Non-invasive high-intensity focused ultrasound treatment of the placenta: a preliminary in-vivo study using a simian model.

OBJECTIVE: To demonstrate the feasibility and efficacy of high-intensity focused ultrasound (HIFU) for the non-invasive creation of placental lesions in a simian model. METHODS: Eight pregnant monkeys were exposed to HIFU treatment after anesthesia, using a toroidal HIFU 2.5-MHz transducer with an integrated ultrasound imaging probe. Lesions on the placental tissue were created non-invasively by placing the HIFU probe on the skin surface. Fetal and maternal parameters, such as maternal heart rate, fetal heart rate and subcutaneous and intra-amniotic fluid temperature, were recorded during HIFU exposure. Cesarean section was performed immediately after the procedure to extract the placenta and examine the fetus and the maternal abdominal cavity. Placental HIFU lesions were assessed by ultrasound, gross pathology and histology. RESULTS: The mean gestational age of the monkeys was 72 ± 4 days. In total, 13 HIFU procedures were performed. The acoustic power and exposure time were increased progressively. This gradual increase in total energy delivered was used to determine a set of parameters to create reproducible lesions in the placenta without complications. Five 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 examination of the placentae revealed hyperechoic regions that correlated well with macroscopic analysis of the HIFU lesions. Necrosis of placental tissue exposed to HIFU was confirmed with macroscopic and microscopic analysis. There was no significant variation in maternal and fetal parameters during HIFU exposure. CONCLUSIONS: This study demonstrates the feasibility of HIFU applied non-invasively to the placental unit in an in-vivo pregnant monkey model. The technique is safe in the immediate short term and is potentially translatable to human pregnancy. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

High-intensity focused ultrasound applied to the placenta using a toroidal transducer: a preliminary ex-vivo study.

OBJECTIVE: To demonstrate in an ex-vivo model the feasibility of applying high-intensity focused ultrasound (HIFU) using a toroidal transducer for the creation of placental lesions. METHODS: In this study we used a toroidal transducer, composed of 32 ring-shaped emitters with an ultrasound probe at the center, operating at a frequency of 2.5 MHz. We examined 45 human placentae, following either normal vaginal delivery or medical termination of pregnancy between 17 and 40 gestational weeks. First, the attenuation coefficients of 12 human placentae were measured and integrated into a numerical model for simulating HIFU lesions. Then, using acoustic parameters from this preliminary study, we performed ex-vivo experiments with 33 human placentae, each overlain with an animal abdominal wall to simulate the maternal wall. We created single HIFU lesions in 25 of these placentae, and a series of six juxtaposed lesions in eight, studying these both sonographically and macroscopically. RESULTS: Human placental attenuation coefficients of the 12 human placentae ranged from 0.072 to 0.098 Np/cm/MHz, according to gestational age. The 25 single HIFU lesions created had an average diameter of 7.1 ± 3.2 mm and an average depth of 8.2 ± 3.1 mm. The average diameter of the eight series of six juxtaposed HIFU lesions was 23.0 ± 5.0 mm and the average depth was 11.0 ± 4.7 mm. The average thickness of the abdominal walls was 10.5 ± 1.8 mm. No lesions or damage were observed in intervening tissues. CONCLUSION: This study demonstrates, using an ex-vivo model, the feasibility, reproducibility, harmlessness and effectiveness of HIFU applied to the human placenta.

A weighted and directed interareal connectivity matrix for macaque cerebral cortex.

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 <1 log unit, range 5 log units). The connectivity profile for each area conformed to a lognormal distribution, where a majority of projections are moderate or weak in strength. In the G29 × 29 interareal subgraph, two-thirds of the connections that can exist do exist. Analysis of the smallest set of areas that collects links from all 91 nodes of the G29 × 91 subgraph (dominating set analysis) confirms the dense (66%) structure of the cortical matrix. The G29 × 29 subgraph suggests an unexpectedly high incidence of unidirectional links. The directed and weighted G29 × 91 connectivity matrix for the macaque will be valuable for comparison with connectivity analyses in other species, including humans. It will also inform future modeling studies that explore the regularities of cortical networks.

Weight consistency specifies regularities of macaque cortical networks.

To what extent cortical pathways show significant weight differences and whether these differences are consistent across animals (thereby comprising robust connectivity profiles) is an important and unresolved neuroanatomical issue. Here we report a quantitative retrograde tracer analysis in the cynomolgus macaque monkey of the weight consistency of the afferents of cortical areas across brains via calculation of a weight index (fraction of labeled neurons, FLN). Injection in 8 cortical areas (3 occipital plus 5 in the other lobes) revealed a consistent pattern: small subcortical input (1.3% cumulative FLN), high local intrinsic connectivity (80% FLN), high-input form neighboring areas (15% cumulative FLN), and weak long-range corticocortical connectivity (3% cumulative FLN). Corticocortical FLN values of projections to areas V1, V2, and V4 showed heavy-tailed, lognormal distributions spanning 5 orders of magnitude that were consistent, demonstrating significant connectivity profiles. These results indicate that 1) connection weight heterogeneity plays an important role in determining cortical network specificity, 2) high investment in local projections highlights the importance of local processing, and 3) transmission of information across multiple hierarchy levels mainly involves pathways having low FLN values.

Cell-cycle kinetics of neocortical precursors are influenced by embryonic thalamic axons.

Thalamic afferents are known to exert a control over the differentiation of cortical areas at late stages of development. Here, we show that thalamic afferents also influence early stages of corticogenesis at the level of the ventricular zone. Using an in vitro approach, we show that embryonic day 14 mouse thalamic axons release a diffusable factor that promotes the proliferation of cortical precursors over a restricted developmental window. The thalamic mitogenic effect on cortical precursors (1) shortens the total cell-cycle duration via a reduction of the G(1) phase; (2) facilitates the G(1)/S transition leading to an increase in proliferative divisions; (3) is significantly reduced by antibodies directed against bFGF; and (4) influences the proliferation of both glial and neuronal precursors and does not preclude the action of signals that induce differentiation in these two lineages. We have related these in vitro findings to the in vivo condition: the organotypic culture of cortical explants in which anatomical thalamocortical innervation is preserved shows significantly increased proliferation rates compared with cortical explants devoid of subcortical afferents. These results are in line with a number of studies at subcortical levels showing the control of neurogenesis via afferent fibers in both vertebrates and invertebrates. Specifically, they indicate the mechanisms whereby embryonic thalamic afferents contribute to the known early regionalization of the ventricular zone, which plays a major role in the specification of neocortical areas.

A linkage disequilibrium map of the MHC region based on the analysis of 14 loci haplotypes in 50 French families.

A sample of 100 individuals from 50 French families of known pedigrees were typed for 14 loci of the HLA region (DPB1, DQB1, DQA1, DRB1, DRB3, 4, 5, C4B, C4A, Bf, C2, TNFa, TNFb, B, Cw, A). Linkage disequilibrium in each pair of loci was investigated by an exact test using a Markov chain algorithm. The results indicate no disequilibrium between DPB1 and the other loci, whereas the other class II genes are all significantly linked to each other. Linkage disequilibrium is also detected between some pairs of class I and class II-class I loci despite the long physical distance separating the loci (e.g. A-B, Cw-DRB1). On the other hand, some contiguous loci of the class III region are found to be in equilibrium with each other. Several hypotheses including selection, but also unequal allelic diversity at different MHC loci are discussed to explain this complex pattern of linkage disequilibrium.

Bihemispheric Collateralization of the Cortical and Subcortical Afferents to the Rat's Visual Cortex.

A fluorescent dye (usually fast blue or rhodamine tagged latex microspheres) was injected into cortical area 17 (or area 17 and the lateral part of area 18b) of adult and juvenile (15 - 22 day old) Sprague-Dawley albino rats. Another fluorescent dye (usually diamidino yellow) was injected into cortical areas 17, 18a and 18b of the opposite hemisphere. The injections involved only the cortical grey matter. After postinjection survival of 2 - 14 days the distribution of retrogradely labelled mesencephalic and prosencephalic cells was analysed. Both small and large injections labelled retrogradely a substantial number of cells in specific and nonspecific dorsal thalamic nuclei (lateral geniculate, lateral posterior, ventromedial, several intralaminar nuclei and nucleus Reuniens) as well as a small number of cells in the preoptic area of the hypothalamus and the mesencephalic ventral tagmental area (VTA). While labelled thalamic cells contained only the dye injected into the ipsilateral cortex, a small proportion of hypothalamic and VTA cells was labelled with the dye injected into the contralateral cortex. Virtually none of the cells in these areas were double labelled with both dyes. Both small and large injections labelled cells in the ipsilateral telencephalic magnocellular nuclei of the basal forebrain and the caudal claustrum. A substantial minority of labelled cells in these structures was labelled by the dye injected into the contralateral cortex. Furthermore, a small proportion (about 1%) of claustral cells projecting to the ipsilateral cortex were double labelled with both dyes. In several cortical areas ipsilateral to the injected area 17, associational neurons were intermingled with commissural neurons projecting to the contralateral visual cortex. A substantial proportion of associational neurons projecting to ipsilateral area 17 also projected to the contralateral visual cortex (associational-commissural neurons). Thus, in visual area 18a, the associational-commissural neurons were located in all laminae, with the exception of lamina 1 and the bottom of lamina 6, and constituted about 30% of the neurons projecting to ipsilateral area 17. In paralimbic association area 35/13, associational-commissural neurons were located in lamina 5 and constituted about 20% of neurons projecting to ipsilateral area 17. In the limbic area 29d, the associational-commissural neurons were located in laminae 4, 5 and the upper part of lamina 6 and constituted about 10% of the associational-commissural neurons projecting to ipsilateral area 17. In oculomotor area 8, double-labelled neurons were located in lamina 5 and constituted about 10% of the neurons projecting to ipsilateral area 17. Thus, it appears that the axons of mesencephalic and diencephalic neurons projecting to the visual cortex do not send collaterals into both hemispheres. The bihemispheric projection to the rat's visual cortex originates almost exclusively in the retinotopically organized cortical area 18a and in integrative cortical areas 35/13, 29d and 8.

Bihemispheric Axonal Bifurcation of the Afferents to the Visual Cortical Areas during Postnatal Development in the Rat.

Numerous cortical neurons in the juvenile and adult rat project to visual areas of both hemispheres whereas the vast majority of subcortical structures projecting to the visual cortex send strictly ipsilateral projections (Dreher et al., 1990). In the present study, the authors have sought to determine whether this pattern of axonal bifurcation in the connectivity of the visual areas undergoes a change during postnatal development. Two retrograde fluorescent dyes were used, fast blue (FB) and diamidino yellow (DY). Large multiple injections of one of the dyes were placed in all visual areas of one hemisphere and a small injection of the other dye was placed in area 17 of the opposite hemisphere. Labelled neurons were observed in subcortical and cortical structures on the side of the small injection. The experiments were performed on ten neonatal albino rat pups aged between 3 and 12 postnatal days (p.n.d.) at the time of injection and the results were compared with those obtained in the juvenile and adult animals, as reported in the preceding paper. In the thalamus of newborn animals, neurons belonging to nuclei located away from the midline send strictly ipsilateral cortical projections. However, in the midline nuclei of the intralaminar thalamic complex, a small region of overlap was observed between neurons projecting ipsilaterally and neurons projecting contralaterally in animals aged less than 9 postnatal days. In addition, in these neonatal animals a small number of bilaterally projecting neurons was detected in this region of overlap. In all other subcortical structures examined (ventral tegmental area, diagonal band of Broca, claustrum), the laterality of the projection was the same in the newborn and the adult animals. In particular, in the claustrum of neonatal animals, as in adult animals, there was a large contingent of contralaterally projecting neurons and only a very small number of bilaterally projecting neurons. The results in the cortex contrast with those observed in subcortical structures. Whereas ipsilaterally projecting neurons were distributed in a broadly similar way in newborn and adult animals, the laminar and areal distribution of contralaterally projecting neurons in newborn animals clearly differed from those observed in the adult animals. Furthermore, double labelled neurons were more numerous in animals aged less than 12 days than in adults. The proportions of such bilaterally projecting neurons were computed with respect to the numbers of neurons sending ipsilateral projections to area 17. These proportions are constant at all ages in the claustrum and cortical area 8. In areas 18a, 29 and 35 on the other hand, the proportions of bilaterally projecting neurons increase after 5 days and reach a peak in the period extending from 9 to 11 days of age when more than half of the neurons projecting ipsilaterally also send an axonal branch to the contralateral cortex. In cortical areas 29 and 35, this peak is followed by a sudden drop to the adult level at 12 postnatal days, whereas the return to the adult level is gradual in area 18a. These results demonstrate that, in subcortical structures and in cortical area 8, the laterality of the afferent connections to the visual cortex does not change during postnatal development. By contrast, cortical areas 18a, 29 and 35 go through a stage when numerous cells send bifurcating connections to both hemispheres. The timing of the decrease in proportions of bilaterally projecting neurons in these areas suggests that numerous neurons retract their callosal axonal branch when the adult pattern of callosal connectivity is established at 9 - 11 days of age.

The timetable of laminar neurogenesis contributes to the specification of cortical areas in mouse isocortex.

In the primate visual cortex, the birthdate of neurons in homologous layers differ on either side of the 17-18 border suggesting that there might be different timetables of laminar histogenesis in these two areas (Dehay et al. [1993] Nature 366:464-466 and Kennedy et al. [1996] Soc. Neurosci. Abst. 22:525). Because of the potential importance of these findings for understanding mechanisms that generate areal identity, we have developed an experimental approach that makes it possible to accurately compute the timetable of laminar histogenesis from birthdating experiments. Here we report the results of an exhaustive examination of the tempo of layer production in five cortical areas of the mouse. Tritiated thymidine pulse injections were made during embryonic development and labeled neurons were examined in three frontoparietal areas (areas 3, 4, and 6) and two occipital areas (areas 17 and 18a) of the adult cortex. The correlation between the radial distribution of neurons and the intensities of labeling enabled us to reliably identify first generation neurons (i.e., those neurons that quit the cell-cycle in the first round of mitosis after injection). For each cortical layer, the percentage of first generation neurons with respect to the total number of neurons defined a laminar labeling index. Changes of the laminar labeling index over time determined the timetable of layer formation. The onset and duration of layer formation was identical in the two occipital areas. This finding contrasted with the frontoparietal areas where there were important differences in the timing of infragranular and granular layer formation and noticeably production of layers VIa, V, and IV occurs earlier in area 3 than in area 6. The timing of laminar production of areas 17 and 18a resembles more that of area 3 than that of area 6. With respect to areas 3 and 6, area 4 shows an intermediate but significantly different timetable of layer production. These marked areal differences in the timetable of laminar histogenesis contrasted with the relative homogeneity within areas so that we have been able to demonstrate that the interareal differences are not merely the expression of known neurogenic gradients. These results suggest that in the mouse frontoparietal isocortex, neighbouring regions of the ventricular zone that will give rise to distinct areas follow distinct programs of layer production. These areal differences occur before thalamic innervation and suggest an early regionalisation of laminar histogenesis.

Characterization of transient cortical projections from auditory, somatosensory, and motor cortices to visual areas 17, 18, and 19 in the kitten.

We have examined the anatomical features of ipsilateral transient cortical projections to areas 17, 18, and 19 in the kitten with the use of axonal tracers Fast Blue and WGA-HRP. Injections of tracers in any of the three primary visual areas led to retrograde labeling in frontal, parietal, and temporal cortices. Retrogradely labeled cells were not randomly distributed, but instead occurred preferentially at certain loci. The pattern of retrograde labeling was not influenced by the area injected. The main locus of transiently projecting neurons was an isolated region in the ectosylvian gyrus, probably corresponding to auditory area A1. Other groups of transiently projecting neurons had more variable locations in the frontoparietal cortex. The laminar distribution of neurons sending a transient projection to the visual cortex is characteristic and different from that of parent neurons of other cortical pathways at the same age. In the frontoparietal cortex, transiently projecting neurons were located mainly in layer 1 and the upper part of layers 2 and 3. In the ectosylvian gyrus, nearly all the neurons are located in layers 2 and 3. In addition, a few transiently projecting neurons are found in layer 6 and in the white matter. Transiently projecting neurons have a pyramidal morphology except for the occasional spindle-shaped cell of layer 1 and multipolar cells observed in the white matter. Anterograde studies were used to investigate the location of transient fibers in the visual cortex. Injections of WGA-HRP at the site of origin of transient projections gave rise to few retrogradely labeled cells in areas 17, 18, and 19, demonstrating that transient projections to these areas are not reciprocal. Although labeled axons were found over a wide area of the posterior cortex, they were more numerous over certain regions, including areas 17, 18, and 19, and absent from other more lateral cortical regions. Transient projecting fibers were present in all cortical layers at birth. Plotting the location of transient fibers in numerous sections and at all ages showed that these fibers are not more plentiful in the white matter than they are in the gray matter. We found no evidence that the white/gray matter border constituted a physical barrier to the growth of transient axons. Comparison of the organization of this transient pathway to that of other transient connections is discussed with respect to the development of the cortex.

Organization of the callosal connections of visual areas V1 and V2 in the macaque monkey.

The interhemispheric efferent and afferent connections of the V1/V2 border have been examined in the adult macaque monkey with the tracers horseradish peroxidase and horseradish peroxidase conjugated to wheat germ agglutinin. The V1/V2 border was found to have reciprocal connections with the contralateral visual area V1, as well as with three other cortical sites situated in the posterior bank of the lunate sulcus, the anterior bank of the lunate sulcus, and the posterior bank of the superior temporal sulcus. Within V1, callosal projecting cells were found mainly in layer 4B with a few cells in layer 3. Anterograde labeled terminals were restricted to layers 2, 3, 4B, and 5. In extrastriate cortex, retrograde labeled cells were in layers 2 and 3 and only very rarely in infragranular layers. In the posterior bank of the lunate sulcus, labeled terminals were scattered throughout all cortical layers except layers 1 and 4. In the anterior bank of the lunate sulcus and in the superior temporal sulcus, anterograde labeled terminals were largely focused in layer 4. Callosal connections in all contralateral regions were organized in a columnar fashion. Columnar organization of callosal connections was more apparent for anterograde labeled terminals than for retrograde labeled neurons. In the posterior bank of the lunate sulcus, columns of callosal connections were superimposed on regions of high cytochrome activity. The tangential extent of callosal connections in V1 and V2 was found to be influenced by eccentricity in the visual field. Callosal connections were denser in the region of V1 subserving foveal visual field than in cortex representing the periphery. In V1 subserving the fovea, callosal connections extended up to 2 mm from the V1/V2 border and only up to 1 mm in more peripheral located cortex. In area V2 subserving the fovea, cortical connections extended up to 8 mm from the V1/V2 border and only up to 3 mm in peripheral cortex.

Callosal connectivity of areas V1 and V2 in the newborn monkey.

The callosal connectivity of areas V1 and V2 in the newborn monkey has been investigated with the neuroanatomical tracers wheat germ agglutinin conjugated to horseradish peroxidase and free horseradish peroxidase. In the adult, callosal projecting neurons in cortex subserving the lower parafoveal visual field were found to extend from the V1/V2 border for a distance of 1-2.5 mm into V1 and 8 mm into V2. In the newborn, the tangential extent and total number of callosal projecting neurons were the same as in the adult. Within area V1, callosal projecting neurons in the adult and newborn were limited to supragranular layers. In the adult, axon terminals of callosal projections were located in layers 4B and 5 and were excluded from layer 4C. In the newborn, axon terminals were more extensively distributed than in the adult and invaded layer 4C. In area V2, the laminar distribution and the patchy location of callosal connections in regions of high cytochrome oxidase activity were similar in the newborn and adult animals. In both newborns and adults, the patchy distribution of callosal projections persisted when the neuroanatomical tracers were injected over extensive regions of the contralateral striate and extrastriate cortex. In the adult, area V1 and V2 project contralaterally to two heterotopic sites located in the fundus of the lunate sulcus and the superior temporal sulcus. This was also found to be the case in the newborn. In the adult the terminals of these heterotopic projections were focused in layer 4. This was not the case in the newborn, where after injection limited to the contralateral V1/V2 border they were more evenly distributed among the different cortical layers. Following extensive contralateral injection of tracer, terminals in cortex anterior to V2 were focused over layer 5 and the bottom of layer 4.

Transient cortical pathways in the pyramidal tract of the neonatal ferret.

Anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to study transient axons from the visual cortex in the pyramidal tract. Injections at birth restricted to the visual cortex labeled axons in the vicinity of the pontine nuclei. Two to eight days after birth, axons from the occipital cortex were found posterior to the pontine nucleus, their caudalmost stable target. Transient corticospinal axons from the presumptive primary visual cortex did not grow caudal to the pyramidal decussation. Innervation of more distal targets preceded innervation of proximal targets. Innervation of the pontine nucleus is initiated around 68 hours after birth, when the transient extension in the medullary pyramidal tract has attained its maximum caudal extent. Innervation of the superior colliculus begins 9 days after birth. Retrograde tracers were used to follow the developmental changes in the cortical distribution of the parent neurons giving rise to axons in the pyramidal tract. In the adult, labeled neurons following injection of retrograde tracer in the pyramidal tract occupied less than a third of the neocortex and were centred on the anterior part of the coronal and spleniocruciate gyri. In the immature brain, labeled neurons covered more than two-thirds of the neocortex. Areal density measurements in the neonate showed that peak labeling was centred in the anterior coronal and spleniocruciate gyri, where corticospinal cells in the adult are located. There was a marked rostral-caudal gradient so that labeled neurons were very scarce towards the occipital pole. These results, showing transient neocortical axons in the pyramidal tract in a carnivore, suggest that this may be a common feature of mammalian development. The finding that the adult pattern of corticospinal projections does not emerge from a uniform distribution is discussed with respect to the areal specification of cortical connectivity.

Area-specific laminar distribution of cortical feedback neurons projecting to cat area 17: quantitative analysis in the adult and during ontogeny.

Corticocortical pathways can be classified as feedback and feedforward, in part according to the laminar distribution of the parent cell bodies. Here, we have developed exhaustive sampling procedures to determine unambiguously this laminar distribution. This shows that individual extrastriate areas in the adult cat have highly stereotyped proportions of supragranular layer neurons with respect to the total population of neurons back-projecting to area 17. During development, these adult laminar patterns emerge from an initially uniform radial distribution through a process of selective reorganization, which is highly specific to each area. Injections of fluorescent retrograde tracers were made in area 17. In areas 19, 20, posteromedial lateral suprasylvian area, and anteromedial lateral suprasylvian area, we defined a projection zone as the region containing retrogradely labeled neurons. In the neonate, counts of labeled neurons throughout the projection zones show constant percentages of 40% in the supragranular layers. During development, there is an area-specific reduction in the percentage of supragranular labeled neurons generating the laminar distributions characteristic of each area. Numbers of labeled neurons were estimated at different eccentricities of the projection zone. This finding indicates that during development there is a relative decrease in the numbers of labeled neurons of the periphery of the projection zone in the supragranular layers but not in the infragranular layers. This decrease is accompanied by a relative decrease in the dimensions of the supragranular projection zone with respect to the infragranular projection zone. These findings suggest that each extrastriate area precisely adjusts the proportions of supragranular layer neurons back-projecting to striate cortex in part by developmental changes in the divergence-convergence values of individual neurons. This shaping of corticocortical connectivity occurs relatively late in postnatal development and could, therefore, be under epigenetic control.

Role of directed growth and target selection in the formation of cortical pathways: prenatal development of the projection of area V2 to area V4 in the monkey.

In experiments combining retrograde tracers and histochemistry, we have looked at the prenatal development of the cortical pathway linking areas V2 and V4. Transient expression of acetylcholinesterase in fetal area V2 reveals the separate compartments that project to V4 (temporal directed pathway) and V5 (parietal directed pathway). During early stages of pathway formation, V2 neurons projecting to area V4 are clustered in the appropriate compartments. During the phase of rapid axonal growth, there is a selective increase of connections originating from the appropriate compartments leading to a strongly clustered organization at the peak of connectivity. During this phase, injections involving the white matter also showed clustering, but this was somewhat reduced in comparison to that of gray matter injections. The growth phase is followed by an elimination phase during which there is a tendency for a preferential loss of intercluster connections, which may sharpen the early formed pattern. These results demonstrate the primary role of axonal guidance and target recognition mechanisms followed by a limited extent of selective elimination during the formation of functional cortical pathways in the primate isocortex. Compared to previous findings, these results suggest that the developmental restriction of callosal connections is not a universal model of cortical development. In the present report, the directed growth and early specification of feed-forward connections contrast with the prolonged remodelling of monkey feedback projections, suggesting two distinct developmental strategies of pathway formation in the monkey.

Contribution of thalamic input to the specification of cytoarchitectonic cortical fields in the primate: effects of bilateral enucleation in the fetal monkey on the boundaries, dimensions, and gyrification of striate and extrastriate cortex.

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 nonstriate 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.

Phenotypic characterisation of respecified visual cortex subsequent to prenatal enucleation in the monkey: development of acetylcholinesterase and cytochrome oxidase patterns.

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 (CO). 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 CO 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.

Binocular competition does not regulate retinogeniculate arbor size in fetal monkey.

Binocular interactions play a prominent role in shaping the axonal arbors of geniculocortical fibers and the arbors of Y cells in the retinogeniculate pathway of the fetal cat. Fiber interactions between the two eyes have also been suggested to regulate the formation of retinal projections to the dorsal lateral geniculate nucleus (dlgn) of the fetal monkey, but whether this reflects structural refinements of retinal arbors has not been established. To address this issue, we quantified the morphologic properties of individual fibers in two macaque monkeys at embryonic day (E) 110 and E121 that had an eye removed at E69 and E61, respectively. Fibers were labeled by DiI crystals into the fixed optic tract and were visualized by confocal microscopy. Three measurements were made: the number of branch points within the axon terminal arbor, the total arborization length, and the incidence of axonal side branches on the preterminal axon within the confines of the geniculate. There were no significant differences with respect to these parameters between the prenatal enucleates and normal monkeys of comparable age. This was the case for retinal fibers innervating the magnocellular and the parvocellular segments of the dlgn. The arbors stemming from the remaining eye were widely distributed in the dlgn, with some terminating in territories normally innervated by the other (enucleated) eye. These results lend support to the hypothesis that the expanded projection from the remaining eye to the lateral geniculate nucleus of the prenatally enucleated monkey is due to the maintenance of a contingent of retinal fibers normally eliminated by ganglion cell death.

Synaptic connections of physiologically identified geniculocortical axons in kitten cortical area 17.

Single geniculocortical axons were recorded in the cortical white matter of kittens and adult cats by using micropipettes filled with horseradish peroxidase (HRP). Of 41 axons recovered in 4-5 week old kittens, three well-filled axons arborized in area 17; the remainder were incomplete or arborized in area 18. One axon had Y-like physiological properties, two were X-like. They were recovered from two 34-day-old kittens. All three axons formed clustered arborizations, mainly in layer 4A. Electron microscopic (EM) analysis of 50 boutons from kitten and 38 boutons from adult controls revealed that the boutons from kitten made synapses more frequently on spines (91% of targets) than did the boutons from the adult (71%). One X-like axon in kitten also had a collateral projection that made synapses in layer 1; this has not been seen in adult cats. In overall extent, the axons from kitten fell within the adult range.

Functional implications of the anatomical organization of the callosal projections of visual areas V1 and V2 in the macaque monkey.

The efferent and afferent connections of the V1/V2 border with the contralateral hemisphere have been examined using anatomical tracers. The V1/V2 border was found to exchange connections with the contralateral V2 area as well as a restricted strip of V1 lying adjacent to the V1/V2 border. Besides these homotopic projections, two heterotopic projections were found to V3/V3A and V5. Anterograde tracing of callosal connections showed that terminals in these heterotopic sites were focused in layer 4, the recipient layer of projections originating from the ipsilateral V1/V2 border. Bilateral injections of fluorescent dyes showed that these heterotopic targets of the V1/V2 border are connected to the homologous ipsilateral V1/V2 border region. The laminar location of callosal projecting neurons as well as their terminals were characteristic for each cortical region. The laminar pattern of callosal connectivity was found to differ markedly from that of associational visual pathways. Two principal hypotheses are suggested by these results. First, the fact that V1 in part is reciprocally callosally connected in all mammals supports the notion that this interhemispheric pathway completes long-range intrinsic cortical connections. Second, the convergence of inter- and intrahemispheric pathways could provide the anatomical basis for the modulation of the sensory processing within one hemisphere by ongoing activity in the contralateral hemisphere.