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Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo., (© 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.)

The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.

Most pulpal afferent neurons have cytochemical features in common with the class of nociceptors that express neuropeptides and respond to NGF, while very few bind the plant lectin IB4, a widely used marker for the class of nociceptors that respond to the GDNF family of neurotrophic factors. The present study was undertaken to determine whether the GDNF receptor, GFRalpha-1, is expressed by pulpal afferents, and, further, to determine whether tooth injury evokes changes in expression of the GDNF and NGF receptors among pulpal afferents. The tracer, fluoro-gold (FG), was applied to shallow cavities in dentin of first and second maxillary molars. After 4 weeks, the molars of one side received a test injury consisting of a deeper cavity that exposed pulp horns. Animals were perfusion fixed 2 days later, and sections of the trigeminal ganglia were double immunostained with combinations of antibodies against GFRalpha-1, and TrkA. Under control conditions, GFRalpha-1 immunostaining was observed in 72% of neurons that projected to the molar pulp, TrkA in 78%, and immunostaining for both receptors was observed in 65% of pulpal afferents. Tooth injury evoked up-regulation of GFRalpha-1 expression (to 93%) and a slight down-regulation of TrkA expression (67%) among tooth afferents, while there was no discernable change in the proportion of pulpal afferents that expressed both TrkA and GFRalpha-1 (to 61%).

The majority of complex congenital heart defects occur in individuals who are afflicted by laterality disease. We hypothesize that the prevalence of valvuloseptal defects in this population is due to defective left-right patterning of the embryonic atrioventricular (AV) canal cushions, which are the progenitor tissue for valve and septal structures in the mature heart. Using embryos of the frog Xenopus laevis, this hypothesis was tested by performing left-right lineage analysis of myocytes and cushion mesenchyme cells of the superior and inferior cushion regions of the AV canal. Lineage analyses were conducted in both wild-type and laterality mutant embryos experimentally induced by misexpression of ALK4, a type I TGF-beta receptor previously shown to modulate left-right axis determination in Xenopus. We find that abnormalities in overall amount and left-right cell lineage composition are present in a majority of ALK4-induced laterality mutant embryos and that much variation in the nature of these abnormalities exists in embryos that exhibit the same overall body situs. We propose that these two parameters of cushion tissue formation-amount and left-right lineage origin-are important for normal processes of valvuloseptal morphogenesis and that defective allocation of cells in the AV canal might be causatively linked to the high incidence of valvuloseptal defects associated with laterality disease., (Copyright (c) 2005 Wiley-Liss, Inc.)

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

The dental pulp is a favorable model for studies of interactions between nociceptive sensory neurons and their peripheral target tissues. In the present study, we retrogradely labeled pulpal afferent neurons with an improved method that permits monitoring of changes in neuronal phenotype in response to controlled tooth injuries. The capacity of retrograde neuronal tracers to diffuse through dentinal tubules was exploited, thereby avoiding the severe injury to the pulp associated with previous tracer application methods. The strategy was to apply the durable fluorescent tracer, Fluoro-gold (FG), to exposed dentin in the floor of shallow cavities in molars, in order to pre-label pulpal neurons in trigeminal ganglia of young adult Sprague-Dawley rats. A high percentage of pupal afferent neurons were retrogradely labeled by application of FG to exposed dentin and the FG fluorescent signal persisted in most labeled neurons for at least 8 weeks. Following tracer application to dentin, the pulp tissue appeared normal histologically, with the exception that a layer of reactive dentin was deposited at the pulp-dentin border beneath the shallow cavities. Assessment of expression of calcitonin gene-related peptide (CGRP) and brain derived neurotrophic factor (BDNF) indicated that pulpal neurons remained in a quiescent, baseline condition cytochemically following application of tracer to cavities in dentin and upregulation of these markers could be detected in neurons that projected to teeth that received a test injury subsequent to tracer application. Thus, labeling of trigeminal neurons via dentinal tubules provides the basis for a useful model for precisely assessing properties of pulpal afferents in both quiescent and activated states.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

The rodent dentition has become an important model for investigations of interactions between dental tissues and peripheral neurons. Although experimental nerve injury has been widely used for such studies, there is uncertainty about the courses of nerve fibers supplying the mandibular teeth. In order to clarify this, we used a mixture of monoclonal antibodies against neurofilament proteins to enhance demonstration of nerve fibers so that small nerves could be readily traced in serial frozen sections of mandibles of Sprague Dawley rats ranging in age from embryonic day (E) 18 to postnatal day (P) 90. The 1st molar and anterior portion of the 2nd molar were innervated by small nerves that emerged as distinct branches of the IAN trunk at or near the mandibular foramen. In contrast, the nerve supply to the 3rd molar and posterior part of the 2nd molar was a branch of the lingual nerve that bypassed the mandibular canal altogether. The IAN trunk split into the mental nerve and a large branch to the incisor about 2 mm anterior to the mandibular foramen. Thick branches of the incisor nerve descended into the incisor socket to form a dense plexus of nerve fiber bundles extending along the length of the incisor periodontium. The sparse pulpal innervation of the incisor was provided by a few thin fascicles that emerged from the caudal portion of the periodontal plexus to enter the incisor apex. The dental branches of the IAN and lingual nerve seen in the adult were well established and readily identifiable at age E18 even though their targets were limited to the follicles of the developing teeth. These studies show that the trigeminal branches that supply the mandibular teeth can be identified at a wide range of ages as distinct nerves at a considerable distance proximal to their targets. This detailed information on the courses taken by the dental nerves can provide an anatomical basis for increased precision in characterization and perturbation of neural pathways from the molars and incisor., (Copyright 1999 Wiley-Liss, Inc.)

The function of acetylcholinesterase (AChE) is to terminate the action of acetylcholine at the cholinergic synapse. Recent evidence suggests additional roles for acetylcholinesterase as a peptidase and/or a protease which is expressed by growing neurites as part of their invasion of developing neural structures. We report the localization of acetylcholinesterase in developing ferret retina. AChE histochemical staining is seen in the developing inner plexiform layer (IPL) of ferret retina at birth (post-natal day zero, PO), the earliest developmental stage examined. Transient expression is seen at the border between the ganglion cell layer and the nerve fiber layer at P14 and P21. A small amount of transient expression is seen in the outer plexiform layer (OPL) at this age as well. By P28, the transient expression in the OPL is at its peak, and is found at photoreceptor terminals and associated with apparent horizontal cell axons. Labeling is also seen intracellularly in the inner nuclear layer (INL), at the OPL/INL border, suggesting that horizontal cells are the source of the transient AChE expression in the OPL. Overt synaptic profiles also appear in the inner plexiform layer (IPL) at P21 and P28. About 2 days layer, the eyes open and the photoreceptor outer segments are fully developed. By 2 weeks later, at P42, the AChE staining pattern in the retina has taken on its adult appearance: no reaction product in the outer retina; intracellular reaction product in the Golgi apparatus of a subset of amacrine and displaced amacrine cells which manufacture AChE; and extracellular reaction product at both synaptic and non-synaptic sites in the IPL. These data are consistent with a role for AChE as a peptidase early in development, and as an enzyme essential in the termination of synaptic action at mature synapses.

The present study was undertaken to test the hypothesis that development of the pulpal innervation is dependent on nerve growth factor (NGF). Newborn rats were given subcutaneous injections of a rabbit anti-mouse NGF serum on alternate days for the first 24 days postnatally. Control animals were untreated and normal rabbit serum-treated litter mates. The animals were deeply anesthetized on postnatal day 26, perfused with fixative and the first mandibular molars were processed for transmission electron microscopy to obtain a complete census of axons entering the four roots. The composition of the mental nerve was also examined. Compared to control animals, the apical innervation of molars from anti-NGF-treated rats had only 62% as many myelinated fibers and 41% as many unmyelinated axons. Those myelinated fibers present in antiserum-treated animals were slightly, but significantly, smaller in average diameter than controls. In teeth of control animals, about 20% of all unmyelinated axons were located in fibers coursing outside of nerve fascicles; these isolated fibers were disproportionately rare after antiserum exposure. The average number of unmyelinated axons per Schwann cell unit was also significantly lower. Postnatal exposure to anti-NGF had milder effects on mental nerve composition compared to the tooth innervation. Numbers of myelinated fibers were 83% of controls, unmyelinated axons were 74% of controls and there was no change in the average number of unmyelinated axons per Schwann cell unit. We conclude that development of dental innervation is highly susceptible to postnatal NGF deprivation. This may be a consequence of the mostly nociceptive composition of dental nerves and their late development.

The rat molar has become an important model for studies of interactions between nerves and the pulp-dentin complex, yet there is only limited quantitative information on the number and size distribution of axons entering the roots of this tooth. This study was undertaken to provide such a detailed characterization of the apical innervation of the rat molar. An additional objective was to compare the apical nerve composition of young, recently erupted rat molars with that of mature teeth in order to determine whether there is ongoing maturation of the innervation after the teeth have attained functional occlusion. A complete census was made of the nerve fibers entering the roots of both mature and recently erupted juvenile mandibular first molars in Sprague-Dawley rats. Each of the four roots of the first molars was processed for electron microscopy of thin sections near the apex. The majority of intradental nerve fibers entered the molar via the two larger (mesial and distal) roots. Within the apical root pulp, most, but not all, axons occurred within well-defined fascicles associated with blood vessels. Molars from adult animals (age 4 months) had a mean total of 232 (S.D. = 49, N = 7 teeth) myelinated fibers and 806 (S.D. = 143) unmyelinated axons entering the four roots. Fibers exceeding the A delta size range (circumference > or = 19 microns) accounted for only 4% of the myelinated axons at the apex. Molars from juvenile animals (age 4 weeks) had fewer myelinated fibers (mean 176, S.D. 18, N = 8), but more unmyelinated axons (mean 1,174, S.D. 160) than adults. The mean ratio of unmyelinated axons to myelinated axons was 6.6:1 for juveniles compared to 3.5:1 for adults. Juvenile teeth contained no myelinated fibers that exceeded 19 microns in circumference. These results indicate that the innervation of the rat molar resembles that of teeth of non-rodent mammals in that (1) innervation density is high, (2) there is a high ratio of unmyelinated axons, and (3) most of the myelinated fibers are of thin caliber. Furthermore, it appears that after the molar erupts, maturation of the nerve fiber composition continues with processes that include both a marked decrease in the number of unmyelinated axons and an increase in the number and size heterogeneity of myelinated fibers.

Intramuscular injections of botulinum toxin type A (Oculinum) is used to treat strabismus and focal dystonias affecting orofacial muscles. However, the toxin-induced morphological changes that underlie the therapeutic alterations of tone in the muscles of mastication have not been described. In this study, paired intramuscular injections of botulinum toxin (10 units) were made in three adult monkeys (Macaca fascicularis) allowed to survive 14, 28 and 63 days. Another monkey received multiple injection-pairs over 84 days. Animals were killed by deep pentobarbital anaesthesia before transcardiac perfusion-fixation. Tissue sampled from comparable regions of the injected masseter, the uninjected masseter and an uninjected animal was processed for ultrastructural analysis. Few changes were found 14 days post-injection. However, muscle fibres showed myofibrillar dissolution, aberrations in the Z-line, and enlarged mitochondria in the region of the I-band by 28 days. In the 63-day and 84-day animals, the injected muscle was considerably smaller than the uninjected, contralateral muscle. Regions of the injected muscle contained fibres with markedly reduced cross-sectional area. Internalization of myonuclei, loss of myofibrillar organization, and helical complexes were common. Toxin-induced changes, though similar to those that follow denervation by axotomy, were not accompanied by degeneration of neuromuscular junctions. Instead, morphological evidence for axonal sprouting in the region of the neuromuscular junction, possibly contributing to functional recovery, was seen as early as 14 days in toxin-treated muscles.