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Distribution and synaptology of dopaminergic fibers in the mature and developing lateral septum of the rat.

The dopamine innervation of the adult and developing lateral septum of the rat was investigated with light and electron microscope immunocytochemistry using anti-dopamine antibodies. Light microscopic analysis showed that the pattern of innervation of the lateral septum exhibited a marked reorganization during the first 2 postnatal weeks, when it acquired features comparable to the adult. Ultrastructural analysis suggested that there may be two different dopamine inputs in the lateral septum. The first develops earlier in life and, through symmetrical axodendritic synapses, affects remote parts of neurons and may cause inhibition. The second develops later and, through asymmetrical axosomatic synapses, affects neuronal somata and may cause excitation. These findings may explain the reported contradictory results concerning the physiological role of dopamine in neurons of the lateral septum.

Animals↗

Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression.

Development of kindling and mossy fiber sprouting, and changes of gene expression were studied after 40 seizures produced during about 3 h by electrical stimulation every 5 min in the ventral hippocampus. As assessed by 5 test stimulations, enhanced responsiveness was present already after 6-24 h but from 1 week post-seizure increased gradually up to 4 weeks without additional stimuli. Sprouting of mossy fibers in the dentate gyrus was demonstrated only at 4 weeks with Timm's staining. In situ hybridization showed a transient increase (maximum at 2 h) of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), TrkB and TrkC mRNA levels and reduction (maximum at 12-24 h) of neurotrophin-3 (NT-3) mRNA expression in dentate granule cells after the seizures. In addition, BDNF mRNA levels were elevated in CA1 and CA3 regions, amygdala and piriform cortex. Marked increases of mRNA for growth-associated protein (GAP-43), with maximum expression at 12-24 h, were observed in dentate granule cells and in amygdala-piriform cortex. Dynorphin mRNA levels showed biphasic changes in dentate granule cells with an increase at 2 h followed by a decrease at 24 h. No long-term alterations of gene expression were observed. These findings indicate that increased responsiveness develops rapidly after recurring seizures but that the kindled state is reached gradually in about 4 weeks. Mossy fiber sprouting occurs in parallel to epileptogenesis and may play a causative role. Short-term changes of neurotrophin and Trk, GAP-43 and dynorphin mRNA levels and the assumed alterations of the corresponding proteins could trigger structural rearrangements underlying kindling but might also contribute to the initial increase of seizure susceptibility.

Analysis of Variance↗

[Age-related development of the arrangement of connective tissue fibers in the lamina propria of the human vocal folds--scanning electron microscopic examination with digestion method].

The lamina propria of the human vocal fold consists of a superficial, intermediate, and deep layer. This stratified structure is thought to facilitate phonation. Each layer has different physical properties based on different alignment and distribution of collagen and elastic fibers. In the present study, developmental changes in vocal fold structure were studied in human fetuses, infants, and children, with special reference to the pattern of distribution of collagen and elastic fibers. Vocal fold specimens were obtained at autopsy from 5 fetuses, 7 neonates, 3 infants, 3 children at the age of 1 year, 3 children at 3 years, 3 children at 5 years, 3 children at 12 years, and 5 subjects at ages ranging from 15 to 22 years. Prior to the examination of collagen fibers, elastic fibers and cells were dissolved with 10% sodium hydroxide treatment. Prior to the examination of elastic fibers, collagen fibers and cells were dissolved by treatment with 90% formic acid. The specimens were then dehydrated, dried, ion-coated with platinum, and examined with a scanning electron microscope. In fetuses and infants, thin, coiled fibers were found distributed densely in the anterior, posterior, and deep parts of the lamina propria, while irregular thick fibers were sparsely seen in the superficial layer of the vocal fold. In children aged 1 to 3 years, the dense fibers in the deep part decreased, and the longitudinal fibers in the superficial layer increased. In children at 5 years of age, longitudinal collagen and elastic fibers were noted in all of the layers of the vocal fold. The distribution of fibers was uniform irrespective of the depth. At 12 years of age, thin, coiled fibers were noted in the superficial layer, while thin, irregular fibers were found in the deep layer. At 17 years, differentiation of the superficial and deep layers was more evident. In male subjects after adolescence the curvature of curly collagen fibers decreased, and the diameter of fibers increased. The present findings suggest that the development of the vocal fold in childhood occurs in two steps. In the first step, dense fibers in the anterior, posterior, and deep parts of the lamina propria found in fetuses and infants shift to the anterior and posterior ends of the vocal fold, between which longitudinal fibers appear. During this step only simple phonation is possible. In the second step, differentiation of the superficial and deep layers occurs, and the stratified structure of the vocal fold appears in the teens. This step is probably related to the complicated modality of phonation in this age group. In males, the development of the vocal fold is completed after changes in collagen fibers during the mutation.

Adolescent↗

Development of macroglial cells in the embryonic chick optic nerve.

Macroglia development in the embryonic chick optic nerve was immunohistochemically examined. The astrocytes with glial fibrillary acidic protein immunoreactivity were initially restricted to the retinal end of the optic nerve at stage 40, but had widely dispersed within the optic nerve in an out-side-in manner by stage 44. Oligodendrocytes with myelin basic protein immunoreactivity appeared at stage 38, and were widely distributed at stage 40. Electron microscopic observation confirmed the presence of intermediate filaments in the glial fibers and developing myelin at stages 40-41. The present results suggest that oligodendrocyte precursors undergo terminal differentiation slightly earlier than astrocyte precursors with respect to the expression of marker proteins.

Animals↗

Feedback controlled nerve fiber impalement.

A device has been described for the automatic impalement of nerve fibers. Although developed specifically for that task, the technique can be applied for intracellular recording from other cells or processes. The basis of the techniques is to use the cell membrane potential to stop the advance of a fast moving microelectrode. With slight modification it can be used to stop microdrive advance upon receipt of an action potential. The circuit was constructed by interconnecting commercially available instruments found in most electrophysiology laboratories. Use of the device for recording from the dorsal column fibers of the cat increased the number of stable cell penetrations per unit of time by between one and two orders of magnitude.

Animals↗

Expression of gap junction genes, connexin40 and connexin43, during fetal mouse development.

The expression patterns of the gap junction genes connexin40 and connexin43 have been analyzed during late mouse fetal development, i.e., at embryonic days 14.5 and 16.5, by in situ hybridization and immunofluorescence. Connexin40 was found in endothelial cells of vessels, cardiomyocytes and in developing myoblasts and myotubes. Expression of connexin40 in developing muscle fibers was strong in the back muscles and weaker in the muscles of the limbs. The number of labeled cells in the back muscle decreased with ongoing differentiation of myoblasts, in accordance with the idea that connexin40 is only expressed in the early stages of muscle cell differentiation. Within a muscle bundle, connexin40 expression was predominantly found at the outermost side where myoblasts fuse to multinucleated myotubes. In contrast, connexin43 exhibits a wide and complex pattern of expression in fetal mouse development. It is found in organs originating from all three germ layers, such as epidermis, heart, lung, muscle, kidney and gut. Connexin43 transcript and protein were very abundant in tissues that had been undergoing inductive interactions, e.g., the inner enamel epithelium of the teeth, the glomeruli of the kidneys and the infundibulum forming the neural part of the pituitary gland. Very high connexin43 expression was found in the embryonic meninges (dura mater) and in the fetal adrenal cortex. During keratinocyte differentiation connexin43 mRNA expression decreased, being much stronger in the stratum basale than in stratum granulosum. No obvious discrepancy between the amount of mRNA and protein of either connexin was noticed, suggesting that there is no specific translational regulation at these developmental stages.

Animals↗

Reversibility of electrophysiologic abnormalities of subendocardial Purkinje fibers induced by ischemia.

INTRODUCTION: During the subacute phase of infarction in the canine heart, the subendocardial Purkinje fibers subtended by the infarct show depolarization greater than can be accounted for by the decrease in [K+]i, and generate abnormal action potentials and spontaneous rhythms due to abnormal automaticity. We have used pinacidil to hyperpolarize these fibers and evaluate the extent to which an increase in resting potential can normalize action potential generation. METHODS AND RESULTS: Twenty-four hours after two-stage ligation of the canine left anterior descending coronary artery, preparations of subendocardial Purkinje fibers were studied in vitro by recording transmembrane potentials through standard microelectrodes and exposing the preparation to pinacidil and increases in [K+]o. Pinacidil increased resting potential to the estimated value of EK, abolished the abnormal automaticity, and restored action potentials of normal amplitude with normal values of Vmax. This effect often persisted after washout of pinacidil. Elevation of [K+]o from 4.0 to 20.0 mM slightly increased maximum diastolic potential, suggesting that the excess (over the change in EK) depolarization was caused by a decrease in gK1. CONCLUSION: The ventricular arrhythmias seen during the subacute stage of infarction probably are caused by abnormal automaticity. Our findings support the conclusion that this abnormal automaticity arises in partially depolarized subendocardial Purkinje fibers. This loss of resting potential is due in large part to a decrease in gK1. Restoration of resting potential to the value of EK permits the Purkinje fibers to develop essentially normal action potentials. An agent capable of reversing the partial block of IK,1 thus might be an effective drug for some types of arrhythmias.

Adenosine Triphosphate↗

[Secondary development of minced muscle tissue of amphibia during transplantation and cultivation in diffusion chambers].

The development of the skeletal muscular tissue from the frog Rana ridibunda was studied under autotransplantation and cultivation in diffusion chambers of the minced gastrocnemius muscle implanted intraperitoneally to other species of the same family. The material was examined by light or scanning electron microscopy. In both series of experiments, the muscular tissue from adult frogs retained its viability and reconstructive activity for a long time. The muscle material underwent reconstruction starting from myoblast formation by means of their separation and migration from disintegrating muscle fibers. The reconstruction eventuated in the formation of differentiated muscle fibers. The development of the muscular tissue proceeded more rapidly during cultivation in diffusion chambers under homo-implantation as compared with the process occurring in the minced muscle autotransplant.

Animals↗

Hairs from patients with maple syrup urine disease show a structural defect in the fiber cuticle.

Models for the surface of cuticle cells in hair fibers consist of a monolayer of fatty acids covalently bound to the underlying protein membrane by thioester linkages. The most prominent of these fatty acids is 18-methyleicosanoic acid (C21a), the synthesis of which requires the oxidative decarboxylation of isoleucine. Maple syrup urine disease (MSUD) is caused by an inherited deficiency in the enzyme branched chain 2-oxo acid dehydrogenase, which leads to the accumulation of branched chain alpha-keto-acids derived from the amino acids, leucine, isoleucine, and valine. Transmission electron microscopy studies of developing hair fibers show a structural defect in the fiber shaft in hair from patients with MSUD. This defect is confined to the cuticle of the fiber, where the cuticle membrane directly apposes the intercellular material. Thus, the defect indicates that C21a is located exclusively on the upper surface of fiber cuticle cells. Lipid analysis of MSUD hairs has demonstrated significant changes in the relative abundance of the covalently bound fatty acids and an almost complete absence of C21a, whereas there was little difference in the amino acid composition compared with normal hair. These results provide further evidence for the existence of the surface lipid monolayer and its crucial role in cellular adhesion.

Amino Acids↗

Possible electrophysiological mechanisms for lethal arrhythmias accompanying myocardial ischemia and infarction.

Ventricular arrhythmias are probably the cause of sudden death due to myocardial ischemia or infarction. Such arrhythmias result from alterations in the transmembrane potentials of ventricular muscle and Purkinje fibers. Most of our knowledge concerning mechanisms for these arrhythmias has been derived from experimental studies on the canine heart. In this model, the early ventricular arrhythmias which occur within minutes after coronary artery occlusion probably have different electrophysiological mechanisms than the late arrhythmias which occur several or more hours after the onset of ischemia. The early arrhythmias probably result from reentry in ventricular muscle due to slow conduction in ischemic or infarcting muscle cells. The later arrhythmias most likely arise in subendocardial Purkinje fibers which develop spontaneous diastolic depolarization and abnormally prolonged action potential duration. It is important for future investigations to determine the relevance of electrophysiological studies on the canine heart to human ischemic and infarction arrhythmias.

Action Potentials↗

An embryological study of fetal development of the rectourethralis muscle--does it really exist?

PURPOSE: The so-called rectourethralis muscle is widely described in the urological literature. However, its description is subject to variations concerning extent and morphology. Moreover, little is known about its fetal development, which would allow a better understanding of the adult anatomy. The aim of this study was to investigate the morphology, extent and development of the rectourethralis muscle in fetal specimens. MATERIALS AND METHODS: A total of 15 normal human male fetuses were included in the study. Age ranged from 13th week of gestation to term. The histological study used plastination and standard and immunohistochemical techniques to identify the developing muscular structures in serial sections of the rectourethral space. Investigations in all 3 planes (coronal, sagittal and transverse) were performed. RESULTS: In all sections of the proximal aspect of the rectourethral space no developing muscle fibers could be identified leaving the anterior wall of the rectum to join the rhabdosphincter or the apical prostate. In the distal aspect of the rectourethral space an independent muscular structure located between the external muscular layer of the anorectal canal and the dorsal bundles of the rhabdosphincter could be clearly demonstrated. This structure consisted of connective, smooth and striated muscle tissue, and was deeply anchored to the apical perineal body. CONCLUSIONS: This study suggests that the so-called rectourethralis muscle is a misnomer in the urological literature. In the fetal period a developing muscular structure could be clearly identified as a part of the apical perineal body, which would correspond to the rectoperinealis muscle in the adult.

Humans↗

[Prevention of postherpetic neuralgia: does it exist?].

THEORETICAL CONSIDERATIONS: Several pathophysiological mechanisms may be responsible for initiation and maintenance of chronic postherpetic pain. (1) Peripheral nociceptive fibers can develop abnormal sensitization. Secondary to this, central nociceptive "second-order" neurons in the spinal cord dorsal horn can also be sensitized, i.e. they become hyperexcitable and start responding to non-noxious stimuli. (2) Degeneration of nociceptive neurons may trigger anatomical sprouting of low-threshold mechanosensitive terminals to form connections with central nociceptive neurons and may subsequently induce functional synaptic reorganization in the dorsal horn. According to these mechanisms theoretical possibilities of therapeutical interventions to prevent postherpetic neuralgia are (1) adequate analgesia in the acute phase (analgesics, antidepressants, sympathetic blocks) and (2) prevention of C-fiber degeneration by reducing the inflammatory reaction (antiviral drugs, corticosteroids, neurotrophins). CLINICAL EXPERIENCE: The present clinical trials concerning prevention of postherpetic neuralgia were analyzed. Only for the antiviral drugs have valid clinical studies been performed. Using acyclovir a considerable reduction of acute pain and time to healing could clearly be demonstrated. However, there was no significant effect on prevention of postherpetic neuralgia. The second-generation antiviral drugs valaciclovir and famciclovir may be more effective when applied early in the disease course. Preliminary studies indicate that the early onset of therapy with the antidepressant agent amitriptyline may reduce the incidence of postherpetic neuralgia. These results need to be confirmed in further studies. CONCLUSION: Although there is no clear evidence in favor of a prevention of postherpetic neuralgia for any of the interventions, it is definitely reasonable to perform the best analgesia possible during the acute phase of herpes zoster.

English Abstract↗

Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.

Animals↗

The expression of myocilin during murine eye development.

PURPOSE: To study the expression and localization of myocilin in the developing mouse eye. Myocilin is a 55- to 57-kDa secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma. METHODS: The eyes of NMRI mice were studied from embryonic day (E) 14.5 to postnatal day (P) 21, and at 2-3 months of age. Immunohistochemistry was performed with antibodies against myocilin. The specificity of the antibodies was checked by two-dimensional gel electrophoresis. RNA was isolated from eyes at various ages, and the presence of myocilin mRNA was analyzed by northern blot hybridization. RESULTS: No immunostaining for myocilin was seen before E16.5. At around E17.5, a distinct positive immunoreactivity of optic nerve axons in the developing nerve fiber layer of the retina was observed. At P5-6, immunostaining appeared in perikarya of optic nerve ganglion cells. In the anterior eye, no immunoreactivity was observed until P10. At P12-14, the cells of the epithelial layers of ciliary body and iris, as well as the cells of the trabecular meshwork and iris stroma, became immunoreactive for myocilin. At that time, positive staining for myocilin was also seen in the corneal endothelium and in keratocytes of the corneal stroma. An essentially similar staining pattern was seen in adult eyes. Northern blot analysis for myocilin mRNA in RNA from developing mouse eyes was negative until P9. At P12, a distinct band was observed. A band with similar mobility, but somewhat more intense, was detected in mRNA from adult mouse eyes 2-3 months of age. CONCLUSIONS: The onset of immunoreactivity for myocilin in the retina occurs in parallel with the maturation of optic nerve ganglion cells. In the anterior eye, the expression of myocilin is associated with the final development of those tissues that are directly involved in aqueous humor dynamics. The presence of myocilin might be important for proper function and structure of mature optic nerve ganglion cells and aqueous humor outflow.

Animals↗

Development of junctions during differentiation of lens fibers.

Throughout the differentiation of eye lens epithelium into fibers, an extensive system of intercellular junctions develops. The junctional assembly is initially characterized by the accumulation of 9.0-nm intramembranous particles, forming linear rows in the matching plasma membranes of adjoining fibers. At the final stage of the fiber differentiation, the junctional particles are assembled in geometrically packed arrays. The formation of linear rows and bidimensional lattices of intramembranous particles probably favors reciprocal recognition of cell surfaces and specific cell-to-cell interlocking. Moreover, the existence of a rather rigid lipid core of the plasma membrane of eye lens fiber may promote the clustered distribution of intramembranous particles and facilitate the junctional assembly.

Animals↗

The rostrocaudal organization in the dorsal root ganglia of the rat: a consequence of plexus formation?

The dorsal root ganglia (DRGs) of the rat have a rostrocaudal organization. This organization can most easily be demonstrated in fetal and neonatal rats because the spatial relationships of their DRGs are maintained better in tissue sections than those of mature rats. This review is concerned with the way in which the rostrocaudal organization of the DRGs is generated. Wheat germ agglutinin--horseradish peroxidase/horseradish peroxidase labeling of peripheral nerves of the brachial and lumbar plexuses shows that the position of the somata of the sensory neurons of the labeled nerves can be restricted to rostral or caudal halves of DRGs. Labeling of the thoracic nerve or its branches always results in labeling throughout the entire thoracic DRG. After application of the marker to forelimb nerves, it was observed that whenever a DRG is labeled only partially, its spinal nerve is correspondingly labeled partially as well. These data suggest that the rostrocaudal organization in the DRG is related to the formation of the plexuses. During development nerve fibers can be segmentally labeled, using the subdivision of the DRGs into a rostral and a caudal half to keep together as they find their way through the plexus. Application of label to forelimb skin, hindlimb skin and even thoracic skin can result in labeling of rostral or caudal halves of a DRG. A possible explanation might be that each dermatome can be divided into a skin area innervated by the rostral half of a DRG and a skin area innervated by the caudal half of the same dorsal root ganglion. In the rat, the segmental sensory innervation of muscles during development has not yet been investigated. The question of whether the segmental unit of innervation of a muscle is a whole DRG or half a DRG therefore still remains unanswered.

Animals↗

Development of histamine-immunoreactive neurons in the rat brain.

This study was undertaken to reveal the cellular stores of histamine in developing rat brain and to determine the stage of development during which the histamine-immunoreactive neurons can first be detected. Rats from embryonal day 12 to postnatal day 14 were studied. The brains were fixed in 4% 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and standard immunofluorescence technique was used. The first histamine-immunoreactive neurons were seen on embryonic day 13 in the border of mesencephalon and metencephalon. On embryonic day 15 immunoreactive neurons were detected in ventral mesencephalon and rhombencephalon. In caudal, tuberal, and postmammillary caudal magnocellular nuclei histamine-immunoreactive neurons were first detected on embryonic day 20 while those in the hindbrain had disappeared. Histamine-immunoreactive nerve fibers were first detected on embryonic day 15 in rhombencephalon and mesencephalon and in some areas of diencephalon including the mammillary bodies and frontal cortex. On embryonic day 18 the number of immunoreactive nerve fibers in the hindbrain had decreased considerably, but the olfactory bulb, septal and hypothalamic area, and the cerebral cortex showed immunoreaction in fibers. The density of histamine-immunoreactive fiber networks increased until postnatal day 14 when an adultlike pattern of neurons and fibers had developed. Histamine-immunoreactive neurons are present in embryonal CNS and they develop extensive projections to various brain areas.

Aging↗