Effects of a brief mindfulness intervention on negative affect and urge to drink among college student drinkers.

Several theories have proposed that negative affect (NA) plays a large role in the maintenance of substance use behaviors - a phenomenon supported in laboratory-based and clinical studies. It has been demonstrated that mindfulness meditation can improve the regulation of NA, suggesting that mindfulness may be very beneficial in treating problematic substance use behavior. The current study tested whether a brief mindfulness meditation would lower levels of NA, increase willingness to experience NA, lower urges to drink, and increase time to next alcoholic drink in a sample of at-risk college student drinkers (N = 207). Participants were randomized to one of three brief interventions (mindfulness, relaxation, or control) followed by an affect manipulation (negative or neutral stimuli). Affect and urge were measured prior to intervention (Time 1 [T1]), after intervention but prior to affect manipulation (Time 2 [T2]), and immediately after the affect manipulation (Time 3 [T3]). Levels of mindfulness and relaxation were assessed from T1-T3. The additional measures of willingness to continue watching NA images and time to next alcoholic drink were examined at T3. Results indicated that the mindfulness intervention increased state mindfulness and relaxation, and decreased NA immediately following the mindfulness intervention. However, the mindfulness intervention did not influence responses to NA induction on any of the outcome variables at T3. One potential explanation is that the mindfulness intervention was not robust enough to maintain the initial gains made immediately following the intervention.

An essential role for connexin43 gap junctions in mouse coronary artery development.

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.

Conotruncal myocardium arises from a secondary heart field.

The primary heart tube is an endocardial tube, ensheathed by myocardial cells, that develops from bilateral primary heart fields located in the lateral plate mesoderm. Earlier mapping studies of the heart fields performed in whole embryo cultures indicate that all of the myocardium of the developed heart originates from the primary heart fields. In contrast, marking experiments in ovo suggest that the atrioventricular canal, atria and conotruncus are added secondarily to the straight heart tube during looping. The results we present resolve this issue by showing that the heart tube elongates during looping, concomitant with accretion of new myocardium. The atria are added progressively from the caudal primary heart fields bilaterally, while the myocardium of the conotruncus is elongated from a midline secondary heart field of splanchnic mesoderm beneath the floor of the foregut. Cells in the secondary heart field express Nkx2.5 and Gata-4, as do the cells of the primary heart fields. Induction of myocardium appears to be unnecessary at the inflow pole, while it occurs at the outflow pole of the heart. Accretion of myocardium at the junction of the inflow myocardium with dorsal mesocardium is completed at stage 12 and later (stage 18) from the secondary heart field just caudal to the outflow tract. Induction of myocardium appears to move in a caudal direction as the outflow tract translocates caudally relative to the pharyngeal arches. As the cells in the secondary heart field begin to move into the outflow or inflow myocardium, they express HNK-1 initially and then MF-20, a marker for myosin heavy chain. FGF-8 and BMP-2 are present in the ventral pharynx and secondary heart field/outflow myocardium, respectively, and appear to effect induction of the cells in a manner that mimics induction of the primary myocardium from the primary heart fields. Neither FGF-8 nor BMP-2 is present as inflow myocardium is added from the primary heart fields. The addition of a secondary myocardium to the primary heart tube provides a new framework for understanding several null mutations in mice that cause defective heart development.

Gap junction communication and the modulation of cardiac neural crest cells.

The analyses of transgenic and knockout mice with perturbations in alpha 1 connexin (Cx43) function have revealed an important role for gap junctions in cardiac development. This likely involves the modulation of cardiac crest migration and function. Studies carried out with these mouse models suggest that clinically there may be a novel category of cardiac defects involving crest perturbations that do not include outflow septation defects, but rather involve more subtle defects in the pulmonary outflow tract.

A novel role for cardiac neural crest in heart development.

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Connexin 43 expression reflects neural crest patterns during cardiovascular development.

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.

A novel role for cardiac neural crest in heart development.

It is well known that cardiac neural crest participates in development of the cardiac outflow septation and patterning of the great arteries. Less well known is that ablation of the cardiac neural crest leads to a primary myocardial dysfunction. Recent data suggests that the myocardial dysfunction occurs because of the absence of an interaction of neural crest and pharyngeal endoderm to alter signaling from the endoderm. Continuation of an FGF-like signal from the endoderm past a precise time in development appears to be detrimental to myocardial maturation.

Gap junction-mediated cell-cell communication modulates mouse neural crest migration.

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.

Cardiac neural crest cells provide new insight into septation of the cardiac outflow tract: aortic sac to ventricular septal closure.

A great deal is unclear about the process of cardiac outflow septation. Much controversy exists regarding the precise details of tissue origins and movements of various components. The contribution of the cardiac neural crest to aorticopulmonary and distal truncal septation has been described; however, the distribution of the neural crest in the proximal outflow and heart is unknown. The present study describes the movement of cardiac neural crest cells from the caudal pharyngeal arches into the outflow tract and base of the heart during the period of outflow septation. Using quail-chick chimeras we found that the cardiac neural crest was distributed to all levels of the outflow tract and into the base of the heart. Septation of the outflow tract lumen occurred by two different processes that involved the cardiac neural crest directly. Cardiac neural crest cells were also distributed to regions of the outflow tract that correlated with sites of remodeling, such as the aortic sac as it was remodeled into the base of the ascending aorta and pulmonary trunk, the distal truncus that was patterned into the two semilunar valves and in the proximal conotruncus where muscularization of the ridges and septum occurred. Additionally, cardiac neural crest cells were found at the site of closure of the ventricular septum, in the wall of the pulmonary infundibulum, and transiently in the wall of the aortic vestibule. Contrary to current thinking, not all of the condensed mesenchyme in the outflow tract during septation was derived from neural crest.

Establishing an interdisciplinary patient care team: collaboration at the bedside and beyond.

The authors describe how an interdisciplinary team used skills in communication and collaboration to improve patient care on a busy surgical service. A major goal was to establish and maintain continuity of care in the face of decreasing lengths of stay and increasing patient acuity. The authors share their insight about designing and supporting a successful interdisciplinary patient care team and discuss how their experiences relate to concepts such as case management and career development.

Cardiac neural crest is essential for the persistence rather than the formation of an arch artery.

Double-label immunohistochemistry was used to compare early aortic arch artery development in cardiac neural crest-ablated and sham-operated quail embryos ranging from stage 13 to stage 18. The monoclonal antibody QH-1 labeled endothelial cells and their precursors, and HNK-1 labeled migrating neural crest cells. In the sham-operated embryos, the third aortic arch artery developed from a lumenizing strand of endothelial precursors that became separated from the pharyngeal endoderm by migrating cardiac neural crest cells as they ensheathed the artery. The arch artery of the neural crest-ablated embryos lumenized but failed to become separated from the pharyngeal endoderm, indicating that neural crest is unnecessary for the early formation of the aortic arch artery. However, once blood flow was initiated through the third arch artery of crest-ablated embryos at stage 16, the artery became misshapen and sinusoidal. By embryonic day 3, abnormal connections to the dorsal aorta occurred and bilateral symmetry was lost, suggesting that the loss of neural crest-derived ectomesenchyme destabilizes the nascent artery. Although here we show no loss of the third arch artery, past studies have reported hypoplasia or missing carotids in older neural crest-ablated embryos (Bockman et al. [1987] Am. J. Anat. 180:332-341; Bockman et al. [1989] Anat. Rec. 225:209-217; Nishibatake et al. [1987] Circulation 75:255-264; Tomita et al. [1991] Circulation 84:1289-1295). We suggest that the cardiac neural crest is essential for the persistence of an arch artery, but not its formation. Furthermore, since changes in the development of the arch artery are seen prior to the formation of the tunica media, it is suggested that a critical period is reached in the development of the arch artery, after lumenization, but prior to the formation of the tunica media, which necessitates the presence of the cardiac neural crest.

Visual understanding of cardiac development: the neural crest's contribution.

Understanding normal and abnormal cardiovascular development is of interest to basic scientists as well as to clinicians taking care of infants with heart defects. This article presents a visual overview of cardiac development. It provides a framework on which to understand how abnormal cardiac development leads to groups of cardiovascular defects requiring clinical care. Human heart development is presented schematically and is correlated with similar points in chick cardiac development. Studying both normal and abnormal cardiac development in neural crest-ablated embryos has highlighted two major themes of cardiac development: there is a mechanism of differential growth in the developing cardiovascular system that is not seen to a major extent after birth and cardiac defects can be pictured as arrested stages of normal development. At a particular stage of development, it is normal to have a certain relationship between developing structures. However, if the development is arrested and this relationship of structures is allowed to persist, it then becomes abnormal. Visualizing heart defects as arrested points in normal development is better used as a tool to categorize defects than as a causative mechanism. The exact mechanisms of how abnormal development results in cardiac defects is not well understood. Study of the neural crest model of cardiac defects suggests possible mechanisms.

Association of the cardiac neural crest with development of the coronary arteries in the chick embryo.

BACKGROUND: Chick coronary arteries originate as penetrating channels from a subepithelial peritruncal ring into the wall of all three aortic coronary sinuses. Two of these capillaries develop a muscular wall and become the definitive coronary arteries. Since cardiac neural crest (CNC) contributes ectomesenchyme to the tunica media (TM) of the aortic arch vessels, we wished to learn if the CNC also contributes to the media of the coronary arteries and if CNC plays an inductive role in determining the site of aortic penetrations and influences which channels persist to hatching. METHODS: Quail-to-chick chimeras were made by bilaterally removing the chick CNC and replacing it with quail CNC. The chimeras and unoperated controls were collected on embryonic days (ED) 7-18, fixed in Carnoy's fixative, serially sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. Several ED 18 controls and chimeras were also stained with Gomori's trichrome stain, or labeled with antineurofilament or antivascular smooth muscle alpha actin. RESULTS: The TM of the coronary arteries and the aortic coronary sinuses did not consist of CNC cells. The media of the surviving coronary arteries was disrupted by clusters of CNC cells scattered in the wall of the base of the coronary artery on ED 14 and 18. Persisting coronary arteries were always associated with large neural crest-derived parasympathetic ganglia near their origin. Branches from parasympathetic nerves entered the base of the coronary arteries where the clusters of neural crest cells were located. Quail cells were also associated with tiny vessels exiting the ostia of the coronary arteries and traveling in the outer aortic wall. Labeling with antibodies confirmed a disruption of the TM at the base of the coronary arteries, and showed innervated clusters of quail cells in the disrupted part of the TM. CONCLUSION: Although the TM of the coronary arteries and the aortic coronary sinuses contained no CNC cells, clusters of innervated quail cells disrupted the TM at the base of the coronary arteries. CNC does not appear to induce capillary penetration directly; however, the exclusive association of CNC-derived parasympathetic ganglia and nerves with persisting coronary arteries suggests that the presence of parasympathetic ganglia is essential to the survival of the definitive coronary arteries. CNC cells may also be associated with the development of the aortic vasa vasorum.

Cardiac neural crest contribution to the pulmonary artery and sixth aortic arch artery complex in chick embryos aged 6 to 18 days.

Previous studies of cardiac neural crest (CNC) migration in early chick embryos demonstrated CNC cells in the media of pharyngeal arch arteries three, four, and six, and in the most proximal part of the developing pulmonary arteries. The objectives of this study were to learn 1) to what extent the CNC is involved in the later development of the pulmonary arteries, 2) how the CNC cells are distributed in the sixth aortic arch artery including the wall of the ductus arteriosus in the older embryo, and 3) what happens to the CNC as the pulmonary artery/sixth arch complex grows into its adult configuration. Quail-to-chick chimeras were used to study CNC distribution in embryos aged 6 to 18 days. Controls (undisturbed chick embryos) were collected with chimeras. Each was fixed, processed, sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. The results demonstrated that CNC disappeared from the proximal pulmonary arteries by embryonic day 9 and played no further role in pulmonary artery development. With the exception of the endothelium, CNC completely filled the wall of the sixth aortic arch artery as far distally as its junction with the dorsal aorta in younger embryos and with the aorta in older embryos, thus suggesting the possibility of proximodistal migration of CNC along the sixth aortic arch. The ductus wall, filled with CNC, was intimately associated with the recurrent laryngeal nerve, also filled with CNC, thereby strongly suggesting a role for CNC in ductal closure.

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.

Development of the musculoelastic septation complex in the avian truncus arteriosus.

It is now well established that cells from the cardiac neural crest (CNC) are essential for normal conotruncal septation. The truncal septation complex consists of the aorticopulmonary (AP) septum and the myocardial sheath of the truncus. The principal role of the CNC cells during septation appears to be their differentiation into the elastogenic smooth muscle that forms the AP septum proper. The objective of this study was to integrate serial reconstruction and specific histochemical markers in order to provide a unified analysis of the relationships between the CNC and the other components of the truncal septation complex. The development of the septation complex was compared normal embryos vs. embryos from which the CNC had been surgically ablated. Embryos from each group were harvested after incubation periods of 4-8 days (Hamburger-Hamilton stages 23-34). Histochemical procedures were performed for positive identification of the elastic matrix and smooth muscle alpha-actin; the presence of these proteins was used as the criterion for "septal cells" and to define the boundaries of the septum. The results indicate that the shape, components, boundaries, and degree of organization of the septation complex may be different from previous descriptions. Furthermore, all of the components of the truncal septation complex are dysgenic in the absence of the CNC. Of special significance in the absence of CNC. Of special significance in the absence of CNC are: 1) the failure of the myocardial sheath to retract; 2) the apparently random distribution of surrogate ectomesenchyme; and 3) the impairment of truncal elastogenesis. These results indicate that the cells of neural crest origin interact with the surrounding mesenchyme during septation and that the entire septation complex depends upon the presence of the neural crest cells for normal development.

Origin of the proximal coronary artery stems and a review of ventricular vascularization in the chick embryo.

The objective of this study was to determine how the coronary artery stems develop in the chick embryo. The hearts of 51 ink-injected and cleared chick embryos, aged embryonic days 6, 6.5, 7, 7.5, 9, and 10, were dissected, examined, and selectively photographed. Two representative hearts from each group were paraffin embedded, serially sectioned at 10 microns, and examined for aortic endothelial budding. We found that the proximal coronary artery did not appear to grow outward from the aorta as commonly described in the literature. It appeared to originate from a capillary ring which encircled the aortic and pulmonary outflow tracts. On embryonic day 7.5, one to three channels arising from this ring penetrated each aortic sinus, in an area of darker textured endothelium. Histologically and grossly, multiple channels were still apparent on day 9, particularly in the left coronary artery. One of these channels always became dominant to form the stem. Each stem, which varied in length from embryo to embryo, always ended in a plexus of sinusoidal endothelial tubes. By day 10, the coronary artery stems were longer, with many major branches. Histologically, evidence of multiple channels still was visible. It is significant that channels from the bulbar vascular ring penetrated the aorta at very specific points in the aortic sinuses and did not penetrate the pulmonary trunk or other aortic sites. We believe this fact indicates that the penetration of the aortic sinuses by channels from the bulbar vascular ring represents a controlled invasion of the aorta.