Connectivity of neuronal populations within and between areas of primate somatosensory cortex.

Functions of the cerebral cortex emerge via interactions of horizontally distributed neuronal populations within and across areas. However, the connectional underpinning of these interactions is not well understood. The present study explores the circuitry of column-size cortical domains within the hierarchically organized somatosensory cortical areas 3b and 1 using tract tracing and optical intrinsic signal imaging (OIS). The anatomical findings reveal that feedforward connections exhibit high topographic specificity, while intrinsic and feedback connections have a more widespread distribution. Both intrinsic and inter-areal connections are topographically oriented across the finger representations. Compared to area 3b, the low clustering of connections and small cortical magnification factor supports that the circuitry of area 1 scaffolds a sparse functional representation that integrates peripheral information from a large area that is fed back to area 3b. Fast information exchange between areas is ensured by thick axons forming a topographically organized, reciprocal pathway. Moreover, the highest density of projecting neurons and groups of axon arborization patches corresponds well with the size and locations of the functional population response reported by OIS. The findings establish connectional motifs at the mesoscopic level that underpin the functional organization of the cerebral cortex.

Vascular distribution patterns in monochorionic twin placentas.

Several recent publications have focused on the association between the occurrence of twin-to-twin transfusion syndrome (TTTS) in diamniotic-monochorionic twins and the presence of a number of selected anatomic placental characteristics (distribution of vascular territory, cord insertion, type and number of inter-twin anastomoses). In contrast, the potential importance of the vascular distribution patterns of the individual twins remains to be elucidated. Based on its gross architectural distribution pattern, chorionic vasculature is traditionally described as disperse, magistral or mixed. The aim of this study was (1) to determine the relative prevalence of these vascular distribution patterns in monochorionic twin placentas, and (2) to correlate these patterns with the presence of TTTS and known anatomic placental features linked to TTTS. The placentas of 89 consecutive diamniotic-monochorionic twins (15 with TTTS, 74 without TTTS), examined at Women and Infants Hospital, were studied. Disperse vascular patterns were seen in 53% of twins, and magistral or mixed patterns in 47%. The prevalence of magistral/mixed vascular patterns was significantly higher in TTTS gestations than in non-TTTS gestations (60% versus 44%, P<0.05) and, in TTTS gestations, much higher in donor twins than in recipient twins (87% versus 33%, P<0.005). A strong association was found between the presence of magistral/mixed patterns and marginal/velamentous cord insertion, low number of inter-twin anastomoses, and uneven distribution of the vascular territories. These findings suggest that the magistral/mixed vascular distribution pattern may represent an important placental architectural feature contributing to the complex pathophysiology of TTTS.

Transformation of revertant murine cells by 5-azacytidine results in rapid inhibition of lysyl oxidase expression.

Lysyl oxidase (LO) is synthesized intracellularly as a proenzyme that is secreted and then processed extracellularly to a mature form. LO is expressed in NIH3T3 cells, but only very low levels are observed after NIH 3T3 is transformed by c-H-ras or one of several other oncogenes. LO functions as a tumor suppressor. Treatment of ras-transformed cells with interferon-alpha with or without retinoic acid results in their persistent reversion to a non-transformed state that is dependent on the restoration of LO expression. When such revertant cells are treated with 5-azacytidine (5-azaC), they undergo rapid morphological retransformation. Within one passage after addition of 5-azaC, there was a down regulation of LO mRNA and proenzyme protein. These data suggest a direct relationship between the transformed state and LO expression.

Fine-scale organization of SI (area 3b) in the squirrel monkey revealed with intrinsic optical imaging.

Optical imaging of intrinsic cortical activity was used to study the somatotopic map and the representation of pressure, flutter, and vibration in area 3b of the squirrel monkey (Saimiri sciureus) cortex under pentothal or isoflurane anesthesia. The representation of the fingerpads in primary somatosensory cortex was investigated by stimulating the glabrous skin of distal fingerpads (D1-D5) with Teflon probes (3-mm diam) attached through an armature to force feedback-controlled torque motors. Under pentothal anesthesia, intrinsic signal maps in area 3b obtained in response to stimulation (trapezoidal indentation) of individual fingerpads showed focal activations. These activations (ranging from 0.5 to 1.0 mm) were discrete and exhibited minimal overlap between adjacent fingerpad representations. Consistent with previously published maps, a somatotopic representation of the fingerpads was observed with an orderly medial to lateral progression from the D5 to D1 fingerpads. Under isoflurane anesthesia, general topography was still maintained, but the representation of fingerpads on adjacent fingers had higher degrees of overlap than with pentothal anesthesia. Multi- and single-unit recordings in the activation zones confirmed the somatotopic maps. To examine preferential inputs from slowly adapting type I (SA) and rapidly adapting type I (RA) and type II (PC) mechanoreceptors, we applied stimuli consisting of sinusoidal indentations that produce sensations of pressure (1 Hz), flutter (30 Hz), and vibration (200 Hz). Under pentothal anesthesia, activation patterns to these different stimuli were focal and coincided on the cortex. Under isoflurane, activation zones from pressure, flutter, and vibratory stimuli differed in size and shape and often contained multiple foci, although overall topography was maintained. Subtraction and vector maps revealed cortical areas (approximate 250-microm diam) that were preferentially activated by the sensations of pressure, flutter, and vibration. Multi- and single-unit recordings aided in the interpretation of the imaging maps. In conclusion, the cortical signals observed with intrinsic signal optical imaging delineated a somatotopic organization of area 3b and revealed different topographical cortical activation patterns for pressure, flutter, and vibratory stimuli. These patterns were dependent on anesthesia type. Possible relationships of these anesthesia effects to somatosensory cortical plasticity are discussed.

Measurement of antiviral activity induced by interferons alpha, beta, and gamma.

The Basic Protocol in this unit describes an assay for murine IFN-induced antiviral activity and employs vesicular stomatitis virus (VSV) and IFN-sensitive fibroblasts. Support Protocol describes the preparation of VSV cultures and the calculation of multiplicity of infection (MOI; i.e., concentration of viral particles required to infect cells). Support Protocol describes the antibody neutralization assay, which can be used to identify the bioactive species of IFN in a sample, or to test the potency of an antibody preparation against a particular species of IFN. Alternate Protocol 1 covers measurement of human IFN-induced antiviral activity; while the steps are quite similar to Basic Protocol, different viral and cell cultures are described.

Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production.

Measles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-alpha/beta. This could have important implications in the virulence and spread of MV.

Patency of different arterial and venous end-to-side microanastomosis techniques in a rat model.

Microvascular anastomotic patency is the most important factor in determining a successful outcome in free-flap transfers. End-to-end and end-to-side techniques have been shown to provide equivalent arterial patency rates in clinical and basic science studies, and end-to-side anastomoses have been used extensively in microsurgical reconstruction. Nevertheless, the effect of venotomy shape on the patency of venous end-to-side anastomoses has not been previously reported. The purpose of this study was to compare the patency rates of end-to-side anastomoses using different techniques in both arteries and veins. In total, 104 Sprague-Dawley rats were subdivided into four groups. The rats were anesthetized, and anastomosis was performed on either the femoral artery or vein on the right with the left used as control. Vesselotomy was varied between an end-to-side hole and an end-to-side slit with patency measured immediately following surgery and at 2 weeks. No significant difference in patency or histology between these techniques was demonstrated in any group. We conclude there is no difference in patency rate between the two techniques in arterial or venous vesselotomies; however, in small vessels < 1.5 mm, the slit technique is technically easier, and clinical recommendations are given.

Effects of sacrocaudal spinal cord transection and transplantation of fetal spinal tissue on withdrawal reflexes of the tail.

Reflex responses to electrocutaneous stimulation of the tail were characterized in awake cats, before and after transection of the spinal cord at sacrocaudal levels S3-Ca1. Consistent with effects of spinal transection at higher levels, postoperative cutaneous reflexes were initially depressed, and the tail was flaccid. Recovery ensued over the course of 70-90 days after sacrocaudal transection. Preoperative and chronic postlesion reflexes elicited by electrocutaneous stimulation were graded in amplitude as a function of stimulus intensity. Chronic postlesion testing of electrocutaneous reflexes revealed greater than normal peak amplitudes, peak latencies, total amplitudes (power), and durations, particularly for higher stimulus intensities. Thus, sacrocaudal transection produced effects representative of the spastic syndrome. In contrast, exaggerated reflex responsivity did not develop for a group of cats that received transplants of fetal spinal cord tissue within sacrocaudal transection cavities at the time of injury, in conjunction with long-term immunosuppression by cyclosporine. We conclude that gray matter replacement and potential neuroprotective actions of the grafts and/or immunosuppression prevent development of the spastic syndrome. This argues that the spastic syndrome does not result entirely from interruption of long spinal pathways.

Sensory nerve endings in the hard palate and papilla incisiva of the goat.

The sensory innervation of the papilla incisiva in the hard palate of the domestic goat was studied with light and electron microscopy, supplemented by electrophysiological studies of free nerve endings. The goat lacks incisor teeth. Grass and leaves are not bitten, but pulled off by pressing them between the tongue and papilla incisiva. Thus, the masticatory mucosa is subject to particularly heavy mechanical loads requiring functional specialization of the horny epithelium in the form of thickening, i.e., the papilla incisiva and 12-14 pairs of rugae palatinae. A thin layer of firm connective tissue (lamina propria) attaches the mucosa to the periost of the hard palate. Sensory nerve fibers were found most abundantly in the papilla incisiva. Their number decreased drastically in aboral direction. A section through the first four rugae palatinae contains only about 10% of the number of free nerve endings found in the same area of mucosa from the papilla incisiva. Four types of sensory nerve endings were found. Free nerve endings were seen ubiquitously in the epithelium and superficial layer of the lamina propria. Merkel nerve endings were found in the bases of the epithelial thickenings in the papilla incisiva and rugae palatinae. Few Ruffini corpuscles were found in the deeper layer of the lamina propria, while lamellated corpuscles were seen just below the basement membrane of the epithelial pegs. Thus, a variety of sensory nerve endings were found in the hard palate, especially in those areas that are in close contact with the tongue during chewing of food. This rich innervation suggests an important role in monitoring the mechanical properties of food. Recordings were made from cell bodies supplying these terminals. Classic low-threshold, slowly adapting responses were observed in Ass afferent populations. This activity was probably mediated by Merkel type endings. Alternately, high-threshold and suprathreshold responses obtained from Adelta category afferents were likely to be nociceptive. In support of this, threshold and suprathreshold sensitization was observed following injection of serotonin into the receptive field of Adelta populations. This activity was likely to be derived from the aforementioned free nerve endings.

Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene.

Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential transcription of lysyl oxidase was not due to regulation of IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region that includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar promoter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control region was confirmed by DNase footprinting. Lysyl oxidase transcription was stimulated by dexamethasone treatment of cells, but this effect could not be assigned within the approximately 3 kb region tested in reporter gene constructs. The promoter activity of the lysyl oxidase reporter gene construct was completely abolished by in vitro DNA methylation, suggesting that the transcriptional suppression after transformation by the ras oncogene may involve DNA methylation.

Lesions and identification of crystalline precipitates of glycoprotein IIb-IIIa antagonists in the rat kidney.

Two glycoprotein IIb-IIIa antagonists (xemilofiban, SC-54684A, and orbofiban, SC-57099B), which are platelet aggregation inhibitors, caused crystalline precipitates in the kidney tubules of rats at high dosages. Dogs were not affected. Depending on the degree of the precipitation, which was dosage dependent, and the location, which differed somewhat between the two compounds, the lesions varied from acute obstruction with tubule cell necrosis, nephron dilation, and sudden death with no inflammation to severe chronic pyogranulomatous inflammation. In order to understand the relevance of the lesions, it was important to identify the precipitates. This was technically challenging because the crystals were water soluble (dissolving in routine fixing and staining techniques) and were present in insufficient quantity to physically isolate. Techniques were devised to evaluate the crystals in situ in unstained frozen sections prepared without directly embedding the tissues in supporting medium, which interfered with the analyses. The crystals were analyzed in situ by infrared and Raman spectroscopy and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). Uroliths found in the renal pelvis of one animal were analyzed by liquid chromatography/mass spectrometry. The resulting spectra showed that the crystals were the de-esterified acids of the parent compounds. This knowledge allowed us to predict that the crystalline precipitates would not be a hazard to humans because of the large multiples of the human dosage at which they occurred and because of differences in renal physiology between rats, dogs, and humans.

Raised object on a planar surface stroked across the fingerpad: responses of cutaneous mechanoreceptors to shape and orientation.

The representations of orientation and shape were studied in the responses of cutaneous mechanoreceptors to an isolated, raised object on a planar surface stroked across the fingerpad. The objects were the top portions of a sphere with a 5-mm radius, and two toroids each with a radius of 5 mm along one axis and differing radii of 1 or 3 mm along the orthogonal axis. The velocity and direction of stroking were fixed while the orientation of the object in the horizontal plane was varied. Each object was stroked along a series of laterally shifted, parallel, linear trajectories over the receptive fields of slowly adapting, type I (SA), and rapidly adapting, type I (RA) mechanoreceptive afferents innervating the fingerpad of the monkey. "Spatial event plots" (SEPs) of the occurrence of action potentials, as a function of the location of each object on the receptive field, were interpreted as the responses of a spatially distributed population of fibers. That portion of the plot evoked by the curved object (the SEPc) provided a representation of the shape and orientation of the two-dimensional outline of the object in the horizontal plane in contact with the skin. For both SAs and RAs, the major vector of the SEPc, obtained by a principal components analysis, was linearly related to the physical orientation of the major axis of each toroid. The spatial distribution of discharge rates [spatial rate surface profiles (SRSs), after plotting mean instantaneous frequency versus spatial locus within the SEPc] represented object shape in a third dimension, normal to the skin surface. The shape of the SA SRSs, well fitted by Gaussian equations, better represented object shape than that of the RA SRSs. A cross-sectional profile along the minor axis [spatial rate profile (SRP)] was approximately triangular for SAs. After normalization for differences in peak height, the falling slopes of the SA SRPs increased, and the base widths decreased with curvature of the object's minor axis. These curvature-related differences in slopes and widths were invariant with changes in object orientation. It is hypothesized that circularity in object shape is coded by the constancy of slopes of SA SRPs between peak and base and that the constancy of differences in the widths and falling slopes evoked by different raised objects encodes, respectively, the differences in their sizes and shapes regardless of differences in their orientation on the skin.

The myoadipose flap: a new composite.

A prefabricated composite fat flap consisting of muscle woven into an anatomically distinct fat pad was studied in a rabbit model. In 17 rabbits, a 2-cm strip of latissimus dorsi was woven into the parascapular fat pad on one side, with the contralateral fat pad serving as a control. At 3 weeks, the endogenous blood supply of both the control and the experimental fat pads was isolated and ligated, and the composite fat/muscle flap was transferred to the chest wall. At 6 weeks, animals were killed, and flaps were analyzed for length, width, and weight; perfused with fluorescein or lead oxide; and examined histologically. Significant differences were found between the control and experimental fat pads with regard to weight and length. Experimental flaps were found to be perfused fully with fluorescein and lead oxide; control fat pads were found not to be perfused. The lead oxide group revealed extensive growth of blood vessels from the latissimus graft into the experimental fat pad. No vessels were visualized in the controls. Finally, sections of the control and experimental flaps were analyzed histologically. A preponderance of viable fat, with evidence of neovascularization, was found in experimental flaps, compared with the necrotic fat that characterized the controls. We conclude that prefabrication of a fat flap is possible and may have extensive application in various areas of plastic surgery.

Encoding of shape and orientation of objects indented into the monkey fingerpad by populations of slowly and rapidly adapting mechanoreceptors.

The peripheral neural representation of object shape and orientation was studied by recording the responses of a spatially distributed population of rapidly and slowly adapting type I mechanoreceptors (RAs and SAs, respectively) to objects of different shapes and orientations indented at a fixed location on the fingerpad of the anesthetized monkey. The toroidal objects had a radius of 5 mm on the major axis, and 1, 3, or 5 mm on the minor axis. Each object was indented into the fingerpad for 4 s at orientations of 0, 45, 90, and 135 degrees using a contact force of 15 gwt. Estimations of the population responses (PRs) were constructed by combining the responses of 91 SA and 97 RA single afferents at discrete times during the indentation. The PR was composed of the neural discharge rates (z coordinate) plotted at x and y coordinates of the most sensitive spot of the receptive field. The shapes of the PRs were related to the shapes of the objects by fitting the PRs with Gaussian surfaces. The orientations of the PRs were determined from weighted principal component analyses. The SA PR encoded both the orientation and shape of the objects, whereas the RA PR did neither. The SA PR orientation was biased toward the long axis of the finger. The RA PR encoded orientation only for the object with the highest curvature but did so ambiguously. Only the SA PR was well fit by a Gaussian surface. The shape of the object was discriminated by the SA PR within the first 500 ms of contact, and the form of the SA PR remained constant during the subsequent 3.5 s. This was manifested by constant widths of the PR along the major and minor axes despite a peak response that decreased from its maximum at 200 ms to an asymptotic value starting at 1 s. Thus the shape and orientation of each object were coded by the shape and orientation of the SA PR.

State health care reforms: how they affect children and adolescents with emotional disorders and their families.

This article reports on the Health Care Reform Tracking Project, a national study designed to describe and analyze state health care reforms and their impact on children and adolescents with emotional disorders and their families. It summarizes the results of the baseline survey of states conducted in 1995, exploring the nature and extent of the reforms in which states are engaged, most of which involve applying managed care technologies to their Medicaid programs. Trends across states are identified with respect to mental health service delivery, particularly with respect to children and adolescents. The article concludes with a discussion of issues and concerns related not only to mental health service delivery for children and adolescents with emotional disorders and their families but also to the systems of care that have been developing over the past decade to serve them. Some of these concerns include the lack of pilots or demonstrations, limited mental health coverage in some reforms, the lack of integration between mental health and substance abuse systems, the lack of special provisions for children, the need for more reliable bases for deriving capitation rates, the limited incorporation of systems of care, the need to incorporate interagency treatment planning and service delivery approaches, the lack of outcome measures specific to and appropriate for children, and the need for greater family involvement in the planning and implementation of these reforms.

Reversion by deletion of transforming oncogene following interferon-beta and retinoic acid treatment.

We have previously shown that prolonged interferon-beta (IFN-beta) treatment of RS485 cells (NIH3T3 cells transformed with multiple copies of an LTR-cHa-ras oncogene) resulted in the phenotypic reversion of 1%-5% of the culture, depending on the conditions used. This reversion persisted after IFN-beta was discontinued, although the revertants retained the LTR-cHa-ras and continued to express ras mRNA and p21. Clones were prepared of such persistent revertant cell lines (PRs). Expression of lysyl oxidase (LOX), which appears to act as a suppressor of ras transformation, was downregulated in RS485 and upregulated in the PRs. When retinoic acid (RA) was combined with IFN-beta treatment of the RS485 cultures, a different mechanism of reversion predominated. Following 60 days of treatment with 20 IU/ml of IFN-beta and 10 microM RA, all of the multiple (3-5) copies of the transforming LTR-c-Ha-ras originally present in RS485 cells were deleted from the genome in 72% of 54 revertant cell lines isolated. As in the case of revertants observed after treatment with IFN-beta alone, LOX mRNA expression was upregulated in all of the revertants that resulted from the treatment with IFN plus RA. The level of LOX mRNA expression acts, therefore, as an indicator of transformation in this system.

Internal stabilization of autogenous rib cartilage grafts in rhinoplasty: a barrier to cartilage warping.

Autogenous rib cartilage grafts have gained more widespread use in rhinoplasty as dorsal onlay grafts and columellar struts. However, the usefulness of rib as a donor site has been limited by difficulties with postoperative cartilage warping. We hypothesized that the internal stabilization of rib cartilage grafts with Kirschner wires would prevent warping. The costochondral cartilages of a fresh cadaver were harvested and carved into 4 x 10 x 40 nm blocks. A single 0.035-in K-wire was placed longitudinally into the center of each of the study specimens (n = 9), whereas no internal stabilization was utilized in the control group (n = 9). Over a 10-day study period, a mean of 2.2 degrees of warping was observed in the grafts with K-wires as compared to 8.9 degrees in the control group. This indicates that internal stabilization of rib grafts significantly reduces warping (p < 0.001). In a subsequent clinical study, 28 patients underwent placement of internally stabilized columellar struts (n = 19) and/or dorsal nasal grafts (n = 12) using autogenous rib cartilage. At a mean follow-up of 13.5 months (range 3 to 36 months), graft warping was not observed in any patient. Satisfactory aesthetic results were achieved in all but one patient, in whom mild displacement of a dorsal onlay graft occurred. Palatal extrusion of the K-wire occurred in 3 of the first 9 columellar struts. This prompted an alteration in technique with no subsequent extrusions. We conclude that the internal stabilization of autogenous rib cartilage grafts with K-wires effectively prevents graft warpage.