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Objective To demonstrate the feasibility and efficacy of high-intensity focused ultrasound (HIFU) to non-invasively treat placental tissues in a monkey model of pregnancy. Materials and methods A toroidal HIFU transducer working at 2.5 MHz with an integrated ultrasound-imaging probe was used. Eight pregnant monkeys were exposed to HIFU treatment after anesthesia. Lesions on placental tissues were performed non-invasively by placing the HIFU probe on the skin. Fetal and maternal parameters, such as maternal heart rate, fetal heart rate, and subcutaneous and intra-amniotic fluid temperature, were recorded during HIFU exposure. A C-section was performed immediately after insonification to extract the placenta, inspect the fetus and inspect the maternal abdominal cavity. Placental HIFU lesions were studied using ultrasound images, gross pathology and histology. Results The average gestational age of the animals was 72 ± 4 days. Thirteen HIFU exposures were performed on placentas. The parameters used in this study were acoustic powers of 65, 80, 110 and 120 W applied for 30, 15, 20 and 20 seconds, respectively. This gradual increase in the total energy delivered was used to determine a set of parameters to create reproducible lesions in the placentas without any complications. Six placental lesions were observed with average diameters of 6.4 ± 0.5 mm and 7.8 ± 0.7 mm and an average depth of 3.8 ± 1.5 mm. Ultrasound images revealed a hyperechoic region that was well correlated with the macroscopic analyses of the HIFU lesions. Necrosis of placental tissues exposed to HIFU was confirmed with macroscopic and microscopic analyses. No significant variation in maternal and fetal parameters was observed during HIFU exposure. Conclusion This study demonstrates the feasibility of HIFU applied non-invasively to the placental unit within an in vivo pregnant monkey model. The technique is safe in the very short term and is potentially translatable to human pregnancy.

There is an extensive modification of the functional organization of the brain in the congenital blind human, although there is little understanding of the structural underpinnings of these changes. The visual system of macaque has been extensively characterized both anatomically and functionally. We have taken advantage of this to examine the influence of congenital blindness in a macaque model of developmental anophthalmia. Developmental anophthalmia in macaque effectively removes the normal influence of the thalamus on cortical development leading to an induced “hybrid cortex (HC)” combining features of primary visual and extrastriate cortex. Here we show that retrograde tracers injected in early visual areas, including HC, reveal a drastic reduction of cortical projections of the reduced lateral geniculate nucleus. In addition, there is an important expansion of projections from the pulvinar complex to the HC, compared to the controls. These findings show that the functional consequences of congenital blindness need to be considered in terms of both modifications of the interareal cortical network and the ascending visual pathways.

There is extensive modification of the functional organization of the brain in the congenital blind human, although there is little understanding of the structural underpinnings of these changes. The visual system of macaque has been extensively characterized both anatomically and functionally. We have taken advantage of this to examine the influence of the congenital blindness in macaque resulting from the removal of the retina during in utero development. Developmental anophthalmia in macaque effectively removes the normal influence of the thalamus on cortical development leading to an induced hybrid cortex (HC) combining features of primary visual and extrastriate cortex. Here we show that retrograde tracers injected in early visual areas including hybrid cortex reveals a drastic reduction of cortical projections of the reduced lateral geniculate nucleus. In addition, there is an important expansion of projections from the pulvinar complex to the hybrid cortex, compared to the controls. These findings show that the functional consequences of congenital blindness need to be considered in terms of both modifications of the inter-areal cortical network and the ascending visual pathways.

Perturbation of the developmental refinement of the corticospinal (CS) pathway leads to motor disorders. While non-primate developmental refinement is well documented, in primates invasive investigations of the developing CS pathway have been confined to neonatal and postnatal stages when refinement is relatively modest. Here, we investigated the developmental changes in the distribution of CS projection neurons in cynomolgus monkey (Macaca fascicularis). Injections of retrograde tracer at cervical levels of the spinal cord at embryonic day (E) 95 and E105 show that: (i) areal distribution of back-labeled neurons is more extensive than in the neonate and dense labeling is found in prefrontal, limbic, temporal, and occipital cortex; (ii) distributions of contralateral and ipsilateral projecting CS neurons are comparable in terms of location and numbers of labeled neurons, in contrast to the adult where the contralateral projection is an order of magnitude higher than the ipsilateral projection. Findings from one largely restricted injection suggest a hitherto unsuspected early innervation of the gray matter. In the fetus there was in addition dense labeling in the central nucleus of the amygdala, the hypothalamus, the subthalamic nucleus, and the adjacent region of the zona incerta, subcortical structures with only minor projections in the adult control.

Perturbation of the developmental refinement of the corticospinal pathway leads to motor disorders. In non-primates developmental refinement is well documented, however in primates invasive investigations of the developing corticospinal pathway have been confined to neonatal and postnatal stages when refinement is relatively modest.Here, we investigated the developmental changes in the distribution of corticospinal projection neurons in cynomolgus monkey. Injections of retrograde tracer at the cervical levels of the spinal cord at embryonic day (E) 95 and E105 show that (i) areal distribution of back-labeled neurons is more extensive than in the neonate and dense labeling is found in prefrontal, limbic, temporal and occipital cortex; (ii) distributions of contra- and ipsilateral projecting corticospinal neurons are comparable in terms of location and numbers of labeled neurons, in contrast to the adult where the contralateral projection is an order of magnitude higher than the ipsilateral projection. Findings from one largely restricted injection suggest a hitherto unsuspected early innervation of the gray matter.In the fetus there was in addition dense labeling in the central nucleus of the amygdala, the hypothalamus, the subthalamic nucleus and the adjacent region of the zona incerta, subcortical structures with only minor projections in the adult control.

The laminar location of the cell bodies and terminals of interareal connections determines the hierarchical structural organization of the cortex and has been intensively studied. However, we still have only a rudimentary understanding of the connectional principles of feedforward (FF) and feedback (FB) pathways. Quantitative analysis of retrograde tracers was used to extend the notion that the laminar distribution of neurons interconnecting visual areas provides an index of hierarchical distance (percentage of supragranular labeled neurons [SLN]). We show that: 1) SLN values constrain models of cortical hierarchy, revealing previously unsuspected areal relations; 2) SLN reflects the operation of a combinatorial distance rule acting differentially on sets of connections between areas; 3) Supragranular layers contain highly segregated bottom-up and top-down streams, both of which exhibit point-to-point connectivity. This contrasts with the infragranular layers, which contain diffuse bottom-up and top-down streams; 4) Cell filling of the parent neurons of FF and FB pathways provides further evidence of compartmentalization; 5) FF pathways have higher weights, cross fewer hierarchical levels, and are less numerous than FB pathways. Taken together, the present results suggest that cortical hierarchies are built from supra- and infragranular counterstreams. This compartmentalized dual counterstream organization allows point-to-point connectivity in both bottom-up and top-down directions.

Regionalization of cell cycle kinetics of cortical precursors has been described in nonhuman primates and rodents indicating a fate map of areas as distinct proliferative programs in the germinal zones of the neocortex. It remains to be understood how proliferative gradients during corticogenesis are transcribed into a stepwise function to form adult areal borders. Here we have used the monkey areas 17 and 18, which show striking cytoarchitectonic differences, as a model system for studying how developmental events establish areal boundaries in the adult. We present data indicating that the events that are involved in the formation of a sharp border separating 2 areas involve an orchestration of diverse phenomena including differential rates of proliferation, migration, and tangential expansion.

Retrograde tracer injections in 29 of the 91 areas of the macaque cerebral cortex revealed 1,615 interareal pathways, a third of which have not previously been reported. A weight index (extrinsic fraction of labeled neurons [FLNe]) was determined for each area-to-area pathway. Newly found projections were weaker on average compared with the known projections; nevertheless, the 2 sets of pathways had extensively overlapping weight distributions. Repeat injections across individuals revealed modest FLNe variability given the range of FLNe values (standard deviation

We investigated the influence of interareal distance on connectivity patterns in a database obtained from the injection of retrograde tracers in 29 areas distributed over six regions (occipital, temporal, parietal, frontal, prefrontal, and limbic). One-third of the 1,615 pathways projecting to the 29 target areas were reported only recently and deemed new-found projections (NFPs). NFPs are predominantly long-range, low-weight connections. A minimum dominating set analysis (a graph theoretic measure) shows that NFPs play a major role in globalizing input to small groups of areas. Randomization tests show that ( i ) NFPs make important contributions to the specificity of the connectivity profile of individual cortical areas, and ( ii ) NFPs share key properties with known connections at the same distance. We developed a similarity index, which shows that intraregion similarity is high, whereas the interregion similarity declines with distance. For area pairs, there is a steep decline with distance in the similarity and probability of being connected. Nevertheless, the present findings reveal an unexpected binary specificity despite the high density (66%) of the cortical graph. This specificity is made possible because connections are largely concentrated over short distances. These findings emphasize the importance of long-distance connections in the connectivity profile of an area. We demonstrate that long-distance connections are particularly prevalent for prefrontal areas, where they may play a prominent role in large-scale communication and information integration.

Prenatal bilateral enucleation induces cortex, which normally would have become striate cortex, to follow a default developmental pathway and to take on the cytoarchitectonic appearance of extrastriate cortex (default extrastriate cortex, Dehay et al. [1996] J. Comp. Neurol. 367:70-89). We have investigated if this manipulation influences the cortical expression of acetylcholinesterase (AChE) and cytochrome oxidase (GO). Early enucleation (before embryonic day 81; E81) had only minor effects on the distribution of AChE and CO in the striate cortex. In animals that underwent operation, the striate cortex CO blobs were significantly more closely spaced on the operculum compared with the calcarine. After early enucleation, there was a periodic distribution of CO dense patches in default extrastriate cortex. These CO patches had a center-to-center spacing that was considerably smaller than that of CO stripes in normal area V2, but was somewhat larger than that of the CO blobs in striate cortex. Although the GO stripes characteristic of normal area V2 could not be detected, there were some high-frequency CO patches, similar to those found in default extrastriate cortex. Early enucleation caused a failure to form the transient AChE bands running perpendicular to the striate border, which are normally present in the fetus and early neonate. Late enucleation did not alter AChE expression in extrastriate cortex. The relatively minor effects of early enucleation in the reduced striate cortex contrast with the changes in expression of these enzymes in extrastriate cortex, which accompany large shifts in the location of the striate border. This suggests a massive reorganisation of cortical phenotype in extrastriate cortex. a 1996 Wiley-Liss, Inc. Indexing terms: primate, plasticity, vision, striate cortex, extrastriate cortex

The operation of real world networks is largely determined by their weighted and spatial characteristics. Surprisingly little is known about these features in cortex. We generated in macaque, a consistent database of inter-areal connections comprising projection densities (link weights) and physical lengths. Contrary to previous assumptions, the cortical connection matrix is dense (66%) and therefore, not a small-world graph. Link weights are both highly specific and heterogeneous and we show that it is these properties that characterize the network. The embedding of this weighted network is governed by a distance rule that predicts both its binary features as well as the global and local communication efficiencies. Analysis of the efficiency of this weighted network suggests that small changes in global communication efficiency are offset by large changes in local efficiency. These findings indicate a weight-based hierarchical layering in cortical architecture and processing.

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain. Disclosure of potential conflicts of interest is found at the end of this article.

SummaryWe have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.

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.

Bilateral enucleation was performed at different fetal ages during corticogenesis, and the brains were prepared for histological examination. Early-enucleated fetuses (operated prior to embryonic day 77) showed morphological changes at the level of the thalamus and the cortex. In the thalamus, there was a loss of lamination and a decrease in size of the lateral geniculate nucleus. There was a decrease in the size of the inferior pulvinar, but there was no change in the lateral pulvinar. The border of striate cortex was as sharp in the enucleates as it was in the normal monkeys. In three of the four early enucleates, we observed an interdigitation of striate and extrastriate cortex. In three of the early enucleates, we observed a small island of nonst,riate cortex near the striate border that was surrounded entirely by striate cortex. Enucleation led to an age-related reduction of striate cortex. This reduction was greater in the operculum than in the calcarine fissure. The reduction of striate cortex was accompanied by an increase in the dimensions of extrastriate visual cortex, so that the overall dimensions of the neocortex remained invariant. The extrastriate cortex in the enucleated animals presented a uniform cytoarchitecture and was indistinguishable from area 18 in the normal animal. There were changes in the gyral pattern that were restricted mainly to the cortex on the operculum. A deepening of minor dimples as well as the induction of a variable number of supplementary sulci led to an increase in the convolution of the occipital lobe. These results are discussed with respect to the specification of cortical areas. They demonstrate that the reduction in striate cortex was not accompanied by an equivalent reduction in the neocortex; rather, there was a border shift, and a large volume of cortex that was destined to become striate cortex appears to be cytoarchitectonically normal extrastriate cortex. ,i 19% Wiley-Liss. Inc.

AN as-yet unresolved issue in developmental neurobiology is whether the discrete areas that form the mammalian cortex emerge from a uniform cortical plate or whether they are already specified in the germinal zone1,2. A feature of the primate striate cortex is that the number of neurons per unit area is twice that of anywhere else in the cerebral cortex3. Here we take advantage of this unique structural feature to investigate whether the extra striate cortical cells are due to increased neuron production during neurogenesis. We labelled precursors undergoing terminal cell division with 3H-thymidine and allowed them to migrate to the cortical plate. Cell counts revealed that their rate of production in the germinal zone of striate cortex is higher than in that giving rise to extrastriate cortex. Also, we used 3H-thymidine pulse injections to investigate cell cycle dynamics and found that this phase of increased production of striate cortical cells is associated with changes in the parameters of the cell cycle. These results show that cortical area identity is at least partially determined at the level of the ventricular zone.