[The neural substrate of biological rhythms]. / Sustrato neural de los ritmos biológicos.

INTRODUCTION AND METHOD: The various bodily functions vary rhythmically along the 24 hour cycle. These circadian rhythms, display common properties and are generated by common cellular and molecular mechanisms. The basic rhythm is endogenously produced in cellular pacemakers in various regions; the most conspicuous are located in the central nervous system. They are composed by cells endowed with the molecular substrate necessary to generate rhythmicity, and to send the circadian time signal to the effectors of the overt rhythms. The rhythmicity in the pacemaker cells is generated by specific genes integrated in a negative feedback double loop. The structure of the genes responsible for the rhythmicity is phylogenetically well preserved. The circadian pacemakers are in turn synchronized by environmental stimuli, being light the best characterized, but other external agents, such as food and some homeostatic factors and products of metabolic activity are also effective. These synchronizing signals are conveyed to the pacemakers via specific receptors and pathways. The role of some neurotransmitters in the synchronizing action has been demonstrated. CONCLUSION: The functional integrity of this complex biochronometrical system is necessary for the maintenance of health. Its alteration results in specific ailments, and enhances the vulnerability to certain diseases

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii.

The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH(2)) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10(-10) to 10(-5)M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.

Red pigment-concentrating hormone induces a calcium-mediated retraction of distal retinal pigments in the crayfish.

The octapeptide red pigment-concentrating hormone is capable of eliciting the aggregation of intracellular pigment granules in distal retinal pigment cells of isolated retinas of the crayfish Procambarus clarkii (Girard). The final level and the time course of pigment aggregation are dose dependent within a range of 10(-10) mol l(-1) to 10(-4) mol l(-1). The effect of red pigment-concentrating hormone is prevented by previous incubation with an anti- red pigment-concentrating hormone antibody; however, application of the antibody after the onset of the red pigment-concentrating hormone effect, does not prevent its full development. A similar effect to that elicited by red pigment-concentrating hormone is induced by the calcium ionophores ionomycin and A-23187. Red pigment-concentrating hormone evokes entry of 45Ca2+ to retinal cells. However, the red pigment-concentrating hormone-induced pigment aggregation persists in the presence of the calcium channel blocker verapamil and in Ca2+-free solutions. Caffeine and thapsigargin, known to release calcium from intracellular stores, elicit distal pigment aggregation, while ryanodine and dantrolene, blockers of intracellular calcium release, as well as the intracellular calcium chelator bapta-AM suppress the effect of red pigment-concentrating hormone. These results suggest that red pigment-concentrating hormone elicits distal retinal pigment aggregation by increasing intracellular calcium concentration, acting via a dual mechanism: (1) promoting calcium entry, and (2) releasing intracellular calcium.

[Bioethics and scientific training of physicians]. / La bioética y la formación científica del médico.

Bioethics is becoming a major current in modern medical thought and action. Given the youth of this field of enquiry, there are still important debates and controversies on its proper role in medicine and in medical education, but the need to foster its integral incorporation into the formative process of the physician is unreservedly accepted, on an equal footing with the scientific, technical, and humanistic components of medical training.

The brain decade in debate: IV. Chronobiology.

The present article is the adapted version of an electronic symposium organized by the Brazilian Society of Neuroscience and Behavior (SBNeC) which took place on June 14, 2000. The text is divided into three sections: I. The main issues, II. Chronodrugs, and III. Methods. The first section is dedicated to the perspectives of chronobiology for the next decade, with opinions about the trends of future research being emitted and discussed. The second section deals mostly with drugs acting or potentially acting on the organism's timing systems. In the third section there are considerations about relevant methodological issues concerning data analysis.

The brain decade in debate: IV. Chronobiology

The present article is the adapted version of an electronic symposium organized by the Brazilian Society of Neuroscience and Behavior (SBNeC) which took place on June 14, 2000. The text is divided into three sections: I. The main issues, II. Chronodrugs, and III. Methods. The first section is dedicated to the perspectives of chronobiology for the next decade, with opinions about the trends of future research being emitted and discussed. The second section deals mostly with drugs acting or potentially acting on the organism's timing systems. In the third section there are considerations about relevant methodological issues concerning data analysis

Serotonin activates a Ca(2+)-dependent K(+) current in identified peptidergic neurons from the crayfish.

The effects of 5-hydroxytryptamine (5-HT) were investigated in red pigment concentrating hormone (RPCH)-containing neurons isolated from the X-organ of the crayfish (Procambarus clarkii). Under current-clamp conditions and using the gramicidin-perforated-patch configuration, 5-HT elicited a prolonged hyperpolarization that suppressed neuronal firing concomitant with an increase in membrane conductance. Under voltage-clamp conditions, 5-HT evoked an outward current at a holding potential of -50 mV. This current reversed at an E(K) of -90 mV, which shifted by 30 mV when the extracellular K(+) concentration was increased from 5.4 to 19 mmol l(-1). The effect of 5-HT was dose-dependent within the range 1-100 micromol l(-1) and followed simple Michaelis-Menten kinetics, with a half-maximal response being elicited at 10 micromol l(-1). Preincubation with charybdotoxin (100 nmol l(-1)), tetraethylammonium (500 micromol l(-1)) or methysergide (100 micromol l(-1)) was effective in blocking the response to 5-HT. These results suggest that 5-HT is an inhibitory mediator of the release of red pigment concentrating hormone by acting on a Ca(2+)-dependent K(+) current.

Circadian clock function in isolated eyestalk tissue of crayfish.

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

[Chronobiological profile of arterial blood pressure and heart rate in a family group determined by automatic monitoring]. / El perfil cronobiológico de tensión arterial y de frecuencia cardiaca en un grupo familiar, determinado mediante monitorizacion automática.

Over a week, blood pressure (systolic, mean and diastolic) and heart rate were determined in a family, by means of automatic, uninterrupted monitoring. The chronobiological profile for each family member was prepared in time series of various periodicities. An ample circadian component and a lesser circaseptan component were apparent. Clear phase differences were identified among the four cardiovascular variables studied. The chronobiological profile of the children was closer to that of the father than to that of the mother.

Regulation of crustacean neurosecretory cell activity.

1. The X organ-sinus gland system is a conglomerate of 150-200 neurosecretory cells in the eyestalk of crustaceans. It is the source of a host of peptide neurohormones which partake in the control of a wide range of physiological functions. Distinct families of X organ peptides have been chemically characterized: (a) two chromatophorotropic hormones of small sizes, one of 8 residues and the other of 15-20 residues; and (b) three metabotropic hormones of high molecular weight (70-80 residues), related to the control of blood sugar levels, molting, and gonad activity. Some of these hormones have been identified only in crustaceans; others are common to various arthropod groups. A number of peptides orginally described in other zoological groups are also present in the X organ-sinus gland system; such is the case for members of the FMRF-amide family, enkephalins, and other peptides. 2. Cells specifically containing each hormone have been located in the X organ and some information is available on the cellular and molecular substrate of the biosynthesis, transport, storage, and release of various hormones. The electrical activity of X organ neurons has been recorded at the cell soma, arborizations, axons, and neurosecretory terminals. Conspicuous regional differences have been defined for the various patterns of activity, as well as the distribution of their underlying ion currents. 3. The release of hormones and the electrical activity of X organ neurons are regulated by environmental and endogenous influences, such as light and darkness, stress, and circadian rhythms. These influences appear to be mediated by a host of neurotransmitters/modulators, most noticeably, gamma-aminobutyric acid, 5-hydroxytryptamine and other amines, and enkephalins. Each of these mediators acts upon a definite ionic substrate(s) and exerts specific regulatory effects on X organ cell activity. A given neuron may be under the control of more than one neurotransmitter, and a transmitter may mediate different and even opposite influences on different neurons.

Seasonal rhythm of red pigment concentrating hormone in the crayfish.

The content of red pigment concentrating hormone (RPCH) in the eye-stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The responsiveness of tegumentary chromatophores to synthetic RPCH varies concurrently.

[Chronobiology in medicine and surgery]. / Cronobiología en medicina y cirugía.

Chronobiology studies predictable variations, rhythms and trends in all forms of life. Each physiologic variable or system in an organism has a time structure which is genetically anchored and capable of being expressed in the absence of external cycles. However, it is normally adjusted by geophysical changes. Chronobiology quantifies rhythms by providing measures of amplitude and timing (phase and period), and a mean more precise than the arithmetic mean. The correct measurement of biological rhythms has led to considerable advances in the understanding of their underlying mechanisms of generation and expression. The use of information on rhythms is opening new opportunities in the prevention, diagnosis and treatment of disease. Chronobiology is also effective in the improvement of the quality of life by teaching self-help to the healthy individual.

Ultrastructural features of neurosecretory cells in the medulla externa of the crayfish eyestalk.

A conglomerate of 8-12 neurons in the medulla externa of the crayfish eyestalk was explored in their reaction to a polyclonal antibody against the tyrosinated octapeptide Red Pigment Concentrating Hormone (Tyr-RPCH). These are large neurons with diameters within a range of 33-43 microns and they were all positively stained with neutral red. By intracellular staining with lucifer yellow, the neurons were found to branch extensively within the medulla externa and the lamina ganglionaris of the eyestalk. Each neurite bifurcates at about 40 microns from the soma. Both branches run to the medial edge of the eyestalk; one proceeds distally to the lamina ganglionaris, while the other runs proximally to the medulla interna. Both end freely in multiple arborizations, covering from the medial to the lateral edges of the eyestalk. No branches were found to the sinus gland, the main neurohaemal organ of the eyestalk. A group of 4 neurons in the conglomerate consistently rendered positive reaction to the anti-Tyr-RPCH antibody (A-RPCH). They are superficially located in the cluster, and at the electron microscope, they showed the usual features of a secretory cell, i.e., clear and dense granules, an active and well-developed Golgi apparatus, and rough endoplasmic reticulum. The dense granules were larger (mean diameter: 101.5 nm) than the clear granules (mean diameter: 90.3 nm). The immunopositive reaction at the electron microscope was found to be largely confined to the dense-cored granules.

Regional distribution and immunocytological localization of red pigment concentrating hormone in the crayfish eyestalk.

A polyclonal antibody was raised against synthetic tyrosinated crustacean red pigment concentrating hormone (RPCH-Tyr) with the sequence Tyr-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2 with a tryptophan amide at the carboxyl terminal end. Its specificity was tested in comparison with peptides of similar structure. It appears to recognize the three to five residues near the carboxyl terminal. Native RPCH in the crayfish eyestalk was determined by two methods: (a) immunoenzymatic assay (ELISA) using the aforementioned antibody; and (b) bioassay on segments of isolated crayfish tegumentary epithelium. The unitary content in whole eyestalks was 5.5 +/- 1.0 nmol for samples (n = 18) taken at night. The regional distribution of RPCH content in the eyestalk was determined. The greatest proportion (40%) was found in the sinus gland, and the lowest in the retina plus lamina ganglionaris (6%). The medulla interna, medulla externa, and medulla terminalis contained similar proportions (about 16% each). The highest specific content was in the sinus gland (65.0 vs 24.4 pmol/micrograms protein for the whole eyestalk). Immunopositive neurons were identified in the various regions of the eyestalk. In 22 preparations, an average of 7 cells were identified in the ventromedial rim of the medulla terminalis, sending axons to the sinus gland, after branching in the neuropil of the medulla terminalis. Dorsally, 2 cells were identified in the medulla interna and 4 large cells and 11 small cells were located in the medulla externa in close proximity to the lamina ganglionaris: none of these cells appeared to project to the sinus gland. Profuse immunopositive fibers were found in the lamina ganglionaris projecting distally toward the base of the retina. Immunopositive axons were also found in the optic nerve.

Excitatory action of gamma-aminobutyric acid (GABA) on crustacean neurosecretory cells.

1. Intracellular and voltage-clamp recordings were obtained from a selected population of neurosecretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of gamma-aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 microM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity. 2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 microns from the cell body. Responses were larger when the application was made at the neuropil level. 3. Topical application of Cd2+ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA. 4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a -50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells. 5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10(-6) M [Ca2+]i, GABA (50 microM) increased the membrane conductance more than threefold and shifted the zero-current potential from -25 to -10 mV. At 10(-9) M [Ca2+]i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential. 6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.

Circadian rhythms.

The neurobiological substratum of circadian rhythmicity encompasses three levels of integration: firstly, generation of time signals by circadian pacemakers; secondly, entrainment of pacemakers by environmental influences; thirdly, coupling of circadian pacemakers among themselves and with target systems responsible for the expression of overt rhythms. From recent contributions, the notion that circadian organization results from the interaction of independent oscillators and pathways has been strengthened. In addition, recent evidence supports the existence of circadian rhythmicity in single isolated neurons. New information was produced on the gene control of circadian rhythm generation in Drosophila, as well as interesting advances in the understanding of neuronal mechanisms involved in the generation, entrainment and coupling of circadian rhythms in various species.

Morphological and biochemical characterization of Procambarus clarki blood cells.

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40-0.78 micron). Granular cell contained large eosinophilic granules (1.79-3.05 microns). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Prophenoloxidase system activation in the crayfish Procambarus clarki.

The prophenoloxidase system (proPO) was studied in primary cultures of hemocytes of the crayfish Procambarus clarki. Both zymosan and lipopolysaccharide (LPS) separately induced rapid degranulation and lysis of semigranular hemocytes, with concurrent release of proPO. ProPO could be demonstrated in the hemocyte lysate supernatant (HLS) obtained by a freeze/thaw method, and was specifically activated by LPS and zymosan. Phenoloxidase activity was blocked by serine protease inhibitors, such as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-sulphonylfluoride (PMSF), and substantially increased by cysteine protease inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activated. Incubation without activators or preincubation with STI prevented the induced enhancement. Electrophoretic analyses of HLS treated with zymosan or LPS showed that three bands at 41, 39, and 37 kDa were specifically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO system in P. clarki, and a mechanism susceptible to cysteine protease inhibitors could be related to its regulation.