Ординатура / Офтальмология / Английские материалы / Progress in Brain Research Visual Perception, Part I Fundamentals of Vision Low and Mid-Level Processes in Perception_2006

method and the general difficulties in staining DBCs, little was known about their detailed distribution and neurochemical characteristics. The introduction of immunocytochemistry for the cal- cium-binding proteins calbindin, parvalbumin and calretinin (Celio et al., 1986; Celio, 1990, reviewed in Baimbridge et al., 1992; Andressen et al., 1993; DeFelipe, 1997) represented an important step in the study of the distribution and biochemical characteristics of interneurons. In particular, it was found that DBCs were among those neurons that were consistently immunolabeled for calbindin (CB) (DeFelipe et al., 1989; Hendry et al., 1989). The advantage of immunocytochemical staining over either the Golgi method or intracellular labeling is that instead of labeling only occasional DBCs, CB immunocytochemistry labeling is far more widespread and homogeneous (Figs. 4 and 5). In addition, CB immunocytochemistry permitted the density and distribution of DBC horsetails to be examined as well as facilitating the analysis of the synaptic connections of large populations of DBCs (see below). Furthermore, their neurochemical characteristics could also be defined by double-labeling immunocytochemical techniques (DeFelipe et al., 1989, 1990, 1999; Hendry et al.,

Fig. 4. Low-power photomicrograph of a CB-immunostained section from the human temporal cortex (area 22) showing the distribution of CB-immunostained cell bodies and DBC horsetails. Note the large number and the regular distribution of DBC horsetails. Arrows indicate some DBC horsetails. Scale bar: 140 mm.

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1989; Del Rio and DeFelipe, 1995, 1997; Peters and Sethares, 1997; Ballesteros-Ya´n˜ez et al., 2005).

Neurochemical characteristics of DBCs

Using double-labeling immunocytochemical techniques as well as correlative light and electron microscopy to examine the synaptic connections of neurochemically defined neurons, DBCs can be considered as interneurons containing GABA and CB, although subpopulations of DBCs have also been shown to express calretinin. The peptides somatostatin and tachykinin are also found in certain cortical areas and species (DeFelipe et al., 1999). In contrast, these cells are never labeled for other peptides or parvalbumin, nor do they appear to express markers found in nitric oxideand tyrosine hydroxylase-expressing neurons (Somogyi et al., 1981; DeFelipe et al., 1989, 1990, 1999; De Lima and Morrison, 1989; DeFelipe and Jones, 1992; Del Rio and DeFelipe, 1995, 1997; DeFelipe, 1997; Benavides-Piccione and DeFelipe, 2003). Therefore, there are specific neurochemical subtypes of DBCs, even though there is little or no information available regarding the possible variation in the neurochemical characteristics of DBCs in different cortical areas and species.

Distribution of DBCs: microcolumnar structure

One of the unique features of DBCs is that while the vast majority of axons from other cortical neurons surpass the size limit for the minicolumn (vertical cylinder of tissue with a diameter of approximately 25–50 mm, basically defined by the space occupied by the small vertical aggregate of pyramidal cells), DBC horsetails fit well within these limits. However, the most striking characteristic of these neurons is that they are so numerous and regularly distributed that the DBC horsetails themselves form a microcolumnar structure (Fig. 4). This microcolumnar organization has been demonstrated in the visual, somatosensorial, auditory and temporal cortex of the macaque monkey as well as in the human prefrontal, motor, somatosensory, temporal and visual cortex (DeFelipe et al., 1990, 1999; Peters and Sethares, 1997; Del Rio and DeFelipe, 1997;

Ballesteros-Ya´n˜ez et al., 2005). For example, it has been shown that in the macaque primary visual and somatosensory cortex, a mean of 10 DBC horsetails was found in a field of 10,000 mm2 from tangential sections through layer III immunostained for CB (range from 7 to 15). The center-to-center spacing of these cells was 15–30 mm and the mean width of the cross-sectioned DBC axonal arborizations was

9 mm (from 5 to 15 mm). The number of axonal collaterals that made up each DBC axon varied depending on the layer examined, ranging from as few as 3 in the deepest part of the axons’ course to as many as 10 or 15 in the upper part (DeFelipe et al., 1990; see also Fig. 5 in Ballesteros-Ya´n˜ez et al., 2005). A similar distribution has been found in the human cerebral cortex. For example, a mean

number of 12 DBC axons can be found in a field of 10,000 mm2 in the temporal cortex, with a mean diameter of 12 mm (range 5–20 mm) and a mean center- to-center spacing of 30 mm (Del Rio and DeFelipe, 1995, 1997). The homogeneous distribution of CB-immunostained DBC horsetails in a tangential section of layer III from area 18 of the human visual cortex is illustrated in Fig. 5. In addition, two morphological types of DBC horsetails have been observed, which in the human cerebral cortex show the following morphometric values (Ballesteros-Ya´n˜ez et al., 2005): the complex type (type I: Fig. 6a) with a mean thickness of 8.9873.27 mm and 5.971.6 axon collaterals; and the simple type (type II: Fig. 6b) with a mean axon thickness of 3.3771.12 mm and 3.971.0 axon collaterals.

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The relationship between DBC horsetails and bundles of myelinated axons

The distribution of DBC horsetails is remarkably similar to the distribution of bundles of myelinated fibers or radial fasciculi that originate from pyramidal cells and that form small vertical aggregates or minicolumns (Del Rio and DeFelipe, 1997; Peters and Sethares, 1997; for a review, see DeFelipe, 2005). Recently, we examined the relationship between bundles of myelinated fibers and DBC horsetails, using dual immunocytochemistry for the myelin basic protein and CB. In these dou- ble-labeled sections, it was clear that DBC horsetails were intermingled with bundles of myelinated axons in all the cortical areas examined (Fig. 7).

Indeed, disregarding some exceptions, there appears to be one DBC horsetail per minicolumn (see the section on Distribution of DBC horsetails in areas 17 and 18).

Synaptic connections of DBCs

In several areas of the monkey cerebral cortex (visual, somatosensory, temporal and auditory), it

has been shown that DBC axons form symmetrical synapses with small dendritic shafts (57–62%) and spines (38–43%) (Somogyi and Cowey, 1981; DeFelipe et al., 1989, 1990; De Lima and Morrison, 1989) (Figs. 8 and 9). Furthermore, it is relatively frequent that the DBC axon terminals establish synapses with two or more postsynaptic elements (multiple synapses). For example, the proportion of multiple synapses formed by DBC horsetails has been estimated to be approximately 16% in the

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Fig. 9. (A): Higher power electron micrograph of the axon terminal a (illustrated in Fig. 8) establishing a symmetrical synapse (arrow) with a dendritic spine. (B): An axon terminal from the tachykinin-ir DBC horsetail illustrated in Fig. 8, which forms symmetrical synapses (arrows) with three different postsynaptic elements (probably dendritic spines). Scale bar: 0.27 mm for (A) and 0.25 mm for (B). From DeFelipe et al. (1990).

primary auditory and somatosensory cortex of the macaque monkey (DeFelipe et al., 1989, 1990; Fig. 9B). Moreover, DBC axons in the human cerebral cortex are virtually identical and establish a microcolumnar structure similar to that found in the monkey. Indeed, with regard to their morphology, distribution and the proportion of synapses, DBC axons establish symmetrical synapses with small dendritic shafts (59%) and spines (41%) in the human temporal cortex (Del Rio and DeFelipe,

1995). Thus, DBCs are likely to participate in similar synaptic circuits in both monkeys and humans (Table 1). The origin of the dendritic shafts that are postsynaptic to DBC axons is unknown. However, the postsynaptic spines belong to pyramidal cells and possibly to spiny stellate cells, which are the two types of spiny neurons found in the cerebral cortex.

Origin of the postsynaptic dendritic shafts of DBCs

In principle, we can assume that the dendritic shafts of both pyramidal cells (excluding their apical dendrites) and interneurons are postsynaptic to DBCs. However, the dendritic shafts of those interneurons with vertically oriented dendritic arbors can be mostly disregarded. Owing to the very narrow extension of the DBC horsetail arbor, the cell body of a presumptive postsynaptic interneuron with vertical dendrites should lie within the axonal arborization of the DBC horsetails, a circumstance that as far as we know has yet to be observed. Thus, the postsynaptic dendritic shafts arise from dendrites crossing the DBC horsetail axonal arbor. Collateral dendritic branches of apical and basal pyramidal cells and the dendrites of some multipolar interneurons, like large basket cells, can run for several hundred micrometers in the monkey and human neocortex. These observations suggest that the synapses of a given DBC horsetail are not restricted to the dendrites of the neurons in the adjacent minicolumn, but rather that they may also establish synapses with dendrites belonging to other surrounding minicolumns, which cross the trajectory of the DBC horsetail.

Origin of the postsynaptic dendritic spines of DBCs

The apical dendrites of pyramidal cells do not lie within the axonal arborization of the DBC horsetails. Hence, it was proposed that the postsynaptic dendritic spines of DBCs arise from collateral branches of apical and basal pyramidal cell dendrites as well as from spiny stellate cells that the DBC horsetails may encounter in their trajectory through the mid-cortical layers (DeFelipe et al., 1989, 1990; Del Rio and DeFelipe, 1995). Furthermore, pyramidal neurons are far more numerous than spiny

stellate cells and, thus, the vast majority of dendritic spines clearly arise from pyramidal neurons. As a result, dendritic spines of pyramidal neurons are one of the major targets of DBC. In addition, these spines establish additional asymmetrical synapses with excitatory axons (DeFelipe et al., 1989; Del Rio and DeFelipe, 1997). Because DBCs are very abundant and establish hundreds of inhibitory synapses within a very narrow column of cortical tissue, it is likely that many spines will form symmetrical synapses. This contrasts with the classic view that the majority of dendritic spines (80–90%) only form one asymmetrical excitatory synapse. Indeed, when spines establish synapses with two separate axon terminals, it is rare that this second synapse is symmetrical (reviewed in DeFelipe and Farin˜as, 1992). However, pyramidal cells display thousands of spines and they are much more numerous than DBCs. Therefore, it is conceivable that in a random examination of dendritic spines, those spines that are not innervated by these neurons would be principally included. All of these observations led us to propose that DBCs form synapses with a special type of spine capable of forming synapses with both excitatory and inhibitory axon

terminals. Furthermore, these must be particularly abundant in the side branches of apical and basal dendrites of pyramidal cells (Del Rio and DeFelipe, 1995), at least in the primate cerebral cortex. Indeed, each DBC horsetail can form several hundreds of synapses within a narrow column of tissue and, thus, they are considered to be a key element in the microcolumnar organization of the cerebral cortex acting on groups of pyramidal cells located in different layers in the minicolumns (DeFelipe et al., 1989, 1990, 1999; Favorov and Kelly, 1994a, b; Del Rio and DeFelipe, 1995; DeFelipe, 1997, 2002, 2005; Jones, 2000; Ballesteros-Ya´n˜ez et al., 2005).

DBC horsetails in areas 17 and 18 of the macaque monkey and human

In general, the density and morphology of DBCs vary in different areas of the macaque and human neocortex (DeFelipe et al., 1999; BallesterosYa´n˜ez et al., 2005). What follows is the description of DBC horsetails in areas 17 and 18 of both the macaque and human (Table 2).

Fig. 10. Percentages of type I and type II DBC horsetails in areas 17 and 18 of the human visual cortex. From BallesterosYa´n˜ez et al. (2005).

type is the most frequent in area 17 where they run from layer II to layer IVB, while the long type runs from layer III to layers V–VI and are the most frequent type found in area 18 (Fig. 11). Assuming that the percentage of multiple synapses made by the single axon terminals in the DBC horsetails (see the section on Synaptic connections of DBCs) is similar in both the complex and simple type of DBC horsetails, the greater complexity of DBC horsetails in area 18 suggests that each single DBC horsetail establishes more synapses. This regional specialization of DBCs is likely to have an important impact on the connectivity of minicolumns.

DBC horsetail density in areas 17 and 18

Morphology of DBC horsetails in areas 17 and 18

There are differences in the morphology of DBC horsetails in area 17 when compared to area 18. First, both complex (type I) and simple (type II) DBC horsetails are found in areas 17 and 18, although the most common type found in area 18 is type I, while in area 17, type II axons are the most abundant (Fig. 10).

Furthermore, there are more short side branches and club-like bouton appendages of the axonal collaterals in area 18 than in area 17, giving DBC horsetails a spiny appearance in area 18. In addition, DBC horsetails can be classified into another two morphological types: short and long. The short

The density of DBC horsetails has been estimated in coronal sections of the human cerebral cortex immunostained for CB. This was achieved by counting the number of DBC horsetails crossing a 500 mm long line in the middle of layer III, parallel to the pial surface (Ballesteros-Ya´n˜ez et al., 2005). We found that the density of CB-immunoreactive (-ir) DBC horsetails was similar in areas 18 and 17 (13.672.9 and 14.472/500 mm, respectively). We also examined the possible correlation between the density of DBC horsetails and the density of CB-ir somata in layers II–IIIA. The density of CB-ir somata in layers II–IIIA was significantly lower in area 17 (46.579.3 somata per 200,000 mm2) than in area 18 (74.35713.6). Therefore, the additional CB-ir neurons in area 18 probably represent other cell types.

Distribution of DBC horsetails in areas 17 and 18

In addition to the aforementioned differences in morphology (Table 2), the most remarkable characteristic that distinguishes DBC horsetails in areas 17 and 18 is related to their distribution. In both areas, CB-ir DBC horsetails are regularly distributed and numerous (Fig. 11). However, while practically the entire extent of area 18 is populated by DBC horsetails, in area 17, DBC horsetails are not present throughout the whole area, but rather are located in groups that occupy cortical segments of a few hundred to several thousand micrometers in width. This differential distribution of DBCs is most dramatic at the border between these two areas, where there were

very few or no CB-ir DBC horsetails in area 17, but there were many in area 18 (Figs. 12 and 13; see also Fig. 1 in DeFelipe et al., 1999). A similar uneven distribution of CB-immunostaining in area 17 has also been described in layer III of the macaque monkey, where a higher density of CB-ir elements were found around cytochrome-oxidase-rich puffs in layer III (Celio, 1986; Van Brederode et al., 1990; Hendry and Carder, 1993; Blu¨mcke et al., 1994; Carder et al., 1996). However, when we compared CB and cyto- chrome-oxidase immunoreactivity in serial sections from the macaque, the distribution of DBC horsetails was not related to the pattern of cytochrome-oxidase staining in layers II and III (unpublished observations; see also Peters and Sethares, 1997).

Are all pyramidal cells of the minicolumns innervated by DBCs?

In sections stained for both the myelin basic protein and CB, it is clear that DBC horsetails are intermingled with bundles of myelinated axons in all the cortical areas of the human cerebral cortex examined (Ballesteros-Ya´n˜ez et al., 2005). As such, it generally appears that there is one DBC horsetail per minicolumn. Whether all pyramidal neurons of the minicolumn or just a fraction of them are innervated by the corresponding DBC horsetail is unknown. However, not all minicolumns are associated with DBC horsetails. For example, adjacent to the numerous consecutive minicolumns that are typically associated with DBC horsetails, one, two or more consecutive minicolumns may not be associated with DBC horsetails. This dissociation of DBC horsetails and minicolumns is the most

evident at the border between areas 17 and 18 where as described above, relatively few DBC horsetails are observed. Of course, the absence of immunostaining may indicate the lack of expression and not the absence of a given type of neuron. Therefore, the lack of labeling of DBC horsetails in certain regions or the lack of association of some minicolumns with DBC horsetails can be interpreted in three ways:

It is possible that DBCs are present in the whole neocortex and that each minicolumn is associated with a DBC horsetail. However, these neurons may be chemically heterogeneous giving rise to an uneven staining. Nevertheless, it should be emphasized that the expression or lack of a given peptide or cal- cium-binding protein in a particular type of