Cerebral Cortex, Vol. 12, No. 5, 453-465,
May 2002
© 2002 Oxford University Press
Early Specification of the Hierarchical Organization of Visual Cortical Areas in the Macaque Monkey
Cerveau et Vision INSERM 371, 18 Avenue du Doyen Lépine, 69675 Bron Cedex and , 1 Faculté de Médecine Lyon Sud, Service de Gynécologie Obstétrique, Centre Hospitalier Lyon Sud, 165 Chemin du Grand Revoyet, 69495 Pierre Bénite Cedex, France
Henry Kennedy, Cerveau et Vision INSERM 371, 18 Avenue du Doyen Lépine, 69675 Bron Cedex, France. Email: kennedy{at}lyon151.inserm.fr.
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Abstract |
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The laminar organization of cortico-cortical projection neurons (expressed by the percentage of supragranular projecting neurons – SLN%) characterizes cortical pathways as feedforward (FF) or feedback (FB) and determines the hierarchical ranking of cortical areas. There is evidence of a developmental reduction in SLN% of pathways to area V1. Here, by analyzing pre- and postnatal projections to area V4, we have been able to address whether developmental reductions of SLN% impact on information processing in the immature cortex. FB pathways to area V4 exhibit 28–84% reduction of SLN%. This contrasts with the FF projections, which show little or no SLN% reduction. However, SLN% values in the immature cortex allocated cortical areas to the same hierarchical levels as in the adult. The developmental reduction of SLN% is a widespread phenomenon in the neocortex and is a distinctive feature of FB pathways. Two mechanisms contribute to developmental changes in SLN%: (i) delayed ingrowth of axons into the cortical target from infragranular layer neurons and (ii) prolonged developmental reduction of the divergence of projections from supragranular layer neurons. The present results show that FF and FB projections exhibit different developmental processes and patterns of connections linking cortical areas and their hierarchical relations are established prenatally, independently of regressive phenomena.
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Introduction |
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Hierarchical organization of information processing in the brain has been an important issue in neurology since the work of Huglins Jackson in the1880s. The work of Hubel and Wiesel formalized the relevance of hierarchical processing for under-standing both the physiology and the connectivity of the visual system (Hubel and Wiesel, 1962
). More recently, a number of conceptually important studies have used mathematical treatment of connectivity data to address the hierarchical organization of the cortex using various combinations of graph theory and non-metric multidimensional scaling (Young, 1992; Jouve et al., 1998; Hilgetag et al., 2000; Sporns et al., 2000). Laminar Patterns of Cortical Connectivity
Rostral directed projections allow outflow of activity away from striate cortex (area V1) towards circumstriate cortex and are thought of as feedforward (FF) pathways. These projections originate largely from supragranular layers, target layer 4 and contrast with the reciprocal, caudal directed projections which in the main originate in infragranular layers, terminate outside of layer 4 and are thought of as feedback (FB) pathways (Lund et al., 1975; Rockland and Pandya, 1979; Maunsell and Van Essen, 1983; Kennedy and Bullier, 1985; Barbas, 1986; Boussaoud et al., 1990; Morel and Bullier, 1990; Webster et al., 1991; Distler et al., 1993; Barone et al., 1995, 2000; Barbas and Rempel Clower, 1997; Felleman et al., 1997b; Gattass et al., 1997; Rempel Clower and Barbas, 2000).
Definitions of Hierarchical Organization, Hierarchical Distance and Hierarchical Rank
The laminar patterns of cortico-cortical connections indicate an anatomical hierarchical ranking of primate cortical areas. For a given cortical area, higher-order areas have FB relations and lower-order areas have FF relations. Pairwise comparisons of the laminar patterns of connectivity have made it possible to determine the hierarchical organization of the visual system which places area V1, V2, V3, etc. on successive hierarchical levels (Maunsell and Van Essen, 1983; Ungerleider and Desimone, 1986; Boussaoud et al., 1990; Felleman and Van Essen, 1991; Webster et al., 1991, 1994; Young, 1992; Distler et al., 1993; Rockland 1997; Barone et al., 2000; Hilgetag et al., 2000).
Individual cortico-cortical pathways exhibit a precise laminar distribution of the parent neurons characterized by the percentage of labeled supragranular layer neurons — SLN%, see Figure 1 (Barone et al., 2000). Following injections of tracers in area V4, SLN% increases at successive lower hierarchical levels, so the SLN% is 60% in area V3, 93% in V2 and 100% in V1. Conversely, there is a progressive decrease in SLN% at successive higher levels. In this way the value of SLN% relates to the number of hierarchical levels separating two cortical areas, which we refer to as the hierarchical distance (Fig. 1). This makes SLN% values extremely powerful in generating hierarchical models of the visual cortex (Barone et al., 2000).
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Experimental Aims of the Present Study
During pre- and postnatal development, all cortical areas which project to area V1 show a 45–90% reduction in SLN% values (Kennedy et al., 1989; Barone et al., 1995). This raises a number of issues concerning the development of association pathways linking cortical areas which we have investigated in the present study.
Firstly, the developmental reduction of the SLN% of area V1 afferents occurs during a period when there is an overall reduction in numbers of connections. This raises the possibility that selective elimination of connections creates the characteristic SLN% differences between areas.
Secondly, by shaping inter-areal connectivity, selective elimination during development could modify the hierarchical organization of the cortex, which in turn might imply differences in the physiological function of the immature cortex (Dehay et al., 1988).
Thirdly, the developmental remodeling of connections might be restricted to projections to area V1, given that this area exhibits a number of unique features (Dehay et al., 1988; Dehay and Kennedy, 1993). Remodeling could be a developmental feature of cortical projections to primary areas, which supposedly receive FF input uniquely via their afferents from the principal thalamic relay nuclei.
Fourthly, earlier studies of the development of projections to area V1 provide no information as to whether there is a developmental remodeling of FF projections. This requires investigating the connectivity of a cortical area such as area V4, which receives both FF and FB cortico-cortical connections.
To address these issues, we have examined the connectivity of the visual area V4 using a method for determining the laminar distribution of projection neurons, which is immune to developmental changes in density.
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Materials and Methods |
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Anesthesia and Surgery
Retrograde tracer experiments were carried out on cynomolgus monkeys, Macaca fascicularis (Table 1). Following premedication with atropine (1.25 mg, i.m.) and dexamethasone (4 mg, i.m.), monkeys were prepared for surgery under ketamine hydrochloride (20 mg/kg, i.m.) and chlorpromazine (2 mg/kg, i.m.). In the case of fetal surgery, the pregnant monkey was premedicated in a similar fashion to the postnatal animals, with the addition of isoxsuprine (2.5 mg i.m.). After intubation, anesthesia was continued with 1% halothane in N2O/O2 (70/30). Heart rate was monitored and respiration adjusted to maintain the end-tidal CO2 at 4.5–6%. The rectal temperature was maintained at 37°C. In the pregnant monkey, a midline abdominal incision allowed uterotomy to be performed over the posterior part of the fetal brain.
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Injection of Retrograde Tracers
Stereotyped injections (3–5 mm) of retrograde fluorescent tracers (0.5–1.5 µl; 3% in H20) were made by means of Hamilton syringes on the prelunate gyrus between the LS the IOS and the STS, in area V4 containing the representation of the central visual field (Gattass et al., 1988). Injection sites spanned the full thickness of the cortex — cortical depth of injection does not influence SLN%, as found previously (Barone et al., 1994; Batardière et al., 1998a) and in the present study (data not shown). Elsewhere, we have characterized the uptake zone of Fb and DY tracers (Kennedy and Bullier, 1985) and reconstructions of injection sites (Fig. 2) showed that of the 15 injections, 11 were successfully confined to the cortical gray matter of presumptive area V4. In three of the fetal injections, the uptake zone extended into the subplate (Kostovic and Rakic, 1990; Smart et al., 2002). In one injection in the newborn, the injection contaminated the underlying white matter (Table 1
). For prenatal material, the fetus was replaced in the uterus and incisions were closed using routine procedures. The pregnant monkey received postoperative medication consisting of a muscular relaxant (isoxsuprine chlorydrate) and an analgesic (tiemonium methylsulfate).
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Histological Processing
Fetuses were delivered by Caesarean section after a 9–13 day survival period. Animals were deeply anaesthetized with a lethal dose of pento-barbital (i.p.) before being perfused transcardially with 200 ml of 2.7% saline, 1–3 l of 8% paraformaldehyde/0.5% glutaraldehyde mixture in phosphate buffer (0.1 M, pH = 7.4), 0.5 l 10% sucrose, 0.5 l 20% sucrose and 1 l 30% sucrose in phosphate buffer (0.1 M, pH = 7.4). Brains were immediately removed, blocked and horizontal 40 m thick sections cut on a freezing microtome. One section in three was mounted in saline onto gelatinized slides. Sections at regular intervals were reacted for cytochrome oxidase activity (Silverman and Tootell, 1987) and acetyl-cholinesterase activity (Mesulam and Geula, 1994).
Examination of Material
Sections were observed in UV light with oil-immersion objectives using a Leitz fluorescent microscope equipped with a D-filter set (355–425 nm). Neurons labeled by DY exhibit a yellow nucleus, while neurons labeled by Fb exhibit a blue coloration in their cytoplasm. An x–y plotter electronically coupled to the microscope stage was used to trace out sections and to record the positions of labeled neurons. After observation, sections were counterstained with cresyl violet and projected on to charts of labeled neurons so as to relate the positions of labeled neurons to histological borders.
Areal and Laminar Distribution of Labeled Neurons
At all ages, injection of tracers into area V4 leads to dense labeling of an extensive region of extrastriate cortex in the occipital, parietal and temporal regions (Tanaka et al., 1990; Baizer et al., 1991; Felleman and Van Essen, 1991; Shipp and Zeki, 1995; Barone et al., 2000), in different known visual areas (V2, V3A, MT, FST, LIP, FEF, TEO, TE) and in TH/TF (Fig. 2D). The areal extent of a population of retrogradely labeled neurons in one cortical area resulting from an injection in the target area is referred to as a projection zone.
The laminar distribution was expressed as the percentage of labeled supragranular layer neurons with respect to the overall population of infra- and supragranular labeled neurons (SLN%) and calculated separately for each projection zone (SLN% = number of labeled supragranular layer neurons/number of labeled supra + number of labeled infragranular layer neurons). SLN% falls off from a peak in the center of the projection zone to minimal values in the periphery (Barone et al., 1995, 2000; Batardière et al., 1998a). Fluctuating densities of supra- and infragranular layer neurons, coupled with the curvature of the cortical layers with respect to the plane of section, mean that stable values of SLN% require high frequency sampling of the entire projection zone.
Criteria for the Location of Cortical Areas
Multiple criteria were used to allocate labeled neurons to one of nine areas, including reference to gross morphological landmarks such as position in a particulars gyrus or sulcus (Barone et al., 2000); see Figure 2D. It was important to optimize the criteria used to distinguish different cortical areas, so as to be able to count neurons throughout a maximum extent of the projection zones in individual areas. Myelinization patterns and the laminar distribution of some histochemical staining in the fetus and neonate are immature and overall cannot therefore be used to identify cortical areas. Some architectonic limits were obtained using acetylcholinesterase histochemistry, which is strongly expressed in area V2 of fetuses and newborn (Barone et al., 1994).
One important criterion is the spatial distribution of labeling itself. Because the injection sites involved area V4 containing the representation of the central visual field, cortical areas which share borders where the far periphery of the visual field is represented show a discontinuous pattern of labeling. This gap in the labeling provides an important indication of the limits of the cortical area.
Area V2 is located in the posterior bank of the LS (Gattass et al., 1981), where it can be identified with cytochrome oxidase histochemistry in the adult (Tootell et al., 1983) and with acetylcholinesterase histochemistry in the fetus (Barone et al., 1994, 1996).
V3A is located in the anterior bank of the LS (Van Essen et al., 1986; Gattass et al., 1988; Felleman et al., 1997a). In most adult cases there is a gap between the labeling in areas V2 and V3A (Barone et al., 2000), while in fetuses this gap is less pronounced, but as in the adult there is a distinct increase in the density of labeling in the infragranular layers of area V3A. As in the adult, no labeling was found in area V3 on the anectant gyrus (Barone et al., 2000). Because of the proximity of the injection sites to area V4t, it was difficult to separate the intrinsic labeling in area V4 from the extrinsic labeling in V4t. Consequently, we have not included V4t projections in the present analysis.
Area MT is located in the posterior bank of the STS and stretches from the fundus to about halfway up the sulcus (Van Essen et al., 1981; Maunsell and Van Essen, 1983; Ungerleider and Desimone, 1986). In the adult, there was a more or less pronounced gap between the labeling of area MT and labeling on the prelunate gyrus. In the fetus, labeling in MT was more continuous with area V4 and a posterior limit of MT was set in the sulcus so as to ensure that no V4t was included in our analysis of MT (Desimone and Ungerleider, 1986; Gattass et al., 1988).
Labeling was found in a visual motion area (FST) in the floor of the STS which is anterior and ventral to area MT (Desimone and Ungerleider, 1986; Ungerleider and Desimone, 1986; Boussaoud et al., 1990). The gap between labeling in area MT and FST was less apparent in the fetus than in the adult and the limit between these two areas was determined with reference to the fundus of the sulcus.
Labeling in the posterior and lateral bank of the IPS was isolated from labeling in other areas and corresponds to the lateral intra-parietal area (LIP) (Andersen et al., 1990; Blatt et al., 1990; Boussaoud et al., 1990; Baizer et al., 1991; Colby et al., 1996; Lewis and Van Essen, 2000a,b).
The major input to area V4 from higher order areas is from the visual areas in the temporal lobe. The temporal occipital area (TEO) is located on the temporal lobe between the IOS and the STS (Ungerleider and Desimone, 1986; Baizer et al., 1991; Boussaoud et al., 1991; Distler et al., 1993). In the adult, labeling was discontinuous between V4 and TEO, whereas in the fetus the limits between these two areas was determined by projecting the location of the gap in the adult on to charts of the cortical labeling in the fetus. Anterior and ventral to TEO in the inferior temporal cortex is the temporal area TE (Webster et al., 1991, 1994).
In the ventral region of the temporal lobe in the parahippocampal cortex are the cortical areas TF and TH, located medial to the rhinal fissure and posterior to the perirhinal cortex (Suzuki and Amaral, 1994). In the adult, SMI32 histochemistry and myelin stains can be used to delimit these temporal areas (Lewis and Van Essen, 2000a,b), but these markers are not expressed in the fetuses. Anteriorly and medially, labeling in TF/TH in the adult showed a gap with labeling in the ventral part of areas TE at the level of the rhinal fissure, while in the fetus labeling was sometimes continuous at this level. When this was the case, the position of the gap in the adult between ventral TE and TF/TH was projected on to charts of labeling in the fetus.
In the frontal cortex, labeled neurons were found systematically in the anterior bank of the AS which is known to house the frontal eye field — FEF or area 8 (Stanton et al., 1989; Schall et al., 1995)
Statistical Tests
A multinomial analysis of variance — ANOVA (Woodward et al., 1990) — was used to test the hypothesis that the SLN% is equal across visual areas. Infra- and supragranular layers were treated as within-subject factors in the analysis. By testing proportions, the problem of the variation in total number of cells was eliminated. The analysis did, however, incorporate the total numbers of labeled cells in the estimates of variance for each proportion, so that proportions based on small total numbers have less precision than those based on larger numbers. When a significant difference between areas was observed, the multinomial ANOVA allowed us to do planned comparisons and to identify the areas that violated the null hypothesis. To test the relationship between SLN% and the number of levels that separate two interconnected areas, derived from the adult hierarchy (Barone et al., 2000), we used the non-parametric Spearman rank correlation test.
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Results |
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Injections in presumptive area V4 at all developmental stages lead to dense retrograde labeling throughout a large extent of extrastriate cortex (Figure 2
). The criteria used for allocating neurons to individual areas are given in the Materials and Methods section and were central to a previous report (Barone et al., 2000). During development, retrogradely labeled neurons in the thalamus are confined to those regions of the lateral and inferior pulvinar which are known to project to area V4 in the adult (Baleydier and Morel, 1992; Adams et al., 2000). Changes in Labeling Density Reflect Timetable of Innervation
So as to detect developmental increases in density of labeling resulting from innovation of the target, we have used a labeling index (LI) to monitor changes in the frequency of labeled neurons. LI is the percentage of labeled neurons with respect to the total population of neurons (Barone et al., 1996) and is not influenced by density changes due to developmental changes in cortical volume. Results for area V2, which is the major source of V4 afferents (Kennedy et al., 2000), are shown in Figure 3A. LI values show that at E106 only few cells have contacted their target (LI < 0.5%). Innervation proceeds steadily up to E129, when peak LI values are obtained (LI5%) before descending to adult-like values in the first postnatal month. This result suggests that the onset of cortical pathway formation is at E106, because this injection returned maximum levels of subcortical labeling coupled with only weak cortical labeling.
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Influence of Injection of Subplate on Density of Labeling
The depth of the injection influences neuron density. This is illustrated in Figure 1 of the Supplementary Material where age-matched plots of retrogradely labeled cells in single sections of area V2 show higher densities of labeled cells in cases where the injection encroaches on the subplate (SP). A quantitative analysis of the effect of the injection extending into the SP at different ages is shown in Figure 3C. This shows that the involvement of the SP increases densities maximally at E123 and that the SP effect has largely disappeared by birth. This result shows that at early stages cortical axons are waiting in the SP.
Quantitative Analysis of Laminar Distributions
As described in the Materials and Methods section, quantification of SLN% within individual areas necessitates extensive sampling of projection zones (Barone et al., 1995, 2000; Batardière et al., 1998a). High-power plots of neuron location are made throughout the maximum extent of labeling at regular intervals (see Supplementary Material). In the adult these charts provide an overview of changes in neuronal labeling density in different cortical areas. Such qualitative comparisons of adult and fetal labeling also give an indication of the developmental reduction of labeled supragranular layer neurons. Counts of numbers of neurons per section in each area make it possible to construct neuron density profiles of the projection zone in each area (Fig. 4) following each injection. This ensures that counts include peak values of the projection zone. Figure 4 illustrates the impossibility of using only two or three sections to estimate SLN%. For example, in the adult the profile for MT (Fig. 4C) returns global values of 55%, whereas individual sections from this injection return values ranging from 6 to 93%. The estimation of SLN% is computed directly by summing the total number of labeled neurons in the density profile for each area and for each injection.
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Developmental Remodeling
The density of labeled neurons at E106 is very low and increases up to E123 (Fig. 3B). This and the fact that distant areas have low numbers of labeled neurons, or in the case of FEF and TE none at all, further supports that cortico-cortical axons begin to innervate their targets some time around E106.
In a first instance we shall consider FB projections obtained at the later fetal stages investigated (i.e. E112, E123, E140). SLN% in higher-order areas (LIP, FST, TEO, TE, TH/TF) tends to fall into one of three groups: high (late fetal ages), medium (neonates) and low (adults and juveniles animals) (Fig. 5D,E and Table 2). The four injections in both of the 2 month old animals give results which are statistically indistinguishable from the adults (multinomial ANOVA, 
2 = 4.75, P = 0.09, n.s.) and these values therefore are pooled. A statistical analysis revealed that values in late fetuses differ significantly from those observed in neonates (2 = 772.3, P < 0.001). Furthermore, except for TE and TH/TF, all the percentages returned by the neonate injections are intermediate between late fetal stages and adult values (2 = 51.14, P < 0.001), showing that cortical connectivity is not fully mature at birth.
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In the FF projections, SLN% in area V2 remains constant at different developmental stages (post hoc comparison, fetus versus adult:
2 = 0.09, P > 0.5; Fig. 5A). This contrasts with a weak (20%) but significant SLN% reduction in area V3A (2 = 24.23, P < 0.001). The lateral connections from area MT in the E112–140 fetuses (Fig. 5B) are concentrated in the supragranular layers (SLN% = 66%), which contrasts with the adult where these connections are more evenly distributed in both layers (SLN% = 47%) so that the development reduction of SLN% is 28%. The FB projections from all areas in both dorsal and ventral streams show important reductions in SLN% (Fig. 5D,E). In the adult and fetus the FEF V4 pathway show stable values (70 versus 73%, 2 = 0.057, P > 0.05).
Because we have results from only one animal per age group, we cannot evaluate variability in SLN% at fetal ages. However, in the adult a statistical analysis did not reveal significant differences in SLN% across subjects (see above, c2 = 4.75, P = 0.09) or because of the type of dyes used when a double injection was performed in the same animal (Fb versus DY, all cases P > 0.05). Furthermore, when individual SLN% are plotted against age (Fig. 5F), the developmental curves follow a regular monotonic decrease from E123 to juvenile–adult values. Taken together, these observations suggest that at each fetal age and in the adult, single-dye injection provides stable SLN%.
Cellular Mechanisms and Timetable of Developmental Remodeling
The cortical and subcortical patterns of labeling at E106 suggest that the great majority of cortical axons have not yet reached their targets at this age. In all areas (except in TH/TF) SLN% of the E106 fetus are lower than those obtained in older fetuses (Fig. 5). The increase in SLN% between E106 and E123 occurs over a time period when there is an important increase in overall numbers of projecting neurons (Fig. 3B). This suggests that it is the consequence of an increase in the number of supragranular rather than a reduction in the numbers of labeled infragranular layer neurons. Hence, it would seem that although development is characterized by excess numbers of SLN, the very early axons to arrive in the cortex at E106 are mostly from infragranular layers (Coogan and Burkhalter, 1988).
At late developmental stages (E123, E140, neonate) a DY and an Fb injection has been made side-by-side in area V4 (see Fig. 2). In each case, the Fb injection involves the underlying SP whereas the DY injection is entirely restricted to the cortical gray matter (GM injections). SP injections label higher numbers of neurons compared to GM injections (Fig. 3C). However, GM injections lead to higher SLN% in all areas (V3A, MT, TEO, TE and LIP) compared to SP injections at the same age (Fig. 6A). The most pronounced increase of SLN% following GM injection is observed at E140. Overall, GM injections give a mean increase in SLN% of ~13% compared to SP injections. These results mean that the SP injections lead to proportionally more axons from infragranular layer neurons capturing and retrogradely transporting the dye than do GM injections. We can deduce, therefore, that from E123 to birth there is a delay in the ingrowth of the infragranular axons into the cortical gray matter of their targets. Note that the developmental SLN% reduction is observed when considering both SP and GM separately (data not shown). Hence, the depth of injection does not influence our results when data from all injections are pooled.
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Side-by-side injections of DY and Fb lead to two populations of single-labeled neurons with a variable degree of spatial overlap, depending on the distance separating the two injection sites. Neurons which project to both injection sites capture both dyes (i.e. are double-labeled) and are located in the overlap zone of the two populations of single-labeled neurons (Kennedy and Bullier, 1985
; Barone et al., 1995). In the adult FB and lateral pathways, maximum numbers of double-labeled neurons are located in the infragranular layers (Fig. 6B–F), which reflects the fact that the spatial extent of the projection zone in the adult is greater in the infragranular layers than it is in the supragranular layers. At fetal stages the majority of double-labeled neurons are located in the supragranular layers, suggesting that at these stages the projection zone in the supragranular layers extends further than those in the infragranular layers (Fig. 4). This finding suggests that supragranular layer neurons undergo a developmental reduction of their divergence (i.e. a reduction of the extent of the target area contacted by individual neurons). Remodeling and Hierarchical Organization
In the adult we have shown that a pathway connecting two areas is characterized by its SLN% and reflects the number of hierarchical levels that separate the two interconnected areas (Barone et al., 2000); for details of calculation see the legends of Figures 1 and 7. In adult FB pathways, increasing the number of levels between interconnected areas decreases SLN%. In adult FF pathways it is the inverse, so that increasing the number of levels between interconnected areas increases the SLN%. This we refer to as a ‘distance rule’. Inspection of the laminar organization of the pooled projections in fetuses (E112–E140, Fig. 7A) suggests that the same distance rule applies during development. As in the adult, SLN% in fetuses are specific for each projection (2 = 24 523; P < 0.001). However, in fetuses differences between SLN% are not as marked and the overall increase of the SLN% means that there is a reduced dynamic range.
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In the adult, SLN% can be used to rank areas on distinct hierarchical levels — see Figure 7B
(Barone et al., 2000). A similar statistical approach was applied to the pooled fetal SLN% values. The paired comparisons of SLN% from each area reveal that 42 out of 45 are significantly different (Table 3). Furthermore, this analysis shows that 45 pairwise comparisons in the fetus reveal 87% homology with the same analysis performed in the adult (39/45 comparisons, Table 3). As in the adult, SLN% in the fetus put V2, V3A as well as ventral areas TEO, TE and TH/TF on successive levels. Furthermore, as in the adult, fetal SLN% values for the FEF place this area on a low hierarchical level, equivalent to that of V3A (2 = 0.61, P = 0.43, n.s.). Similarly, as in the adult, fetal SLN% values place MT and TEO on the same level (2 = 0.76, P = 0.38, n.s.) and put FST and LIP on separate levels (2 = 79.29, P < 0.001). In the fetus there is a strong and significant correlation (Spearman, = –0.83, P = 0.01, Fig. 7B) between SLN% and the hierarchical distance using the hierarchical model of the adult visual system proposed by Barone et al. (Barone et al., 2000) and a somewhat lower correlation (Spearman, 
= –0.78, P = 0.01; r2 = 0.71 versus 0.82). using the hierarchical scheme proposed by Felleman and Van Essen (Felleman and Van Essen, 1991). The fact that the fetal values show a close correlation with the adult hierarchy strengthens the idea that the hierarchical relationships remain constant during development, despite the overall higher SLN% values in the immature cortex.
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Values of SLN% differ significantly between E123 and E140 (multinomial ANOVA,
2 = 217.5, P < 0.001) and from E140 to birth (2 = 269.2, P < 0.001). Using SLN% at individual ages (123, 140, newborn), we obtain adult-like sequences in ventral (TEO > TE > TH) and dorsal (FEF > MT > FST) streams. The progressive emergence of the adult organization during development can be assessed by analyzing the correlation of hierarchical rank and SLN% at different ages (Fig. 8B). At all stages (E106, E123, E140 and neonate), there is a significant correlation between the SLN% and hierarchical rank (Spearman, P < 0.05) except in the case of the gray matter injection at E123 (Spearman, = –1.67, P = 0.09). The fact that the GM injection at E123 gives a weaker correlation than the SP injection at the same age is because of the high number of axons from supragranular layers that have penetrated the target at this age. The SP injection returns a lower SLN% because it recruits the waiting infragranular layer neurons.
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As seen in Figure 8D
, there is a progressive increase in the steepness of the correlation slope from E123 to birth, when adult-like values are observed. Similarly, the correlation factor (r2) increases with development and by birth returns values similar to those obtained in the adult (Fig. 8C). Overall, this analysis shows that hierarchical ranking of visual cortical areas is established in the fetus and, further, that it is only marginally influenced by extending the injection into the SP.
Although an adult-like sequence is present in fetal stages, SLN% are overall significantly higher in the fetus compared to the adult. SLN% reduction influences differentially each FB pathway to area V4. For example, between E123 and adult (Fig. 8E), in the dorsal pathway, the reduction in SLN% is progressively higher going from V3A (–20%), MT (–33%), LIP (–65%) and FST (–71%). A similar increase in reduction is observed in the ventral pathway going from TEO to TE. In Figure 8F, the decrease in SLN% for individual projections to V4 is plotted against the number of hierarchical levels, i.e. hierarchical distance (Barone et al., 2000), separating each area from area V4. In fetuses, the amplitude of reduction (E123 adult, E140 adult) is tightly related to the hierarchical distance (both cases, Spearman, P < 0.01). From birth to adulthood, the remodeling is weaker than in fetuses (see Fig. 5) and is not correlated with the hierarchical distance to V4 (Spearman, = –0.27, P > 0.05).
Altogether, although global SLN% is higher in the fetus compared to the adult, the hierarchical ranks are clearly established in the immature cortex. Hence, differences of SLN% are sharp enough to maintain a distance rule (and therefore hierarchical levels) and thus the relative relations between areas are as in the adult. The adult SLN% is, however, established progressively through a prolonged developmental period that lasts until the first month after birth.
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Discussion |
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Technical Considerations
Because immunohistochemistry and myelin stains can not be used in the fetus to define cortical areas, a major difficulty in a developmental study such as this is the correct allocation of neurons to individual cortical areas. In the present study, we found that projections from individual areas originate from well-defined projection zones showing peak levels of labeling (see Fig. 4). This means that immature cortico-cortical projections do not form a uniform distribution, but instead link spatially defined regions of the cortex which correspond to future cortical areas. Although the immature material did not show clearly defined gaps between labeled regions (see Materials and Methods), it is unlikely that imprecision on the exact position of areal borders significantly influences the present results, given that peak levels were centered in the presumptive cortical areas. Hence, while uncertainty regarding the exact location of areal borders might introduce a small degree of error, in the present findings this does not influence the major result, which is that the number of labeled neurons peaks in presumptive cortical areas and that SLN% are characteristic for each area.
Primate developmental studies such as this invariably suffer from using small numbers of animals at each developmental stage. This means that the variable which is to be measured needs to be highly reliable. This is, in fact, the case. In the adult we have shown that, correctly assessed, SLN% values across individuals are constant and are extremely robust indicators of hierarchical rank (Barone et al., 2000).
Time Course of Remodeling
We are confident that we have encompassed the developmental period during which cortical connections undergo reorganization. Our first injection at E106 showed that few connections from afferent cortical areas had been made with the cortical gray matter of area V4, despite the fact that injections in the SP at this age reveal numerous projections (Coogan and Van Essen, 1996). The present study shows that the laminar distribution for projections to area V4 matures according to a similar time course as those back-projecting to area V1, where the adult configuration is achieved 1–2 months after birth (Barone et al., 1995).
Comparison with other Species
In the kitten, the laminar distribution of FB projections to area 17 is uniform across individual extrastriate areas and the selective reduction of the SLN% generates the laminar distribution characteristic of each area (Batardière et al., 1998a). This contrasts with the primate, where FB projections to area V1 show a rudimentary areal specificity right at the start of cortico-cortical pathway formation, as has been shown in this report and elsewhere (Barone et al., 1995). The present results show that those pathways which project to area V4 and which show a developmental remodeling (i.e. from areas V3A, MT, FST, LIP, TEO, TE, TH/TF) are also specified from the onset of pathway formation and therefore exhibit characteristic SLN% values during early stages of development prior to developmental remodeling of the pathway. In this way, in the primate the reduction of SLN% serves to sharpen an early formed pattern.
Cellular Mechanisms Underlying Developmental Changes in the Laminar Distribution
The developmental reduction of SLN% in FB projections to area V1 has been shown to be accompanied by a larger decrease in the convergence values of supragranular projection neurons compared to infragranular projection neurons (Barone et al., 1995, 1998; Batardière et al., 1998a). In the present study, the switch of double-labeled neurons from a supragranular location in the fetus to an infragranular layer location in the adult suggests that the remodeling of the laminar distribution for projections to area V4 is also due, at least in part, to a reduction of the convergence values of cortico-cortical connections (Kennedy et al., 1994; Price et al., 1994).
Which cortical layer first forms a projection with its cortical target? This question is important because the process of path-finding and target recognition might be expected to be the prerogative of these first-formed connections. The present results, showing that at the very earliest age (E106) the few cortical connections from higher-order areas stem from infragranular layers, suggests that the earliest born neurons might be the first to send an axon to their appropriate cortical target early in development (Coogan and Burkhalter, 1988). Such a developmental strategy would make sense because when the layer 6 neurons have just completed their migration to the cortex at ~E70, the distances separating cortical areas are considerably shorter than at E100 when upper layer neurons begin to form connections. The present results show that after E106, injections involving the SP lead to an appreciably lower SLN% than do injections restricted to the cortical gray matter. This suggests that a fraction of axons from infragranular layer neurons have not yet invaded the cortical gray matter in the immature brain between E120 and birth. Hence, it would be reasonable to conclude that the axons of infragranular neurons are the first to contact the target where one subpopulation remains in the SP while some axons penetrate the GM. Subsequently, axons from the supragranular layers penetrate the cortex in large excess. At this late stage the infragranular axons continue to form an appreciable number of transient connections with the SP. In this way, formation of cortical connections involves two sets of transient connections: the first from the infragranular neurons to the SP of the target and the second from the supragranular neurons with the GM of the target area. Elimination of these transient connections follows different timetables. Elimination to the SP is complete by birth and to the GM 1 month later. Thus, the late invasion of the GM by infragranular axons along with the reduction of convergence of supragranular layer axons, contributes to establishing the mature SLN%.
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Conclusion |
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The development of the cortical connectivity of area V4 illustrates a highly dichotomous strategy in the formation of FF and FB pathways. The development of the FF pathway from area V2 to area V4 has been shown to be complete early in prenatal life, to depend largely on directed growth and target recognition mechanisms and not to involve the large-scale elimination of inappropriate axons (Barone et al., 1996
). Supragranular layer neurons constitute the major component of the FF pathway and their axons accurately target their final destination early in development. This contrasts with the prolonged remodeling of the FB projections, where selection leads to a massive pruning of early formed connections but where late progressive factors may also play a role as has been demonstrated in this study and elsewhere (Rodman, 1994; Barone et al., 1995).
In the adult, SLN% values are highly specific for individual cortical areas (Barone et al., 2000). Because the exact SLN% is related to the hierarchical distance separating cortical areas, it is expected in turn to relate to the physiological role of FF and FB pathways which are beginning to be understood from both ultrastructural and physiological investigations (Ishai and Sagi, 1995; Miyashita, 1995; Vanduffel et al., 1997; Anderson et al., 1998; Gonchar and Burkhalter, 1999; Lamme and Roelfsema, 2000). Throughout development we observed that the relative hierarchical organization of the visual system is similar to that in the adult.
The early prenatal specification of FF connections could provide the neurophysiological substrate for the steady increase of visual capacities observed in infant monkeys during the first postnatal months (Blakemore and Vital-Durand, 1981; Boothe et al., 1985; Rodman et al., 1993; Rodman, 1994; Distler et al., 1999). However, the adult laminar organization of FB pathways is not present before the second postnatal month. This prolonged development of FB cortical connections might be of particular importance in primates, as we also detected this phenomenon in the somatosensory system (Batardière et al., 1998b) and one would predict that this could be further extended in humans (Burkhalter, 1993; Kennedy and Dehay, 1997; Kennedy et al., 1997). Given the evidence of the involvement of FB projections in figure ground discrimination (Zipser et al., 1996; Hupé et al., 1998), it is interesting to note that this psychophysical response emerges at the end of the first year of life and only becomes adult-like at around 8–13 years of age in human (Sireteanu and Rieth, 1992). The searching question that remains is why would FB pathways in the visually experienced infant include 28–84% additional supragranular layer projection neurons?
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Supplementary Material |
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Supplementary material can be found at: http://www.cercor.oupjournals.org
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Notes |
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Financial support was provided by the EEC, Quality of Life and Management of Living Resources (QLG3-1999-01064) and the Human Frontier Science Program Organization (HFSP RG0133/2000-B). A.B. was supported by an MRT grant. We thank Colette Dehay for help with experiments, Brice Ronsin, Antoine Guillarme, Luc Renaud and Christel Merrouche for excellent technical assistance, and Ghislaine Clain for animal care.
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2 Current address: Centre de Recherche Cerveau et Cognition, CNRS–UPS–UMR 5549, Université Paul Sabatier, 133 route de Narbonne, 31062 Toulouse Cedex, France
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Abbreviations |
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| Cortical Visual Areas |
| V1 visual area 1 |
| V2 visual area 2 |
| V3 visual area 3 |
| V4 visual area 4 |
| MT middle temporal area |
| TEO temporal occipital area |
| TE temporal area |
| TF temporal area TF |
| TH temporal area TH |
| FST fu |