Identification of medullary loci critical for neurogenesis of gasping

We hypothesized that a discrete medullary locus, critical for gasping neurogenesis, could be identified. In decerebrate, cerebellectomized, vagotomized, paralyzed, and ventilated cats, activities of phrenic, hypoglossal, and recurrent laryngeal nerves were monitored. Gasping was induced by freezing the brain stem, via a fork thermode, at the pontomedullary junction. By reversible cooling of the medulla, chemical lesions with kainic acid, and radio-frequency lesions, a critical area for gasping neurogenesis was localized bilaterally 2-3 mm rostral to obex, 2.0-2.5 mm lateral to midline, and 3-4 mm ventral to medullary surface. Electrical stimulation in this area elicited premature gasps, whereas unilateral lesions or lidocaine injections eliminated gasping activities in all nerves. These procedures did not cause similar changes during eupnea. In apneusis, however, lidocaine injections markedly altered the pattern or caused apnea. We conclude that discharge of neurons in a discrete portion of the lateral tegmental field of medulla is required for gasping neurogenesis. Our results are consistent with these neurons comprising the central pattern generator for gasping.     

Differing activities of medullary respiratory neurons in eupnea and gasping

Our purpose was to compare further eupneic ventilatory activity with that of gasping. Decerebrate, paralyzed, and ventilated cats were used; the vagi were sectioned within the thorax caudal to the laryngeal branches. Activities of the phrenic nerve and medullary respiratory neurons were recorded. Antidromic invasion was used to define bulbospinal, laryngeal, or not antidromically activated units. The ventilatory pattern was reversibly altered to gasping by exposure to 1% carbon monoxide in air. In eupnea, activities of inspiratory neurons commenced at various times during inspiration, and for most the discharge frequency gradually increased. In gasping, the peak discharge frequency of inspiratory neurons was unaltered. However, all commenced activities at the start of the phrenic burst and reached peak discharge almost immediately. The discharge frequencies of all groups of expiratory neurons fell in gasping, with many neurons ceasing activity entirely. These data are consistent with the hypothesis that brain stem mechanisms controlling eupnea and gasping differ fundamentally.     

Afferent mechanisms underlying stimulation modality-related modulation of acupuncture-related cardiovascular responses.

Despite the use of acupuncture to treat a number of heart diseases, little is known about the mechanisms that underlie its actions. Therefore, we examined the influence of acupuncture on sympathoexcitatory cardiovascular responses to gastric distension in anesthetized Sprague-Dawley rats. Thirty minutes of low-current, low-frequency, (0.3-0.5 mA, 2 Hz) electroacupuncture (EA), at P 5-6, S 36-37, and H 6-7 overlying the median, deep peroneal, and ulnar nerves significantly decreased reflex pressor responses by 40, 39, and 44%, respectively. In contrast, sham acupuncture involving needle insertion without stimulation at P 5-6 or 30 min of EA at LI 6-7 acupoints overlying the superficial radial nerve did not attenuate the reflex. Similarly, EA at P 5-6 using 40- or 100-Hz stimulation frequencies did not inhibit the reflex. Compared with EA at P 5-6, EA at two sets of acupoints, including P 5-6 and S 36-37, did not lead to larger inhibition of the reflex. Two minutes of manual acupuncture (MA; 2 Hz) at P 5-6 every 10 min for 30 min inhibited the reflex cardiovascular pressor response by 33%, a value not significantly different from 2-Hz EA at P 5-6. Single-unit afferent activity was not different between electrical stimulation (ES) and manual stimulation. However, 2-Hz ES activated more somatic afferents than 10- or 20-Hz ES. These data suggest that, although the location of acupoint stimulation and the frequency of stimulation determine the extent of influence of EA, there is little difference between low-frequency EA and MA at P 5-6. Furthermore, simultaneous stimulation using two acupoints that independently exert strong effects did not lead to an additive or a facilitative interaction. The similarity of the responses to EA and MA and the lack of cardiovascular response to high-frequency EA appear to be largely a function of somatic afferent responses.     

Different mechanisms underlying the stimulation of myocardial alpha- and beta-adrenoceptors.

On isolated electrically driven rat left atria (1 Hz) experiments were undertaken in order to characterize further the mechanism of the positive inotropic effect underlying the stimulation of cardiac α-adrenoceptors.— The increase in extracellular Ca²⁺ from 1.25 to 2.5 mM increased the pD₂-value for the α-mimetic effect of phenylephrine by 0.6 log units. Under the same conditions the pD₂-value for its β-mimetic positive inotropic effect increased by only 0.19 log units.—When the amplitude of contraction was diminished by carbachol which is known to inhibit the influx of Ca²⁺ the pD₂-value for the α-mimetic effect of phenylephrine was significantly increased while that for the β-mimetic effect was decreased.—After increasing concentrations of dinitrophenol the pD₂-value for the α-effect of phenylephrine was significantly increased by maximal 0.6 log units whereas that for the β-effect was lowered by 0.25 log units.—In conclusion, all measures favouring the conditions for an enhanced influx of Ca²⁺-elevation of the gradient for Ca²⁺, shortening of the plateau phase of action potential by carbachol, increased efflux of Ca²⁺ by dinitrophenol- are able to increase the pD₂-value, i.e. the affinity of phenylephrine to cardiac α-adrenoceptors, thus demonstrating the importance of an increased Ca²⁺-influx induced by stimulation of α-adrenoceptors.     

Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.     

Molecular mechanisms of early neurogenesis in vertebrates

Recent studies revealed a great variety of genes which control the early development of the central nervous system in vertebrates, including neural induction and differentiation of primary neurons. Most of these genes were first identified inDrosophila melanogaster, then their structural and functional homologs were found in vertebrates. Modern data on the molecular-genetic mechanisms of vertebrate neurogenesis are reviewed. The neurogenetic mechanisms are compared for vertebrates and invertebrates. Widely discussed hypotheses are considered along with the commonly accepted mechanisms.     

Genetic mechanisms of early neurogenesis inDrosophila melanogaster

The neurogenic ectoderm ofDrosophila melanogaster consists of the ventral neuroectoderm and the procephalic neuroectoderm. It is hypothesized that epidermal and central neural progenitor cells separate from each other in three steps: conference on the neuroectodermal cells the capability of producing neural or epidermal progenies, separation of the two classes of progenitor cells, and specification of particular types of neuroblasts and epidermoblasts. Separation of neuroblasts and epidermoblasts in controlled by proneural and neurogenic genes.Delta andNotch serve as mediators of direct protein-protein interactions. E(spl)-C inhibits neurogenesis, creating epidermal cells. The achaete-scute complex (AS-C) controls the commitment of nonoverlapping populations of neuroblasts and leads the development of neuroectodermal cells as neuroblasts.     

Ionic transport mechanisms underlying fluid secretion by the pancreas

The pancreas is a 'leaky' epithelium and secretes a juice in which sodium and potassium have concentrations similar to those of plasma. The characteristic features of the secretion are its isosmolality and its high bicarbonate concentration. It is the latter that has attracted considerable attention. Secretion in the isolated cat pancreas is directly proportional to the bicarbonate concentration in the nutrient fluid. The ability of the gland to secrete weak acids has led to the view that because of the very different chemical nature of the anions, it is most likely that it is a component common to all buffers, the proton, that is subject to active transport. This is supported by the decrease in pH and the increase in rho CO2 of the venous effluent when secretion occurs and the sensitivity of secretion to the pH of the nutritional extracellular fluid. It is proposed that the cellular mechanisms are as follows: CO2 diffuses into the cell and is hydrated to carbonic acid under the influence of carbonic anhydrase. The bicarbonate ion so formed diffused into the ductular lumen and the proton is transported backwards through the epithelium with a proton pump (Mg2+ -ATPase) provisionally located in the luminal membrane and a hydrogen-sodium exchange carrier located in the basolateral membrane. Energy for the latter process is derived from the sodium gradient between extracellular fluid and cell. This gradient is maintained by a (Na+ + K+)-ATPase also located in the basolateral membrane. Chloride appears to be transported partly through a chloride-bicarbonate exchange mechanism but largely passively together with a large sodium and potassium component through the paracellular pathway. Osmotic equilibrium is likely to occur in the small ductules.     

Molecular mechanisms underlying oxygen reception

In this review, modern concepts on molecular mechanisms underlying reception of the oxygen level in natural O₂-sensory structures and cellular in vitro models are considered and discussed.Neirofiziologiya/Neurophysiology, Vol.36, No.4, pp.330–347, July–August, 2004.     

Improving yield by exploiting mechanisms underlying natural variation of photosynthesis

Increasing photosynthesis in C3 species has been identified as an approach to increase the yield of crop plants. Most of our knowledge of photosynthetic performance has come from studies in which plants were grown in controlled growth conditions but plants in natural environments have to cope with unpredictable and rapidly changing conditions. Plants adapt to the light environment in which they grow and this is demonstrated by the differences in anatomy and morphology of leaves in sun and shade leaves. Superimposed on this are the dynamic responses of plants to rapid changes in the light environment that occur throughout the day. Application of next generation sequencing (NGS), QTL analysis and innovative phenomic screening can provide information to underpin approaches for breeding of higher yielding crop plants.     

Signaling mechanisms underlying dendrite formation

During development, the nervous system is confronted with a problem of enormous complexity; to progress from a large number of 'disconnected' neurons to a network of neuronal circuitry that is able to dynamically process sensory information and generate an appropriate output. To form these circuits, growing axons must make synapses with targets, usually the dendrites of postsynaptic neurons. Although a significant amount is known about the signals that regulate and guide developing axons, we are only now starting to understand how environmental cues like growth factors and activity regulate the formation and maintenance of dendrites in the developing and mature nervous system.     

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.     

Synaptic mechanisms underlying auditory processing

In vivo voltage clamp recordings have provided new insights into the synaptic mechanisms that underlie processing in the primary auditory cortex. Of particular importance are the discoveries that excitatory and inhibitory inputs have similar frequency and intensity tuning, that excitation is followed by inhibition with a short delay, and that the duration of inhibition is briefer than expected. These findings challenge existing models of auditory processing in which broadly tuned lateral inhibition is used to limit excitatory receptive fields and suggest new mechanisms by which inhibition and short term plasticity shape neural responses.