Targeting of 3D oral cancer spheroids by αVß6 integrin using near-infrared peptide-conjugated IRDye 680.

BACKGROUND: In the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVß6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVß6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells. METHODS: αVß6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population. RESULTS: αVß6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20. Round shaped spheroids with an average diameter of 400 µm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVß6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts. CONCLUSIONS: The present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers.

Photon correlations in the collective emission of hybrid gold-(CdSe/CdS/CdZnS) nanocrystal supraparticles.

We investigate the photon statistics of the light emitted by single self-assembled hybrid gold-CdSe/CdS/CdZnS colloidal nanocrystal supraparticles through the detailed analysis of the intensity autocorrelation functiong(2)(τ). We first reveal that, despite the large number of nanocrystals involved in the supraparticle emission, antibunching can be observed. We then present a model based on non-coherent Förster energy transfer and Auger recombination that well captures photon antibunching. Finally, we demonstrate that some supraparticles exhibit a bunching effect at short time scales corresponding to coherent collective emission.

Fluorescence decay enhancement and FRET inhibition in self-assembled hybrid gold CdSe/CdS/CdZnS colloidal nanocrystal supraparticles.

We report on the synthesis of hybrid light emitting particles with a diameter ranging between 100 and 500 nm, consisting in a compact semiconductor CdSe/CdS/CdZnS nanocrystal aggregate encapsulated by a controlled nanometric size silica and gold layers. We first characterize the Purcell decay rate enhancement corresponding to the addition of the gold nanoshell as a function of the particle size and find a good agreement with the predictions of numerical simulations. Then, we show that the contribution corresponding to Förster resonance energy transfer is inhibited.

Redundant roles of photoreceptors and cytokinins in regulating photosynthetic acclimation to canopy density.

The regulation of photosynthetic acclimation to canopy density was investigated in tobacco canopies and in tobacco and Arabidopsis plants with part of their foliage experimentally shaded. Both species acclimated to canopy light gradients and partial shading by allocating photosynthetic capacity to leaves in high light and adjusting chloroplast organization to the local light conditions. An investigation was carried out to determine whether signalling mediated by photoreceptors, sugars, cytokinin, and nitrate is involved in and necessary for proper photosynthetic acclimation. No evidence was found for a role for sugars, or for nitrate. The distribution of cytokinins in tobacco stands of contrasting density could be explained in part by irradiance-dependent delivery of cytokinins through the transpiration stream. Functional studies using a comprehensive selection of Arabidopsis mutants and transgenics showed that normal wild-type responses to partial shading were retained when signalling mediated by photoreceptors or cytokinins was disrupted. This indicates that these pathways probably operate in a redundant manner. However, the reduction of the chlorophyll a/b ratio in response to local shade was completely absent in the Arabidopsis Ws-2 accession mutated in PHYTOCHROME D and in the triple phyAphyCphyD mutant. Moreover, cytokinin receptor mutants also showed a reduced response, suggesting a previously unrecognized function of phyD and cytokinins.

Thalamic and brainstem contributions to large-scale plasticity of primate somatosensory cortex.

After long-term denervation of an upper limb in macaque monkeys, the representation of the face in somatosensory cortex expands over many millimeters into the silenced representation of the hand. Various brainstem and cortical mechanisms have been proposed to explain this phenomenon. Reorganization in the thalamus has been largely ignored. In monkeys with deafferented upper limbs for 12 to 20 years, it was found that the brainstem cuneate and the thalamic ventral posterior nuclei had undergone severe transneuronal atrophy, and physiological mapping in the thalamus revealed that the face and trunk representations were adjoined while the normally small representation of the lower face had expanded comparable to the expansion in cortex. Reorganization of brainstem and thalamic nuclei associated with slow transneuronal atrophy is likely to be a progressive process. When coupled with divergence of ascending connections, it is likely to make a substantial contribution to representational changes in cortex.

Physiological evidence for serial processing in somatosensory cortex.

Removal of the representation of a specific body part in the postcentral cortex of the macaque resulted in the somatic deactivation of the corresponding body part in the second somatosensory area. In contrast, removal of the entire second somatosensory area had no grossly detectable effect on the somatic responsivity of neurons in the postcentral cortex. This direct electrophysiological evidence for serial cortical processing in somesthesia is similar to that found earlier for vision and, taken together with recent anatomical evidence, suggests that there is a common cortical plan for the processing of sensory information in the various sensory modalities.

Massive cortical reorganization after sensory deafferentation in adult macaques.

After limited sensory deafferentations in adult primates, somatosensory cortical maps reorganize over a distance of 1 to 2 millimeters mediolaterally, that is, in the dimension along which different body parts are represented. This amount of reorganization was considered to be an upper limit imposed by the size of the projection zones of individual thalamocortical axons, which typically also extend a mediolateral distance of 1 to 2 millimeters. However, after extensive long-term deafferentations in adult primates, changes in cortical maps were found to be an order of magnitude greater than those previously described. These results show the need for a reevaluation of both the upper limit of cortical reorganization in adult primates and the mechanisms responsible for it.

Cortically induced thalamic plasticity in the primate somatosensory system.

The influence of cortical feedback on receptive field organization in the thalamus was assessed in the primate somatosensory system. Chronic and acute suppression of neuronal activity in primary somatosensory cortex resulted in a striking enlargement of receptive fields in the ventroposterior thalamus. This finding demonstrates a dramatic 'top-down' influence of cortex on receptive field size in the somatosensory thalamus. In addition, this result has important implications for studies of adult neuronal plasticity because it indicates that changes in 'higher-order' areas of the brain can trigger extensive changes in the receptive field characteristics of neurons located earlier in the processing pathway.

Progressive transneuronal changes in the brainstem and thalamus after long-term dorsal rhizotomies in adult macaque monkeys.

This study deals with a potential brainstem and thalamic substrate for the extensive reorganization of somatosensory cortical maps that occurs after chronic, large-scale loss of peripheral input. Transneuronal atrophy occurred in neurons of the dorsal column (DCN) and ventral posterior lateral thalamic (VPL) nuclei in monkeys subjected to cervical and upper thoracic dorsal rhizotomies for 13-21 years and that had shown extensive representational plasticity in somatosensory cortex and thalamus in other experiments. Volumes of DCN and VPL, number and sizes of neurons, and neuronal packing density were measured by unbiased stereological techniques. When compared with the opposite, unaffected, side, the ipsilateral cuneate nucleus (CN), external cuneate nucleus (ECN), and contralateral VPL showed reductions in volume: 44-51% in CN, 37-48% in ECN, and 32-38% in VPL. In the affected nuclei, neurons were progressively shrunken with increasing survival time, and their packing density increased, but there was relatively little loss of neurons (10-16%). There was evidence for loss of axons of atrophic CN cells in the medial lemniscus and in the thalamus, with accompanying severe disorganization of the parts of the ventral posterior nuclei representing the normally innervated face and the deafferented upper limb. Secondary transneuronal atrophy in VPL, associated with retraction of axons of CN neurons undergoing primary transneuronal atrophy, is likely to be associated with similar withdrawal of axons from the cerebral cortex and should be a powerful influence on reorganization of somatotopic maps in the somatosensory cortex.

Leaf Respiration in Light and Darkness (A Comparison of Slow- and Fast-Growing Poa Species).

We investigated whether leaf dark respiration (nonphotorespiratory mitochondrial CO2 release) is inhibited by light in several Poa species, and whether differences in light inhibition between the species are related to differences in the rate of leaf net photosynthesis. Four lowland (Poa annua L., Poa compressa L., Poa pratensis L., and Poa trivialis L.), one subalpine (Poa alpina L.), and two alpine (Poa costiniana Vick. and Poa fawcettiae Vick.) Poa species differing in whole plant relative growth rates were grown under identical controlled conditions. Nonphotorespiratory mitochondrial CO2 release in the light (Rd) was estimated according to the Laisk method. Photosynthesis was measured at ambient CO2 partial pressure (35 Pa) and 500 [mu]mol photons m-2 s-1. The rate of photosynthesis per unit leaf mass was positively correlated with the relative growth rate, with the slow-growing alpine Poa species exhibiting the lowest photosynthetic rates. Rates of both Rd and respiration in darkness were also substantially lower in the alpine species. Nonphotorespiratory CO2 release in darkness was higher than Rd in all species. However, despite some variation between the species in the level of light inhibition of respiration, no relationship was observed between the level of inhibition and the rate of photosynthesis. Similarly, the level of inhibition was not correlated with the relative growth rate. Our results support the suggestion that rates of leaf respiration in the light are closely associated with rates in darkness.

Enhancing fluorescence in vivo imaging using inorganic nanoprobes.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

Patterns of thalamocortical degeneration after ablation of somatosensory cortex in monkeys.

We examined the pattern of cytochrome oxidase (CO), Nissl staining, and gamma-amino butyric acid (GABA) immunoreactivity in the ventroposterior lateral nucleus (VPL) of the thalamus in monkeys that received no, total, or subtotal, ablation of the hand representations in postcentral somatosensory cortex. In unoperated animals, the region of VPL representing the hand was characterized by relatively dense and homogeneous CO staining throughout the rostral-caudal extent of VPL. Counts of neurons in the VPL hand representation from adjacent thalamic sections processed for Nissl and GABA immunostaining indicated that there were approximately 261.4 neurons/mm2 of which 78.4/mm2 stained positive for GABA. GABA(+) puncta-like terminals were readily apparent throughout the VPL. By contrast, animals that received total removals of the postcentral hand representations showed a dramatic reduction in CO staining in the VPL, which was confined to the expected location of the thalamic hand representation. Counts of neurons in the affected region from adjacent sections that underwent Nissl staining and GABA immunostaining also revealed a dramatic reduction of Nissl-stained neurons, with a smaller reduction in the number of neurons staining positive for GABA. Specifically, large to medium-sized (> 180 microns 2) GABA(-) neurons were virtually eliminated in the affected portion of the VPL, and the numbers of GABA(+) neurons were significantly reduced. The remaining population of GABA(+) neurons was typically shrunken, and no GABA(+) puncta-like terminals were observed in the affected region. The results obtained after subtotal ablation of the postcentral hand representations (only one postcentral area spared, 3b or 3a) differed from those obtained when total removals were made. Instead of virtually complete degeneration of medium-sized to large neurons throughout the hand representation in VPL, as was the case with total removals, after partial removals, we found alternating regions in the VPL hand representation that appeared qualitatively normal, or dramatically degenerated. Thalamic sections stained with CO revealed light, moderate, and darkly stained patches of label within the hand representation in VP, depending on the type of cortical ablation. The most dramatic reduction of Nissl-stained neurons coincided precisely with the lightest staining CO patches. Interestingly, the only statistically significant reduction in the number of GABA(+) neurons occurred in the light CO patches. In the thalamic regions coincident with the dark and moderately stained CO patches, the number of medium-sized and large neurons decreased, but the number of GABA(+) neurons was comparable to normal. Optical density measurements of the dark patches also indicated a statistically significant difference from normal CO staining in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticocortical connections of area 2 of somatosensory cortex in macaque monkeys: a correlative anatomical and electrophysiological study.

The cortical connections of electrophysiologically identified locations in the body representations in somatosensory cortex of macaque monkeys were investigated after injections of horseradish peroxidase, wheat germ agglutinin (WGA) conjugated with horseradish peroxidase, tritiated WGA, or tritiated proline. After extensive microelectrode mapping of portions of the body representations in areas 3b, 1, 2, and 5 and careful determinations of electrophysiological borders between areas, restricted injections of tracers were placed, usually into the representation of the hand in area 2. Other injections were placed in the foot representation in area 2 or in area 1, in the wrist representation in area 1, and in the forearm and wrist representation in area 5. Connection patterns were related to the physiological mapping results and to cortical cytoarchitecture. Injections confined to a lateral portion of area 2 representing the glabrous digits of the hand revealed reciprocal connections with the digit representations in areas 1 and 3b. Projections to area 2 were largely from layer III neurons in both of these fields, and return projections terminated largely in supragranular layers. Other inputs were from layer III cells in one or more separate locations in area 5 and in one or more closely spaced foci in the expected location of S-II in the lateral sulcus. These connections were also reciprocal with terminations apparent in layers IV and III. A few neurons in area 4 were labeled in some of these cases. Results were similar after an injection in the foot representation in area 2 with the differences that infragranular neurons, in addition to supragranular neurons, formed a substantial part of the projection to area 2, terminations as well as projections were noted from area 4, interconnections were found more rostrally in area 6, and a dense focus of label was apparent in the dorsal bank of cingulate sulcus in the apparent location of the supplementary motor area. Injections in the foot representation in area 1 revealed dense layer IV terminations in the foot representation in area 2, as well as connections with area 3b, the S-II region, and areas 5 and 7. The injection in the wrist representation in area 1 resulted in dense terminations in the portion of area 5 responsive to the distal forearm and hand, sparser connections with a lateral location in part of area 2 related to the hand, and interconnections with 3b and S-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Connections of area 2 of somatosensory cortex with the anterior pulvinar and subdivisions of the ventroposterior complex in macaque monkeys.

The principal goal of the present study was to determine the thalamic connections of area 2 of postcentral somatosensory cortex of monkeys. The placement of injections of anatomical tracers (horseradish peroxidase, wheat germ agglutinin, or 3H-proline) was guided by extensive microelectrode maps of cortex in the region of the injection site. These maps identified the body parts represented in the cortex included in the injection site, and provided information about the physiological boundaries of area 2, which was related later to the cortical architecture. Most injections were placed in the representation of the hand in area 2, which was highly responsive to cutaneous stimuli and could be mapped in detail. Injections were also placed in other parts of area 2, area 1, or area 5, and some injections involved more than one area. As other investigators have determined, regions of retrograde and anterograde thalamic label overlapped, demonstrating that connections with cortex are reciprocal. Injections completely confined to area 2 consistently produced label in two locations: the anterior pulvinar (Pa) and a dorsal capping zone of the ventroposterior complex that we term the ventroposterior superior nucleus (VPS). Single restricted injection sites resulted in one region of label in VPS, and multiple foci of label in Pa. In some cases where the injection was confined to the representation of the hand in area 2, label was also found more ventrally in the ventroposterior complex in ventroposterior nucleus proper (VP). Thus, area 2 receives input from Pa, VPS, and, at least in some locations and individuals, VP. Injections of tracers into area 1 confirmed previous findings that area 1 is densely interconnected with VP. In addition, there appear to be sparse connections with VPS. There was no evidence of connections with Pa. Evidence from injection sites that extended from area 2 into areas 5 and 7, and from injection sites in area 5, indicates that the lateral posterior nucleus (LP) projects to rostral areas 5 and 7. The results support the conclusion that area 2 is a functionally distinct subdivision of somatosensory cortex, and indicate that area 2 has thalamic connections that are characteristic of both "sensory" (VP and VPS) and "association" (Pa) cortical fields.

Changes in glutamate immunoreactivity in the somatic sensory cortex of adult monkeys induced by nerve cuts.

Antibodies to glutamate (Glu) were used to study the effects of reduced afferent input on excitatory neurons in the somatic sensory cortex of adult monkeys. In each monkey, immunocytochemical staining was compared to thionin and cytochrome oxidase (CO) staining in adjacent sections. In the cervical spinal cord, dorsal column nuclei, ventroposterior thalamus, and primary somatic sensory cortex (SI), Glu immunoreactivity (Glu-ir) was analogous to that described in normal animals; regions with reduced or absent Glu-ir were never observed and no appreciable differences were noted between the experimental and normal side. There were also no differences in CO or thionin-stained sections from the affected hemisphere. In the insuloparietal operculum, sections in the hemisphere contralateral to the nerve cut showed that most cortical fields had a normal pattern of Glu-ir (pattern a), some exhibited a reduction of Glu-ir (pattern b), and that in the central portion of the upper bank of the central sulcus, which corresponds to the general location of the hand representation of the second somatic sensory cortex (SII), Glu-ir had virtually disappeared (pattern c). Adjacent sections processed for CO or stained with thionin showed that in the regions corresponding to those characterized by pattern c, CO was slightly decreased and that glial cells had increased in number. In the regions of SII characterized by pattern c, small intensely stained glial cells displayed Glu-ir. These findings indicate that Glu-ir is regulated by afferent activity and suggest that changes in Glu levels in neurons as well as in glial cells may trigger the biochemical processes underlying the functional and structural changes occurring during a slow phase of reorganizational plasticity in the cerebral cortex of adult monkeys.

Serial and parallel processing in rhesus monkey auditory cortex.

Auditory cortex on the exposed supratemporal plane in four anesthetized rhesus monkeys was mapped electrophysiologically with both pure-tone (PT) and broad-band complex sounds. The mapping confirmed the existence of at least three tonotopic areas. Primary auditory cortex, AI, was then aspirated, and the remainder of the cortex on the supratemporal plane was remapped. PT-responses in the caudomedial area, CM, were abolished in all animals but one, in which they were restricted to the high-frequency range. Some CM sites were still responsive to complex stimuli. In contrast to the effects on CM, no significant changes were detectable in the rostral area, R. After mapping cortex in four additional monkeys, injections were made with different tracers into matched best-frequency regions of AI, R, and CM. Injections in AI and R led to retrograde labeling of neurons in all three subdivisions of the medial geniculate (MG) nucleus (MGv, MGd, and MGm), as well as nuclei outside MG, whereas CM injections led to only sparse labeling of neurons in a restricted zone of the lateral MGd and, possibly, MGm, in addition to labeling in non-MG sites. The combined results suggest that MGv sends direct projections in parallel to areas AI and R, which drive PT-responses in both areas. PT-responses in area CM, however, appear to be driven by input relayed serially from AI. The direct input to CM from MGd and other thalamic nuclei may thus be capable of mediating responses only to broad-band sounds.

The relationship of corpus callosum connections to electrical stimulation maps of motor, supplementary motor, and the frontal eye fields in owl monkeys.

Microstimulation and anatomical techniques were combined to reveal the organization and interhemispheric connections of motor cortex in owl monkeys. Movements of body parts were elicited with low levels of electrical stimulation delivered with microelectrodes over a large region of precentral cortex. Movements were produced from three physiologically defined cortical regions. The largest region, the primary motor field, M-I, occupied a 4-6-mm strip of cortex immediately rostral to area 3a. M-I represented body movements from tail to mouth in a grossly somatotopic mediolateral cortical sequence. Specific movements were usually represented at more than one location, and often at as many as six or seven separate locations within M-I. Although movements related to adjoining joints typically were elicited from adjacent cortical sites, movements of nonadjacent joints also were produced by stimulation of adjacent sites. Thus, both sites producing wrist movements and sites producing shoulder movements were found next to sites producing digit movements. Movements of digits of the forepaw were evoked at several locations including a location rostral to or within cortex representing the face. Overall, the somatotopic organization did not completely correspond to previous concepts of M-I in that it was neither a single topographic representation, nor two serial or mirror symmetric representations, nor a "nesting about joints" representation. Instead, M-I is more adequately described as a mosaic of regions, each representing movements of a restricted part of the body, with multiple representations of movements that tend to be somatotopically related. A second pattern of representation of body movements, the supplementary motor area (SMA), adjoined the rostromedial border of M-I. SMA represented the body from tail to face in a caudorostral cortical sequence, with the most rostral portion related to eye movements. Movements elicited by near-threshold levels of current were often restricted to a single muscle or joint, as in M-I, and the same movement was sometimes multiply represented. Typically, more intense stimulating currents were required for evoking movements in SMA than in M-I. A third motor region, the frontal eye field (FEF), bordered the representation of eyelids and face in M-I. Eye movements elicited from this cortex consisted of rapid horizontal and downward deviation of gaze into the contralateral visual hemifield.

The relation of corpus callosum connections to architectonic fields and body surface maps in sensorimotor cortex of new and old world monkeys.

Corpus callosum connections of parietal and motor cortex were studied in New World owl monkeys (Aotus trivirgatus) and Old World macaque monkeys (Macaca fascicularis) after multiple injections of 3H-proline and horseradish peroxidase, HRP, into one cerebral hemisphere, and extensive microelectrode mapping of architectonic Areas 3b, 1, and 2 of the other hemisphere. Results were obtained both from parasagittal brain sections cut orthogonal to the brain surface and from sections from flattened brains cut parallel to the brain surface. Cortical fields varied in density of callosal connections, and the density of connections varied according to body part within sensory representations. Thus, Area 3b had few, Area 1 had more, and Area 2 had relatively dense callosal connections. Within each of these fields, connections were much less dense for the representations of the glabrous hand and foot and much more dense for the representations of the face and trunk. For the representation of the hand, retrogradely labeled cells were extremely sparse in Area 3b, moderately sparse in Area 1, and moderate in Area 2. There were less dense callosal connections in the hand representations of Areas 3b, 1, and 2 in macaque as compared to owl monkeys. Label in posterior parietal cortex was uneven with zones of extremely dense connections. A large region of very dense callosal connections was noted in motor cortex just medial to the probable location of the hand representation. In all regions, callosally projecting cells appeared to be more broadly distributed than callosal terminations. In no region was the discontinuous arrangement of callosal connections obviously organized into an extensive pattern of mediolateral or rostrocaudal bands or strips.