362 ± 6142
77765 ± 6282
122092 ± 7161
120315
Number AS
Human
136
147
144
171
136
127
110
971
Rat
158
206
192
201
172
169
1098
Mouse
199
178
145
125
125
772
Number SS
Number UC
Number all
synapses
% AS
% SS
29
19
19
20
15
14
10
126
176
181
174
186
155
142
84
1098
341
347
337
377
306
283
204
2195
82.4
88.6
88.3
89.5
90.1
90.1
91.7
88.5
17.6
11.4
11.7
10.5
9.9
9.9
8.3
11.5
40
22
17
24
19
10
132
296
241
215
192
184
165
1293
494
469
424
417
375
344
2523
79.8
90.4
91.9
89.3
90.1
94.4
89.3
20.2
9.6
8.1
10.7
9.9
5.6
10.7
35
22
43
23
23
146
264
231
285
191
165
1136
498
431
473
339
313
2054
85.0
89.0
77.1
84.5
84.5
84.1
15.0
11.0
22.9
15.5
15.5
15.9
AS, asymmetrical synaptic profiles; SS, symmetrical synaptic profiles; UC, uncharacterized synaptic profiles.
∗ Values include data from the centers or ”hollows” and the septa of the barrels, according to their relative volumes.
and 0.23 µm, in the human, rat and mouse). No significant differences were found between humans and rats
except in layer V of the human where asymmetrical
synapses were shorter (0.27 µm) compared to layer Va
(0.31 µm) and Vb (0.30 µm) of the rat.
With regards to symmetrical synapses, they were also
generally smaller in the mouse when compared to the
human and rat, and longer in the rat than in the human (mean all layers: 0.25 µm, 0.28 µm and 0.21 µm
in the human, rat and mouse). Significant differences
were found between human, rat, and mouse in all
layers, except in layer I, depending on the layer and
species (Fig. 3). Furthermore, within a given species,
there were significant differences in the size of symmetrical synapses in the human between all layers except
I, IV and VI, and in the size of asymmetrical synapses
in the mouse between all layers except layer V. In summary, laminar-specific differences in the cross-sectional
length of synaptic junctions can be observed between
different species and within certain layers of the same
species.
DENSITY OF SYNAPSES
A pioneer in comparative ultrastructural studies of the
neocortex, Cragg (1967) observed that there was rela-
tively little variation in synaptic density between areas
that were so cytoarchitectonically and functionally different as the motor and visual cortex of the mouse and
monkey (between 6 and 9 × 108 synapses/mm3 ). From
their own data, and that of Cragg and other authors,
Colonnier and colleagues argued that the numerical
density of synapses was relatively constant throughout the cortical layers, as well between different cortical
areas and different species (see O’kusky & Colonnier,
1982; Beaulieu & Colonnier, 1989). Similarly, Schüz and
Palm (1989) reported no systematic differences between
layers or different cortical regions in the mouse, including areas 6, 8 and 17. A remarkable narrow variability, 15 to 20 synaptic profiles per 100 µm2 , was found
in the neuropil of all cortical layers by Rakic and colleagues (considering both asymmetrical and symmetrical synapses) in diverse regions of the rhesus monkey
neocortex, including the motor cortex, somatosensory
cortex, prefrontal cortex and visual cortex (Rakic et al.,
1986; see also Zecevic & Rakic, 1991). Synaptic densities
were also found to be similar in the human visual, auditory and prefrontal cortex Huttenlocher and Dabholkar
(1997).
This uniformity in synaptic density led O’kusky and
Colonnier (1982) to propose that it probably reflects the
optimal number of synapses, and that it may due to
308
D E F E L I P E , A L O N S O - N A N C L A R E S and A R E L L A N O
Fig. 3. Comparison of the mean cross-sectional lengths (± s.e.m.) and densities of synaptic profiles between human, rat and
mouse. Density values obtained in layers II, IIIa and IIIb of human, and layers Va and Vb of rat were recalculated according to
the relative thickness of these layers to estimate the representative values of layers II–III and V, respectively.
Cortical microstructure
309
Table 3. Measured length (µm) of synaptic profiles in the
human, rat and mouse cerebral cortex.
Table 4. Number of synapses (× 108 ) per mm3 (Nv) in the
neuropil in the human, rat and mouse cerebral cortex.
Layer
AS
I
II
IIIa
IIIb
IV
V
VI
I–VI
0.29 ± 0.01
0.31 ± 0.01
0.31 ± 0.01
0.29 ± 0.01
0.29 ± 0.01
0.27 ± 0.01
0.30 ± 0.01
0.30 ± 0.01
I
II–III
IV
Va
Vb
VI
I–VI
0.30 ± 0.01
0.30 ± 0.01
0.29 ± 0.01
0.31 ± 0.01
0.30 ± 0.01
0.29 ± 0.01
0.30 ± 0.01
I
II–III
IV∗
V
VI
I–VI
0.24 ± 0.01
0.26 ± 0.01
0.21 ± 0.01
0.24 ± 0.01
0.20 ± 0.01
0.23 ± 0.01
SS
Human
0.25 ± 0.01
0.30 ± 0.02
0.29 ± 0.02
0.22 ± 0.01
0.23 ± 0.02
0.20 ± 0.02
0.22 ± 0.02
0.25 ± 0.01
Rat
0.27 ± 0.01
0.28 ± 0.02
0.28 ± 0.02
0.30 ± 0.02
0.30 ± 0.04
0.29 ± 0.02
0.28 ± 0.01
Mouse
0.22 ± 0.01
0.23 ± 0.02
0.20 ± 0.01
0.22 ± 0.02
0.18 ± 0.02
0.21 ± 0.01
UC
All synapses
Layer Nv AS
0.25 ± 0.01
0.29 ± 0.01
0.27 ± 0.01
0.29 ± 0.01
0.25 ± 0.01
0.27 ± 0.01
0.27 ± 0.01
0.27 ± 0.01
0.27 ± 0.01
0.30 ± 0.01
0.29 ± 0.01
0.29 ± 0.01
0.27 ± 0.01
0.26 ± 0.01
0.28 ± 0.01
0.28 ± 0.01
I
II
IIIa
IIIb
IV
V
VI
I–VI
4.78 ± 0.65
4.80 ± 0.60
4.61 ± 0.61
5.75 ± 0.65
4.53 ± 0.40
4.65 ± 0.47
3.56 ± 0.43
4.67 ± 0.21
0.27 ± 0.01
0.30 ± 0.01
0.28 ± 0.01
0.29 ± 0.01
0.29 ± 0.01
0.28 ± 0.01
0.28 ± 0.01
0.28 ± 0.01
0.30 ± 0.01
0.29 ± 0.01
0.30 ± 0.01
0.29 ± 0.01
0.28 ± 0.01
0.29 ± 0.01
I
II–III
IV
Va
Vb
VI
I–VI
5.20 ± 0.48
6.57 ± 0.42
6.40 ± 0.57
6.36 ± 0.50
5.52 ± 0.35
5.72 ± 0.45
5.96 ± 0.19
0.21 ± 0.01
0.24 ± 0.01
0.19 ± 0.01
0.21 ± 0.01
0.20 ± 0.01
0.21 ± 0.01
0.22 ± 0.01
0.25 ± 0.01
0.20 ± 0.01
0.22 ± 0.01
0.20 ± 0.01
0.22 ± 0.01
I
II–III
IV∗
V
VI
I–VI
12.72 ± 1.07
10.60 ± 0.77
11.33 ± 0.76
8.16 ± 0.86
9.99 ± 1.09
10.56 ± 0.43
Nv SS
Nv UC
Human
1.18 ± 0.22 6.95 ± 0.58
0.68 ± 0.17 6.17 ± 0.62
0.65 ± 0.15 6.36 ± 0.51
0.91 ± 0.23 6.28 ± 0.46
0.65 ± 0.16 6.03 ± 0.53
0.72 ± 0.21 5.22 ± 0.51
0.45 ± 0.16 3.07 ± 0.39
0.75 ± 0.07 5.73 ± 0.21
Rat
1.52 ± 0.19 10.77 ± 0.60
0.81 ± 0.16 7.77 ± 0.46
0.62 ± 0.15 7.41 ± 0.63
0.82 ± 0.15 6.40 ± 0.45
0.73 ± 0.23 6.25 ± 0.42
0.35 ± 0.10 5.72 ± 0.60
0.80 ± 0.07 7.39 ± 0.25
Mouse
2.51 ± 0.41 18.95 ± 1.53
1.54 ± 0.25 14.84 ± 0.84
3.54 ± 0.52 23.76 ± 1.05
1.67 ± 0.34 13.81 ± 1.43
2.14 ± 0.71 13.04 ± 1.70
2.27 ± 0.22 16.85 ± 0.72
Nv all synapses
12.67 ± 1.13
11.35 ± 0.97
11.44 ± 0.90
12.73 ± 0.92
11.02 ± 0.74
10.34 ± 0.62
7.00 ± 0.54
10.94 ± 0.34
17.23 ± 0.70
15.02 ± 0.76
14.24 ± 0.96
13.43 ± 0.72
12.29 ± 0.62
11.68 ± 0.74
13.97 ± 0.33
33.73 ± 1.77
26.56 ± 1.32
38.44 ± 1.09
23.28 ± 2.26
24.78 ± 2.55
29.31 ± 1.02
AS. asymmetrical synaptic profiles; SS, symmetrical synaptic profiles;
UC, uncharacterized synaptic profiles.
∗ Values were obtained in the centers or “hollows” of the barrels.
AS, asymmetrical synaptic profiles; SS, symmetrical synaptic profiles;
UC, uncharacterized synaptic profiles.
∗ Values were obtained in the centers or “hollows” of the barrels.
some limiting metabolic or structural factor (see also
Rakic et al., 1986). However, the methods used by the
different authors to estimate synapse number are rather
different, including whether or not stereological methods were used. In addition, most comparisons are only
qualitative and not based on statistical analysis. For
instance, Beaulieu et al. (1992, 1994) found significant
differences in the number of synapses per volume between certain layers of both the rat and monkey visual
cortex when using the disector method and statistical
comparisons.
We have found that when considering all types
of synapses, the synaptic density considering all layers was lower in the human (1094 million/mm3 ),
than in the rat (1397 million/mm3 ) and mouse
(2931 million/mm3 ) (Tables 1 and 4). These differences were statistically significant in most layers among
the three species (Fig. 3). Moreover, the high density of synapses found in the mouse barrel cortex
when compared to the rat and human, was not a peculiarity of this cortical region of the mouse, since
the mouse visual cortex also showed a high density (2519 million/mm3 ; Table 1). When asymmetrical and symmetrical synapses were analyzed separately, a significantly higher density was also observed
for asymmetrical synapses in all mouse cortical layers
when compared the rat and human, and in layers IV and
VI of the rat when compared to human. The density of
symmetrical synapses was also significantly higher in
all layers of the mouse compared the rat and human,
but no significant differences were observed between
the human and rat. Layer IV demonstrated the greatest differences in symmetrical synapse density between
humans and rats when compared to the mouse (65
million/mm3 in the human, 62 million/mm3 in the rat
and 354 million/mm3 in the mouse). In conclusion, the
density of synapses is not uniform throughout the cortical layers and there are laminar specific differences between different cortical areas in different species. Thus,
if an optimal number of synapses for cortical circuits
exists, it must be species specific.
NUMBER OF SYNAPSES PER NEURON
Since the thickness and neuronal density in different
layers and species may differ (Table 2, Fig. 4), comparing the density of synapses between species alone
is difficult to interpret in terms of connectivity. Therefore, it is common to divide the synaptic density by the
corresponding neuronal densities in the same brains
310
D E F E L I P E , A L O N S O - N A N C L A R E S and A R E L L A N O
Fig. 4. Comparison of the means of the laminar thickness (± s.d.) and neuronal densities (± s.e.m.) between human, rat and
mouse. The values obtained in layers II, IIIa and IIIb of human, and layers Va and Vb of rat were recalculated according to the
relative thickness of these layers to estimate the representative values of layers II–III and V, respectively.
to estimate the average number of synapses on each
neuron. According to Cragg (1967), this idea originated
from the observations of the histologists, Nissl (1898)
and Von Economo (1926). Nissl pointed out that in the
mole and dog, cortical neurons were more crowded
than in man. Based on this observation Von Economo
proposed that the greater separation between neurons
the richer the fiber plexus will be between them, increasing the opportunity for neuronal interactions. Thus, a
wider separation of neurons in humans compared to
other species could be taken as an indication of a greater
refinement of the connections between neurons. Nevertheless, as dendrites, particularly of pyramidal cells,
may cross several layers, the number of synapses per
neuron in a given layer is not an accurate estimate of the
number of synapses received by neurons in that layer.
The most obvious pitfall can be seen layer I, where the
number of synapses per neuron is very high (Table 5).
However, it is known that most postsynaptic dendrites
in layer I, are the apical dendritic tufts of the underlying
pyramidal cells. In addition, axon terminals in a given
layer may originate from local neurons or from neurons located in other layers or in other cortical areas
or subcortical nuclei. Thus, the synapse/neuron ratio
should be taken as a useful parameter to compare between cortical areas and species in terms of “general’’
connectivity.
Using this approach, Cragg (1967) compared the motor and visual areas of the mouse and macaque monkey, and he found an inverse relationship between neuronal density and the number of synapses per neuron.
Furthermore, he found an opposite tendency in the ratio of synapses per neuron between specific areas in
both species. In the motor cortex, the density of neurons was lower and the number of synapses per neuron was much greater in the monkey than in the mouse
(16.1 × 106 neurons/cm3 ; 60000 synapses/neuron vs.
64.4 × 106 neurons/cm3 ; 13000 synapses/neuron). On
the contrary, in the visual cortex there was a greater density of neurons and a lower synapse/neuron ratio in the
monkey than in the mouse (110.3 × 106 neurons/cm3 ;
5600 synapses/neuron vs. 92.4× 106 neurons/cm3 ; 7000
synapses/neuron). Unfortunately, as pointed out earlier, comparison with other studies is rather difficult.
For example, an inverse relationship between neuronal
density and the number of synapses per neuron was
also observed for individual layers of the macaque visual cortex by O’Kusky and Colonnier (1982), corroborating the findings of Cragg. However, O’Kusky and
Colonnier’s estimates were approximately 60% lower
than Cragg (2300 synapses/neuron) in the macaque visual cortex. In any case, O’Kusky and Colonnier suggested that this inverse relationship was due to a limiting factor such that neurons receiving more synapses
would have a more complex dendritic arborization, increasing the distance between their cell bodies. In contrast, neurons receiving fewer synapses would have a
less complex dendritic arbor, allowing them to be more
densely packed.
We found here that the principle of the inverse relationship between neuronal density and number of
synapses per neuron held true in the human when
compared with the rat and mouse. The density of neurons (neurons/mm3 in layers I–VI) was lower in human (24186) than in the rat (54483) and mouse (120315),
and the number of synapses per neuron was higher in
human (29807) than in rat (18018) and mouse (21133;
Table 5; Fig. 5). However, the rat showed a lower density of neurons and synapses/per neuron than mouse
and thus, this principle does not appear to be generally applicable. Additionally, when specific layers were
compared, this rule was applicable to all layers in the
Cortical microstructure
311
Table 5. Number of synapses per neuron in the human, rat
and mouse cerebral cortex. Values were obtained by dividing
the density of synapses by the density of neurons. Uncharacterized synapses were included in the asymmetrical and
symmetrical types, according to the frequency of both types
of synapses in each layer.
Layer
No. AS
per neuron
I
II
IIIa
IIIb
IV
V
VI
I–VI
83883
14886
32573
50266
14379
26806
25482
26096
I
II–III
IV
Va
Vb
VI
I–VI
286409
15992
10493
19484
22588
12545
16127
I
II–III
IV
V
VI
I–VI
143438
15647
14952
23068
15561
17595
No. SS
per neuron
Human
19188
2007
4436
6951
1806
3561
2811
3711
Rat
76766
1831
969
2428
2734
756
1891
Mouse
26593
2086
4524
4444
3093
3538
No. all synapses
per neuron
103071
16894
37009
57217
16186
30367
28293
29807
363175
17823
11462
21912
25322
13301
18018
170031
17733
19476
27512
18654
21133
AS, asymmetrical synaptic profiles; SS, symmetrical synaptic profiles.
rat, but not in the human or mouse (compare Tables 2
and 5). In addition, the laminar distribution of the number of synapses per neuron did not follow a common
pattern in the three species.
Similarly, there was no common pattern between
species or within a given species with respect to the
distribution of synapses per neuron when considering
asymmetrical and symmetrical synapses (Table 5 and
Fig. 5). In human, the ratio of asymmetrical synapses
per neuron was higher in all layers when compared
to the rat and mouse, except in layer IV of the human which showed a slightly lower synaptic density
per neuron than in the mouse (Fig. 5; Table 5). Rat and
mouse showed similar ratios in layers II–III, but in the
rest of layers the ratio was higher in the mouse than
in the rat, particularly in layer IV (42%). For symmetrical synapses, human and mouse displayed very similar values when considering all layers, approximately
50% higher than the rat. However, there were dramatic
laminar differences not only between certain layers of
the human and mouse when compared to the rat, but
also between the human and mouse (Fig. 5 and Table 5).
Fig. 5. Graphs showing the number of synapses per neuron
in the human, rat and mouse. The values obtained in layers
II, IIIa and IIIb of human, and layers Va and Vb of rat were
recalculated according to the relative thickness of these layers
to estimate the representative values of layers II–III and V,
respectively.
For example, the most noticeable variation between the
three species was found in layer IV, where the mouse
showed a considerably higher number of symmetrical
synapses/neuron than in the human and in the rat. Another interesting difference was found in layers II–III,
where this ratio was by far higher in human than in
mouse and rat (55 and 61%, respectively), but the rodents showed similar values.
In summary, examination of the synaptology of the
human, rat and mouse neuropil revealed dramatic
312
D E F E L I P E , A L O N S O - N A N C L A R E S and A R E L L A N O
differences in the density of synapses between the three
species. Regarding, the proportion, length and density
of asymmetrical and symmetrical synapses, and the
ratios of asymmetrical and symmetrical synapses per
neuron, there were also notable laminar specific differences between human, rat and mouse, which did
not necessarily affect the same layers. Furthermore, the
density of synapses was not inversely correlated with
the density of neurons in the three species. Thus, certain
general features of the neocortical synaptology are applicable to the human, rat and mouse, but we also detect
significant differences and this means that the pattern
of synaptic organization is characteristic of each cortical
area and species.
Functional significance of the species differences
in the synaptology of the neuropil
The main sources of asymmetrical synapses are the
corticortical and thalamocortical axons, and the local
axon collaterals of pyramidal cells and spiny stellate
cells, which are known to be excitatory. In contrast,
the main sources of symmetrical synapses are the inhibitory GABAergic interneurons (Houser et al., 1984;
White, 1989; Peters et al., 1991; DeFelipe & Fariñas,
1992; Peters & Palay, 1996; Conti & Weinberg, 1999;
Jones, 2000b; Amitai, 2001). Furthermore, synaptic size
plays an important role in the functional properties of
synapses (Mackenzie et al., 1999; Schikorski & Stevens,
1999; Takumi et al., 1999; Kubota & Kawaguchi, 2000;
Lüscher et al., 2000). For example, larger synapses
seem to contain a greater number of postsynaptic receptors (Mackenzie et al., 1999) and are associated
with a greater number of docked synaptic vesicles
(Schikorski & Stevens, 1999). We have shown that in
each species, the cortical neuropil has its own characteristic layer-specific synaptology. Thus, differences in
the density, proportion and size of excitatory and inhibitory synapses among cortical areas or species probably reflects the functional differences of cortical the
circuits involved.
Finally, there is approximately a 10% increase in the
proportion of GABA interneurons in primates when
compared to rats (see section “Species-specific variations in neuronal types’’). This must be considered in
conjunction with the fact that certain subtypes of interneurons are lacking or greatly modified in some
species. If the species differences in the number of
synapses per neuron and, therefore, in the synaptic
weights, are also taken into account, then this might
serve to emphasize the variability in the design of microcircuits between cortical areas and species. The laminar specific similarities between the human, rat and
mouse and other species with respect to the percentage, length and density of asymmetrical and symmetrical synapses, and in the number of synapses per neuron, might be considered as basic bricks of cortical
organization. In contrast, the differences probably indicate evolutionary adaptations of excitatory and inhibitory circuits to particular functions.
Methods
Synaptic density per unit area (NA) was estimated from ten
electron microscope samples of neuropil from each layer and
from each species, using a correlative light and electron microscope technique (for a detailed description see DeFelipe et
al., 1999a). The numerical density of synapses per unit volume
of the neuropil was calculated using the formula NV = NA/d
where NA is the number of synaptic profiles per unit area and
d the average cross-sectional length of synaptic junctions. The
cross-sectional lengths of synaptic junctions (synaptic apposition length) of all synaptic profiles were measured in the
prints using a magnetic tablet (SummaSketch III) and the
Scion Image analysis program (Scion corporation, Frederick,
Maryland, USA). Statistical comparisons of the means were
carried out using ANOVA. When the overall ANOVA was significant, a post-hoc Bonferroni’s test was applied. All these
studies were performed with the aid of the SPSS statistical
package (SPSS Science, Chicago, IL, USA).
Neuronal density was estimated in all layers of each cortical area where synaptic counting was performed using optical
disectors as described by West and Gundersen (1990; see also
Williams & Rakic, 1988). Ten optical disectors per layer and
per case were performed in 100 µm Nissl stained sections
adjacent to those used for synaptic counting. Optical disectors were performed using X100 oil objective on a surface of
2400 µm2 with a depth of 40 µm, rendering a study volume
of 96000 µm3 per optical disector. Nucleoli for humans and
rats, and nuclei for mice were counted to obtain an estimation of the number of neurons. The thickness of the layers was
calculated from ten measurements from each layer and case.
The total density of neurons and synapses of each area was
calculated from the relative density of each layer as a function
of its thickness.
Processing the tissue for electron microscopy produced
a greater shrinkage of the tissue compared to Nissl stained
sections. In order to obtain homogeneous estimations of the
density of neurons and synapses, this difference in shrinkage
was evaluated by measuring the cortical surface in adjacent
sections processed for electron microscopy and Nissl, using
Scion Image analysis. The shrinkage of the cortical tissue for
the material processed for electron microscopy was as follows: 32.8% in human; 26.4% in rat, and 8.1% in mouse. The
number of synapses per neuron in each layer was obtained
by dividing the density of synapses by the density of neurons
for each layer after correction considering the shrinkage. For
interspecies comparisons, the values of neuron and synaptic densities of layers II, IIIa and IIIb of human, and layers
Va and Vb of rat were recalculated according to their relative thickness, to obtain values representing layer II–III and
V, respectively.
Acknowledgments
We would like to thank Alberto Muñoz for critically
reading the manuscript, and C. Nilesh Patel and Paul
Manger for kindly supply brain tissue from the giraffe
Cortical microstructure
and platypus, respectively. This work was supported
by grants from the Spanish Ministry of Science and
Technology (DGCYT PM99-0105) and the Comunidad
de Madrid (08.5/0027/2001.1). L A-N is supported by
fellowship from the Spanish Ministry of Science and
Technology (FP2000-4989).
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