Abstract:
Brain-mapping of the congenitally blind human reveals extensive plasticity1. The visual cortex of the blind has been observed to support higher cognitive functions including language and numerical processing2,3. This functional shift is hypothesized to reflect a metamodal cortical function, where computations are defined by the local network. In the case of developmental deafferentation, local circuits are considered to implement higher cognitive functions by accommodating diverse long-distance inputs4–7. However, the extent to which visual deprivation triggers a reorganization of the large-scale network in the cortex is still controversial8. Here we show that early prenatal ablation of the retina, an experimental model of anophthalmia in macaque, leads to a major reduction of area V1 and the creation of a default extrastriate cortex (DEC)9,10. Anophthalmic and normal macaques received retrograde tracer injections in DEC, as well as areas V2 and V4 post-natally. This revealed a six-fold expansion of the spatial extent of local connectivity in the DEC and a surprisingly high location of the DEC derived from a computational model of the cortical hierarchy11. In the anophthalmic the set of areas projecting to the DEC, area V2 and V4 does not differ from that of normal adult controls, but there is a highly significant increase in the relative cumulative weight of the ventral stream areas input to the early visual areas. These findings show that although occupying the territory that would have become primary visual cortex the DEC exhibits features of a higher order area, thus reflecting a combination of intrinsic and extrinsic factors on cortical specification. Understanding the interaction of these contributing factors will shed light on cortical plasticity during primate development and the neurobiology of blindness.
Main text
Early visual cortex deafferentation via bilateral removal of the eyes at early stages of prenatal development in the macaque provides a non-human primate (NHP) model of anophthalmia. In NHP anophthalmics, there is an in-depth modification of the development of the visual system accompanied by characteristic sulci malformations; cortex destined to become striate cortex (area V1) reverts to a default phenotype (Default Extrastriate Cortex DEC)9,10,12. The three anophthalmics used in this study each showed important shifts in the border of striate cortex (area V1) leading to an important reduction in its dimensions (Figure 1). In these animals instead of the typical border between areas V1 and V2 one can detect a large region of interceding cortex where the stria of Genari is absent and its cytoarchitecture can be broadly defined as extrastriate. This stretch of cortex exhibits small islands of striate cortex and the hybrid expression of histochemical phenotypes of striate and extrastriate cortex both during in utero development and postnatally12,13. The cortex between area V2 and the reduced striate cortex area V1 corresponds to the DEC (lower panels of Figure 1A,B). The proportion of area V1 with respect to the total cortex is considerably reduced in anophthalmic brains compared to the normal (Figure 1C). This contrasts with the proportions of total visual cortex (including DEC) with respect to neocortex, which is similar in anophthalmics and normals, therefore coherent with deafferentation causing a border shift rather than merely a shrinkage of area V1. Hence the DEC plus the remaining area V1 in the anophthalmic matches the extent of area V1 in the normal animal14.
We used retrograde tracers that allow exploring the intrinsic labelling of a cortical area, which corresponds to the local connectivity15. In the normal brain intrinsic connectivity corresponds to 80-90% of the total connectivity and exhibits an exponential decline with distance15,16. In the anophthalmic brain the space constant of the exponential decline is significantly larger and intrinsic projections extend over considerable distances (Figure 2A). Hence in the normal V1 the 75% threshold is at 0.25mm, the 80% at 0.35mm and the 95% at 0.80mm. In the DEC these distances are increased 4 to 6 fold (Figure 2B), so that local connectivity extends across a large extent of the DEC on the operculum (Figure S1).
The topography of connectivity in the anophthalmic was overall similar to that in the normal cortex. Following retrograde tracer injection in a target area, the numbers of labelled neurons in a given source area with respect to the total number of labelled neurons in the brain defines the Fraction of Labelled Neurons FLN, a weight index reflecting the strength of the particular pathway15. High frequency sampling of labelled neurons in the cortex allows characterization with a single injection of the weighted connectivity of a pathway linking any two cortical areas17. Injections limited to the grey matter were performed in DEC, V2 and V4 in three anophtalmic brains (Figures S2 Table 1). Inspection of cortical labelling suggested that early visual deafferentation leads to an increase of numbers of labelled neurons in ventral stream areas (Figures S2, Fig S3). Injections in DEC and area V2 show that deafferentation profoundly affects the relative strengths of the dorsal and ventral pathways, as seen after summing the FLN values across all ventral versus dorsal stream areas (Figure 2C, D). Differences between normal and anophthalmic cumulative FLN values were not found to be significant following injections in area V4, suggesting that the effect of deafferentation are restricted to early cortical areas.
The laminar distribution of retrogradely labelled parent neurones of a pathway is defined by its proportion of supragranular labelled neurons or SLN index, which has been shown to be highly consistent across individuals11. The SLN values of a pathway define it as feedforward or feedback and specify a hierarchical distance18. In the absence of the retina there is an increase in numbers of labelled supragranular layer neurons (Figures S4). The SLN value for area V2 projections to DEC is significantly higher than for the V2 projection to V1 and likewise the projection of V3, FST and PIP to DEC have significantly increased SLN values (Fig S4, panels A). Following deafferentation, projections to area V2 showed significant increases in the SLN in areas V1 and V3 as well as the dorsal stream areas MT, V3A, LIP, PIP, STP and PGa as well as an increase in the ventral stream area TEO (Figures S4, panel B). By contrast, deafferentation had little or no effect on the SLN values for any of the projections to area V4 (Figure S4D) with the marked exception of V1 where only infragranular neurons were observed. However, given the very low FLN value, this cannot be considered significant. The most marked change in the SLN is the projection of area V1 to the DEC (Figure 3A). Labelled neurons in area V1 projecting to the DEC are entirely located in the supragranular layers, which makes this projection very different from any projection from area V1 in the normal brain (Figure 3A). Area V1 also projects strongly to area V2, but the V1->V2 pathway originates from both infra- and supragranular layers. A projection of V1 which is entirely from the supragranular layers would be to area V4, but the V1->V4 pathway is considerably weaker than the DEC->V4 pathway.
SLN has been shown to be a robust indicator of hierarchical distance11,19 (see Materials and Methods). When the SLN values extracted from injections at different levels are mapped on to hierarchical space by means of a sigmoid function they display surprisingly good agreement (Figure 3B). This is shown by the probit transformed values of SLN plotted in a pairwise fashion; if the transformation leads to a coherent measure of hierarchical distance the points will cluster around lines of unit slope. Importantly, both the normal and anophthalmic brains display this consistency in laminar organization (Figure, 3B, C, D). All the common projections to areas V1 and V2 in the normal cortex are feedback and, as expected, are observed in the lower left quadrant. This contrasts with the anophthalmic where V1 is in the top right quadrant, indicating it to be feedforward to DEC and V2. Consideration of an ensemble of SLN values following injections in areas V1, V2 and V4 allows fitting a hierarchical model to both the normal and anophthalmic data sets (Figure 4A)11. This shows that the overall layout of the ventral and dorsal stream areas remain approximately similar to that observed in the normal. However, in the anophthalmic brain, DEC and area V2 are considerably higher in the hierarchy than expected. The goodness of fit of the model is shown by close agreement between the empirical and estimated SLN values by source and target areas (Figure 4B, normal r2=0.72; enucleate r2=0.67).
The present results show that in the anophthalmic the topography of connectivity and global organization of the ventral and dorsal streams are largely conserved as has been suggested by imaging studies in the human congenitally blind20–26, in line with the evidence of early developmental specification of the functional streams27,28. Further, in the anophthalmic brain we observe en expansion of the ventral pathway that could reflect cross modal plasticity24
The present findings show that early primate visual cortex in anophthalmia is profoundly modified both in its cytoarchitecture and local connectivity. While the global hierarchy remains largely conserved, there are important local changes in the hierarchical organization. These changes would seem to reflect a persistence of immature features making the congenitally blind ‘visual’ cortex neotenic. Indeed, interareal connectivity during in utero development in the primate undergoes extensive remodelling characterised by a greatly expanded population of supragranular projecting neurons, a global hierarchical organization similar to that found in the adult, an absence of ectopic pathways and finally a markedly extensive local connectivity11,29–31. The relatively high position in the cortical hierarchy and the conservation of an extensive local connectivity in the DEC could ensure the long time constants which would be required for the observed higher cognitive functions of the deafferentated cortex of the blind19,32.
The present findings need to be considered in view of current understanding of the developmental specification of the cortex. Developmental patterning of the neocortex is consequent to an interplay between intrinsic genetic mechanisms based on morphogens and secreted signalling molecules and extrinsic inputs relayed to the cortex by thalamocortical axons33–35. The role of thalamic axons in arealization is a multistep hierarchical process involving events at progenitor and neuronal levels36. A recent spatiotemporal transcriptome analysis of the pre- and postnatal macaque forebrain revealed a small number of genes that have persistent expression across cortical development, suggesting a large potential for extrinsic shaping of the cortex37. Interestingly, this study shows that areal and laminar molecular phenotypes are acquired late postnatally indicating a wide and potentially important role of contextual shaping of the structure and function of the cortex, suggesting that particular attention should be paid to the care of the young congenitally blind.
MATERIALS & METHODS
We examined the connectivity of the cortex in two 25-day old and one 10 month-old macaques that had been enucleated between 92 and 107 days before birth i.e. between embryonic day 58 (E58) and E73 (Table 1). In these three experimental animals we made six tracer injections, a fast blue (FB) and a dyamidino (DY) injection in each and we compared the results to 10 injections made in 8 adult controls.
Anaesthesia and Surgery
The present study is based on observations following bilateral enucleation performed in three monkey foetuses and contrasted to eight normal controls. The enucleated foetuses were carried to term and after birth injected with retrograde tracers (Diamidino Yellow, DY; and Fast Blue, FB) at different postnatal ages (Table 1). Pregnant cynomolgus monkeys (Macaca fascicularis) received atropine (1.25 mg, i.m.), dexamethasone (4 mg, i.m.), isoxsuprine (2.5 mg, i.m.), and chlorpromazine (2 mg/kg, i.m.) surgical premedication. They were prepared for surgery under ketamine hydrochloride (20 mg/kg, i.m) anaesthesia. Following intubation, anaesthesia was continued with 1-2% halothane in a N20/02 mixture (70/30). The heart rate was monitored, and the expired CO2 maintained between 4.5% and 6%. Body temperature was maintained using a thermostatically controlled heating blanket. Between embryonic day 58 (E58) and E73 and using sterile procedures a midline abdominal incision was made, and uterotomy was performed. The foetal head was exposed, bilateral eye removal performed, and the foetus replaced in the uterus after closing the incisions. The mother was returned to her cage and given an analgesic (visceralgine, 1.25 mg, i.m.) twice daily for 2 days. All foetuses were allowed normal development until term (E165).
Injections of Retrograde Tracers
Identical medication, anaesthesia and monitoring procedures were used as described above. Tracer injections were placed in the DEC, area V2 and area V4. Injections were made by means of Hamilton syringes in a stereotypic fashion. Following injections, artificial dura mater was applied, the bone flaps were closed, cemented and the scalp stitched back into position.
The tracer injection sites are shown in Figure 2. Three injections are located in the DEC (top three rows in Figure 2), one in area V2 (fourth row Figure 2) and two in area V4 (last row Figure 2). All injections in the enucleate brain were confined to the cortical grey matter, and except for case BB122 LH DY (third row injection sites). BB122 LH DY injection in the DEC was very small and restricted to upper layers and is only considered for the examination of topography. Side-by-side injections in target areas of retrograde tracers reveal the topology of connectivity in source areas. Such side-by-side injections were made in the DEC in the lower part of the medial operculum in case BB181 corresponding in normal cortex to area V1subserving parafoveal visual field38. In case BB122 a single injection was made in V2 near the lip of the lunate sulcus where foveal visual field is represented in the normal cortex39. Finally, a pair of very large injections were made on the dorsal part of the prelunate gyrus spanning the central and peripheral representation of area V4 in the normal brain40.
The full extent of labelled neurons were charted across the cortex, which was parcellated according to a 91 area atlas17. Injection of retrograde tracer in an area leads to a region of labelling in each afferent area. This region is referred to as the projection zone. So as to obtain reliable counts of labelled neurons it is necessary to chart labelled neurons throughout the full extent of the projection zone in each area11,29. This makes it possible to estimate the fraction of labelled neuron (FLN) and the ratio of supragranular layer neurons (SLN) in each area (see Materials and Methods). FLN is a weight index which allows construction of a weighted and directed matrix of the cortical network 15, while SLN value of interareal pathways constitutes an index of hierarchical distance, allowing areas to be organized in a determinate hierarchy11.
Animal euthanasia
After 10 to 12 days of recovery that allows optimal retrograde labelling of neurons projecting to the pick-up zone, animals were anesthetised with ketamine (20 mg/kg, i.m.) followed by a lethal dose of Nembutal (60 mg/kg, i.p.) and perfused through the heart with a 1.25% paraformaldehyde and 1.5% glutaraldehyde solution. After fixation, perfusion was continued with a 10-30% sucrose solution to provide cryoprotection of the brain.
Data Acquisition
Depending in the enucleation case, parasagital (BB181) or horizontal (BB122 and BB169) sections (40-μm thick) were cut on a freezing microtome and at least 1 in 3 sections were stained for Nissl substance. Normal controls were cut in horizontal and coronal planes. Sections were observed in UV light with oil-immersion objectives using a Leitz fluorescence microscope equipped with a D-filter set (355-425 nm). High precision maps were made using Mercator software running on Exploranova technology, coupled to the microscope stage. Controlled high frequency sampling gives stable neuron counts despite curvature of the cortex and heterogeneity of neuron distribution in the projection zones of individual areas11,41 Characteristics of neurons labelled with FB or DY are described by Keizer and colleagues42. Area limits and layer 4 were marked on the charts of labelled neurons. These neurons were then attributed to areas of our atlas based on landmarks and histology, and counted according to that parcellation17.
Statistical analysis
All statistical analyses were performed in the R statistical environment43 with additional tools from the MASS, aod, and Betareg packages44–46. Each injection gave rise to retrogradely labelled neurons, which were plotted and compared against those of normal animals, injected at anatomically equivalent locations. As previously shown15, the FLN (Fraction of Labelled Neurons), corresponds to the proportion of cells located in a given source area with respect to the total number of labelled neurons in the cortex. The connectivity profile is defined by the FLN values for each of the structures labelled from the injected target area. The SLN measurement of the proportion of supragranular labelled neurons in an area, has been shown to be a stable anatomical assessment of areal hierarchical relationships11,18.
FLN
The distribution of FLN values has been successfully modelled previously by a negative binomial distribution15,17. This can be performed using a Generalized Linear Model (GLM) when the dispersion parameter is fixed. We initially estimated the dispersion parameter for individual areas obtaining values between 10.43 and 11.7, and for subsequent tests used an average value of 4. We then used this model to compare connection strengths (i.e. FLN values) between normal (i.e. non-enucleated) and enucleated animals. As explanatory variables, we used a 2 level factor, Group (Normal/Enclueated) and an 2 level factor, Area for the labelled areas projecting on the target injection. The linear predictor in the GLM includes main effect of both factors and their interaction. In the model, the raw neuron counts enter as the response variable, but a log link is used with the natural log (i.e. base e) of the total number of labelled neurons in each case included as a fixed offset. In this way, the model coefficients estimate FLN values. Confidence intervals were computed to assess the significance of the difference, based on the model fitted estimates. To test the effect of enucleation in either stream, the linear predictor was modified to include the factor Group and the two level factor, Stream (Ventral/Dorsal).
SLN
A similar approach was used to analyse SLNs (Supragranular Labelled Neurons, the proportion of labelled neurons situated above the layer 4 (granular layer) vs. below it, in each area) but using beta regression44 to model the proportions. Since the beta distribution is defined on values in the interval (0, 1), it is useful to analyse proportions. Like the binomial, the parameters of interest are linked to the explanatory values via a linear predictor. In this case a logit link was used. Like the negative binomial distribution on counts, the model includes a dispersion parameter that can account for overdispersion beyond that expected from a purely binomial process. As before, confidence intervals were evaluated and significance was assessed using likelihood ratio tests.