Frequency-specific response facilitation of supra and infragranular barrel cortical neurons depends on NMDA receptor activation in rats.

Sensory experience has a profound effect on neocortical neurons. Passive stimulation of whiskers or sensory deprivation from whiskers can induce long-lasting changes in neuronal responses or modify the receptive field in adult animals. We recorded barrel cortical neurons in urethane-anesthetized rats in layers 2/3 or 5/6 to determine if repetitive stimulation would induce long-lasting response facilitation. Air-puff stimulation (20-ms duration, 40 pulses at 0.5-8Hz) was applied to a single whisker. This repetitive stimulation increased tactile responses in layers 2/3 and 5/6 for 60min. Moreover, the functional coupling (coherence) between the sensory stimulus and the neural response also increased after the repetitive stimulation in neurons showing response facilitation. The long-lasting response facilitation was due to activation of N-methyl-d-aspartate (NMDA) receptors because it was reduced by APV ((2R)-amino-5-phosphonovaleric acid, (2R)-amino-5-phosphonopentanoate) and MK801 application. Inactivation of layer 2/3 also blocked response facilitation in layer 5/6, suggesting that layer 2/3 may be fundamental in this synaptic plasticity processes. Moreover, i.p. injection of eserine augmented the number of layer 2/3 neurons expressing long-lasting response facilitation; this effect was blocked by atropine, suggesting that muscarinic receptor activation favors the induction of the response facilitation. Our data indicate that physiologically repetitive stimulation of a single whisker at the frequency at which rats move their whiskers during exploration of the environment induces long-lasting response facilitation improving sensory processing.

Validation of an original incubator set-up for the exposure of human astrocyte cells to X-band microwaves in a GTEM-chamber.

A current concern about the biological effects of electromagnetic fields (EMF) is increasing with the wide spread use of X-band microwaves (MW, 8-10 GHz range). Gigahertz transverse electromagnetic (GTEM) field flat transmission lines are currently being used for experimental exposure of biological samples to high frequency EMF. Experiments carried out on human cells in culture require optimal growing temperature conditions, i.e. 37 °C, 5% CO2 in a humidified atmosphere. The aim of our work has been: i) to built up an original incubator set-up, the so called GTEM-incubator, for exposure of human cells in culture to MW inside a GTEM-chamber, under optimal growing physical conditions; ii) to make the validation of the GTEM-incubator by growing cell samples inside the non-energized GTEM-chamber (test sample) comparing the results with the ones obtained from cell samples grown inside a standard incubator (control samples). The features for comparison were: cell morphology, expression and distribution of cytoskeleton proteins, genotoxicity, viability and cell cycle progression. Any variation in any of the studied parameters would allow for detecting any possible failure or misconception in our GTEM-incubator working test. The results obtained in control and test incubators showed non-significant differences in the development of both cell populations for any of the studied parameters. Thereby our GTEM-incubator is considered valid for our purposes of human cell exposures to X-band MW.

Exposure to ELF-pulse modulated X band microwaves increases in vitro human astrocytoma cell proliferation.

Common concern about the biological effects of electromagnetic fields (EMF) is increasing with the expansion of X-band microwaves (MW). The purpose of our work was to determine whether exposure to MW pulses in this range can induce toxic effects on human astrocytoma cells. Cultured astrocytoma cells (Clonetics line 1321N1) were submitted to 9.6 GHz carrier, 90% amplitude modulated by extremely low frequency (ELF)-EMF pulses inside a Gigahertz Transversal Electromagnetic Mode cell (GTEM-cell). Astrocytoma cultures were maintained inside a GTEM-incubator in standard culture conditions at 37+/-0.1 degrees C, 5% CO2, in a humidified atmosphere. Two experimental conditions were applied with field parameters respectively of: PW 100-120 ns; PRF 100-800 Hz; PRI 10-1.25 ms; power 0.34-0.60 mW; electric field strength 1.25-1.64 V/m; magnetic field peak amplitude 41.4-54.6 microOe. SAR was calculated to be 4.0 x 10-4 W/Kg. Astrocytoma samples were grown in a standard incubator. Reaching 70-80% confluence, cells were transferred to a GTEM-incubator. Experimental procedure included exposed human astrocytoma cells to MW for 15, 30, 60 min and 24 h and unexposed sham-control samples. Double blind method was applied. Our results showed that cytoskeleton proteins, cell morphology and viability were not modified. Statistically significant results showed increased cell proliferation rate under 24h MW exposure. Hsp-70 and Bcl-2 antiapoptotic proteins were observed in control and treated samples, while an increased expression of connexin 43 proteins was found in exposed samples. The implication of these results on increased proliferation is the subject of our current research.

Development of the dynamic structure (force lines) of the middle ear ossicles in human foetuses.

OBJECTIVES: To study the ontogenic development of the organisation of the human middle ear ossicles structure. MATERIAL AND METHODS: 46 human temporal bones of ages varying from 32 days post-conception to newborns. RESULTS: The development of the structural organisation of the malleus begins at 16 weeks via two cortical fascicles situated in the neck; at 21 weeks they extend towards the head, at 23 weeks to the lateral process and at 24 weeks to the handle. In the handle, the force lines are transmitted via three cardinal fascicles, two of them via the cortical fascicle and one via the centre, which starts after 29 weeks' development and is consolidated after 31 weeks. In the incus the force lines start at 16 weeks via two cortical fascicles situated in the long process, which progressively extend in a rostro-caudal direction between 17 and 20 weeks. At 21 weeks they occupy the whole extension of the long process and at 22 weeks the fusion of both cortical fascicles begins. From 30 weeks onwards it is strengthened by the crossing of bone trabeculae from one cortical to another. Two fascicles come out of the incus body, surrounding the medullary cavity and going in the direction of the short process. In the beginning, the stapes have two cortical fascicles in their crura. The remodelling process makes the internal cortical fascicle disappear and after 31 weeks all the force lines run through the external cortical fascicle. The tympanic membrane of the stapes footplate undergoes a remodelling process and after 28 weeks bony trabeculae are deposited. In newborns (40 weeks), the ossicles' structure is cavitary and has not been completed. The fan-shaped trabecular fascicle, which starts in the articular facets of the malleus and the incus, still has to develop.

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.

Test ergoespirométrico en el caballo pura raza español / No disponible

No disponible

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry.

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

Influence of high-carbohydrate enriched diets on plasma insulin levels and insulin and IGF-I receptors in trout.

Insulin and insulin growth factor-I (IGF-I) binding to skeletal muscle semipurified receptors were assessed in rainbow trout (Oncorhynchus mykiss) fed with different enriched carbohydrate diets. The animals were fed for 2 months, either in spring, summer or autumn with a control diet (C, commercial diet containing 21% raw carbohydrates) or with two diets supplied with highly digestible carbohydrates (E1, 22% expanded wheat; and E2, 37% expanded wheat). Insulin and IGF-I receptors were semipurified by affinity chromatography (WGA-agarose). Fish fed with a carbohydrate enriched diet did not show lower growth rates than those fed with the control diet. Independently of the season, rainbow trout fed E1 and E2 presented higher insulin and glucose plasma levels as well as higher tissue glycogen reserves than fish fed C. An increase in the number of insulin receptors during the diet adaptation was observed especially in fish fed with E2. No differences in the affinity of receptors were observed. IGF-I specific binding in skeletal muscle was higher than that of insulin in all groups and in all seasons. Furthermore, IGF-I receptors showed the same tendency as insulin receptors, with increases in their number in experimentally fed fish, especially those fed with E2. Insulin and IGF-I receptors TKA increased only slightly, as a consequence of E1 and E2 diet adaptation. In conclusion, rainbow trout can be fed high-carbohydrate levels and show good rates of growth. This adaptation determines increases in circulating glucose and insulin, and muscle insulin receptors, which indicate an adaptation of the fish to higher levels of glucose supply. The response of IGF-I receptors also suggests a possible role in the regulation of metabolism.

Surface plasmon resonance analysis of gp17, a natural CD4 ligand from human seminal plasma inhibiting human immunodeficiency virus type-1 gp120-mediated syncytium formation.

We have previously isolated from human seminal plasma a CD4 ligand, the gp17 glycoprotein, which shares sequence identity with three previously identified proteins: secretory actin-binding protein (SABP) from seminal plasma, gross-cystic-disease fluid protein-15 (GCDFP-15) and prolactin-inducible protein (PIP) from breast tumor cells. Functions of these glycoproteins are unknown. To further characterize the physical interaction between gp17 and CD4 we used surface plasmon resonance and demonstrated that gp17-CD4 binding affinity is high. Competition experiments indicated that gp17 interferes with human immunodeficiency virus (HIV) envelope protein/CD4 binding, although it binds to a site distinct from but close to the gp120-binding site. We observed moreover that gp17 inhibits syncytium formation between transfected cells expressing the wild-type HIV-1 envelope glycoprotein and CD4, respectively. Our results suggest that gp17, which may function as an immunomodulatory CD4-binding factor playing a role at insemination, may also play a role in controlling HIV spread in the sexual tract.

Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels.

Insulin and IGF-I binding to semi-purified red muscle receptors was characterized in brown trout, Salmo trutta and the common carp. Cyprinus carpio. The yield of glycoprotein obtained after semipurification of receptors with WGA-agarose affinity chromatography in microgram g-1 initial tissue was 210.6 +/- 21 micrograms g-1 in trout and 108.5 +/- 2.5 micrograms g-1 in carp. IGF-I specific binding (4.72 +/- 0.64%/10 micrograms glycoprotein) was 4-5-times higher than insulin binding (1.04 +/- 0.12%/10 micrograms glycoprotein) in trout red muscle. This difference in binding was due to a higher number and a greater affinity of the IGF-I (Kd, 0.21 +/- 0.03 nM) compared with the insulin (Kd, 0.67 +/- 0.06 nM) receptors in this tissue. Carp red muscle IGF-I binding (9.14 +/- 0.55%/10 micrograms glycoprotein) surpassed insulin binding (2.59 +/- 0.094%/10 micrograms glycoprotein) mainly because of a greater affinity of the IGF-I (Kd, 0.092 +/- 0.027 nM) compared with the insulin (Kd, 0.1515 +/- 0.0285 nM) receptor. IGF-I and insulin binding in carp red muscle were higher than in trout, as a consequence of a higher affinity of carp red muscle receptors. Arginine injection provoked acute hyperinsulinemia in both trout (23.3 +/- 1.01 ng ml-1) and carp (24.3 +/- 1.34 ng ml-1. Specific binding of insulin and IGF-I to the red muscle decreased 4 h after injection. In trout, a decrease of insulin and IGF-I binding of 47.0% and 63.3%, respectively was observed compared with controls, in carp, these values were 44.0% and 45.0%. The number of insulin and IGF-I receptors decreased (42-55%) but affinities did not change suggesting that receptor down-regulation is a consequence of high insulin levels.

Aging and the nigro-striatal pathway.

Aging is associated with a progressive impairment in motor function. This feature, together with the decline in mental function, could be considered as an aging syndrome which may finally compromise the ability of the elderly to maintain an active, independent life-style. In the present paper a wide variety of morphological aspects, which have been classically related to brain aging and others such as cytoskeletal changes, the role of growth factors and molecular changes, will be reviewed focusing on aging of the nigrostriatal pathway. In addition to sharing features of aging common to other structures, it is likely that the nigrostriatal pathway has specific characteristics derived from its particular molecular characteristics and/or from a selective vulnerability to aging. To gain further insight into the aging syndrome, the acquisition of rigorous criteria for selecting control cases is paramount. The improvement of methods for the preservation of human tissue is also crucial.

Insulin and IGF-I binding in isolated trout cardiomyocytes.

Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout, Salmo trutta fario. Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 +/- 0.28%/10 mg cells after overnight incubation at 4 degrees. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 +/- 0.043 nM and a binding site density of 1. 61 +/- 0.19 x 10(8)/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and the Kd value obtained was 4.77 +/- 2.82 nM, 17-fold higher than Kd values for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70 +/- 1.42%/10 mg cells), affinity (Kd = 0.163 +/- 0.023 nM), and binding site density (4.00 +/- 1.13 x 10(8)/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd = 33.6 +/- 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.

Synaptophysin in spinal anterior horn in aging and ALS: an immunohistological study.

Aged-related spinal cord changes such as neuronal loss have been related to the degree of clinical severity of amyotrophic lateral sclerosis (ALS); morphological data on synapses are, however, wanting. Variations in synaptophysin (Sph) expression in aging and ALS were thus studied at the level of lower motor neurons in 40 controls with non-neurological diseases and 11 cases of ALS. Control sections of formalin fixed paraffin embedded cervical (C7/8), thoracic (T10) and lumbar spinal cord (L5) and C6, C7, C8 and L5 of ALS cases were stained with haematoxylin and eosin, luxol fast blue (LFB), and immunostained with a mouse monoclonal antibody against Sph. The neuropil of the anterior horn (AH) in all control cases demonstrated Sph positivity. A dot-like pattern of positivity of presynaptic terminals on soma of motor neurons and fine immunoreactivity along neuronal processes were observed. A significant reduction of Sph immunostaining was observed in the neuropil with increasing age and 3 different somatic patterns were seen: a- well preserved Sph reactivity around the soma and the proximal dendrites of histologically normal neurons; b- few chromatolytic neurons showing large numbers of dot-like presynaptic terminals around the cell body and in a "fused" pattern; c- intense, diffuse, and homogeneous reactivity of some neurons. Attenuation of Sph reactivity in the AH neuropil, to its complete loss, was observed in all ALS cases. In addition to patterns a-c, two additional microscopic findings were noted in ALS: d- chromatolytic neurons showing complete absence of Sph reactivity; e- absence of Sph reactivity around the soma and the proximal dendrites of histologically normal surviving neurons. Our findings demonstrate that there is a decrease in Sph immunostaining with aging, thus suggesting an alteration in dendritic networks of the AH with aging. Changes in the pattern of Sph immunoreactivity in cell bodies may represent synaptic plasticity and/or degeneration. Reinnervation may also be a possible mechanism as a response to neuronal loss in oldest control cases. Sph reactivity results may thus lend support to the presence of superimposed aging components in ALS cases which may give an insight into explaining the increasing severity of the disease which is encountered with advancing age.

Zinc in hypothalamus and hypophysis of the rat.

Zinc has been located using both histochemical and autoradiographic procedures in the neurons of the nuclei of the hypothalamic medial area and in some adenohypophisary cells. Some suggestions about the functional significance of the presence of Zn in these places are made.

[Cognitive functions in multiple sclerosis]. / Funciones cognitivas en la esclerosis múltiple.

Although the presence of intellectual impairment in multiple sclerosis (MS) is known since the first studies of the disease frequently the impairment goes equally undetected for the patients, their family and the physician because the physical dysfunction is much more outstanding. In this study a population of 50 persons suffering clinically definite MS (Poser's criteria) and 50 healthy controls matched with the patients in sex, age and cultural level were submitted to a neuropsychological test battery (NPTB). The existence of a correlation between the punctuation obtained in the NPTB and factors such as the level of disability, the time of evolution, the type of MS and the work status was searched. The results show a greater difficulty in doing tasks requiring attention-concentration skills thus giving the MS patients significantly lower scores in the memory test when compared with the controls. The execution of all tests was worse in those forms of MS with the longest time of evolution, higher EDSS scores and the chronic-progressive types. In our study the loss of the working status was never due to the intellectual impairment, it was because of the physical disability.

Melanin storing cells in anuran gut.

1. The location, distribution and morphological characteristics of the pigment cells found in the frog gut are described. 2. The pigment cells show long and large protoplasmic projections. At the ultrastructural level, the nucleus is elongated with prominent nucleolus and dense marginal chromatin. The cytoplasm is full with pigment granules (2500-7500 A) and typical premelanosome structures have been observed. 3. The pigment cells number is higher in the esophagus and large intestine than in the stomach or small intestine and the pigment cells are always located in close contact with blood vessels and nervous structures (ganglia and fibres). 4. We have observed that the pigment content depends upon seasonal variations, increasing during the cold months. 5. We have demonstrated by histological methods that the cells pigment content is melanin. 6. According to their morphological and tinctorial characteristics the anuran gut melanin storing cells are similar to the skin epidermal melanocytes.

[Fibrous hamartoma of the chest wall: apropos of a case]. / Hamartoma fibroso de pared costal: a propósito de un caso.

Fibrous hamartoma of the child is one of the congenital fibromatosis. It is considered as an expansive, benign process, genuine of the settling site although changing in quantity, disposition and differentiation. The thoracic localization is very seldom found. Authors comment the most important features referring to this tumour.