Effects of standardized Ginkgo biloba extract on the acquisition, retrieval and extinction of conditioned suppression: Evidence that short-term memory and long-term memory are differentially modulated.

Studies in our laboratory have characterized the putative neuromodulatory effects of a standardized extract of the green leaves of Ginkgo biloba (EGb), which comprises a formulation of 24% ginkgo-flavoglycosides and 6% ginkgo-terpenoid lactones, on conditioned suppression. This model comprises a suitable animal model for investigating the behavioral changes and pharmacological mechanisms that underlie fear memory and anxiety. The characterization of the effects on distinct stages of fear memory or fear extinction will help illustrate both the beneficial and harmful effects. Three hundred adult male Wistar rats were randomly assigned to 30 groups according to the treatment as follows: i-ii) control groups (CS-US and CSno-US); iii) vehicle group (12% Tween®80); and iv-vi) EGb groups (250, 500 and 1000mgkg(-1)); or experimental procedures designed to assess the effects of EGb treatment prior to the acquisition (n=20 per group) and retrieval of conditioned fear (n=10 per group) or prior to the extinction training (n=10 per group) and extinction retention test (n=10 per group). Furthermore, to better understand the effects of acute EGb treatment on fear memory, we conducted two additional analyses: the acquisition of within- and between-session extinction of fear memory (short- and long-term memory, respectively). No difference was identified between the control and treatment groups during the retention test (P>0.05), with the exception of the CSno-US group in relation to all groups (P<0.05). A between-session analysis indicated that EGb at 250mgkg(-1) facilitated the acquisition of extinction fear memory, which was verified by the suppression ration in the first trial of extinction training (SR=0.39) and the extinction retention test session (SR=0.53, P<0.05), without impairments in fear memory acquisition, which were evaluated during the retention test (SR=0.79). Moreover, EGb administered at 1000mgkg(-1) prior to conditioning did not enhance the long-term extinction memory, i.e., it did not prevent the return of extinguished fear memory in the extinction retention test, in which the spontaneous recovery of fear was demonstrated (SR=0.63, P<0.05); however, it significantly facilitated short-term memory as verified by data from the within-session extinction (1 to 8-10 trials) during the retention test (SR=0.73 to SR=0.59; P<0.05) and the extinction retention test (SR=0.63 to SR=0.41; P<0.05). Moreover, spontaneous recovery was identified in response to a higher dose of EGb when administered prior to extinction training (SR=0.75, P<0.05) and the extinction retention test (SR=0.70; P<0.05). At dose of 500mgkg(-1) EGb reduced the suppression ratio when administered prior to the retention test (SR=0.57) and extinction training (SR=0.55; P<0.05) without preventing the acquisition of fear memory, which suggests that EGb has anti-anxiety effects. Taken together, the current findings suggest that EGb differentially modulates short- and long-term memory, as well as anxiety-like behavior. The actions of EGb may provide information regarding the beneficial effects in the prevention and treatment of neurocognitive impairments and anxiety disorders. Additional analyses are necessary to facilitate an understanding of these effects; however, previous data from our group suggest that GABAergic, serotoninergic and glutamatergic receptors are potential targets of the effects of EGb on conditioned suppression.

Long-term treatment with standardized extract of Ginkgo biloba L. enhances the conditioned suppression of licking in rats by the modulation of neuronal and glial cell function in the dorsal hippocampus and central amygdala.

Our group previously demonstrated that short-term treatment with a standardized extract of Ginkgo biloba (EGb) changed fear-conditioned memory by modulating gene expression in the hippocampus, amygdaloid complex and prefrontal cortex. Although there are few controlled studies that support the long-term use of EGb for the prevention and/or treatment of memory impairment, the chronic use of Ginkgo is common. This study evaluated the effects of chronic treatment with EGb on the conditioned emotional response, assessed by the suppression of ongoing behavior and in the modulation of gene and protein expression. Male adult Wistar rats were treated over 28days and assigned to five groups (n=10) as follows: positive control (4mgkg(-1) Diazepam), negative control (12% Tween 80), EGb groups (0.5 and 1.0gkg(-1)) and the naïve group. The suppression of the licking response was calculated for each rat in six trials. Our results provide further evidence for the efficacy of EGb on memory. For the first time, we show that long-term treatment with the highest dose of EGb improves the fear memory and suggests that increased cAMP-responsive element-binding protein (CREB)-1 and glial fibrillary acidic protein (GFAP) mRNA and protein (P<0.001) in the dorsal hippocampus and amygdaloid complex and reduced growth and plasticity-associated protein 43 (GAP-43) (P<0.01) in the hippocampus are involved in this process. The fear memory/treatment-dependent changes observed in our study suggest that EGb might be effective for memory enhancement through its effect on the dorsal hippocampus and amygdaloid complex.

Factorial design optimization of experimental variables in the on-line separation/preconcentration of copper in water samples using solid phase extraction and ICP-OES determination.

An on-line preconcentration procedure using solid phase extraction (SPE) for the determination of copper in different water samples by inductively coupled plasma optical emission spectrometry (ICP-OES) is proposed. The copper was retained on a minicolumn filled with ethyl vinyl acetate (EVA) at pH 8.0 without using any complexing reagent. The experimental optimization step was performed using a two-level full factorial design. The results showed that pH, sample loading flow rate, and their interaction (at the tested levels) were statistically significant. In order to determine the best conditions for preconcentration and determination of copper, a final optimization of the significant factors was carried out using a central composite design (CCD). The calibration graph was linear with a regression coefficient of 0.995 at levels near the detection limit up to at least 300 µg L(-1). An enrichment factor (EF) of 54 with a preconcentration time of 187.5 s was obtained. The limit of detection (3σ) was 0.26 µg L(-1). The sampling frequency for the developed methodology was about 15 samples/h. The relative standard deviation (RSD) for six replicates containing 50 µg L(-1) of copper was 3.76%. The methodology was successfully applied to the determination of Cu in tap, mineral, river water samples, and in a certified VKI standard reference material.

On-line preconcentration and determination of mercury in biological and environmental samples by cold vapor-atomic absorption spectrometry.

An on-line procedure for the determination of traces of total mercury in environmental and biological samples is described. The present methodology combines cold vapor generation associated to atomic absorption spectrometry (CV-AAS) with preconcentration of the analyte on a minicolumn packed with activated carbon. The retained analyte was quantitatively eluted from the minicolumn with nitric acid. After that, volatile specie of mercury was generated by merging the acidified sample and sodium tetrahydroborate(III) in a continuous flow system. The gaseous analyte was subsequently introduced via a stream of Ar carrier into the atomizer device. Optimizations of both, preconcentration and mercury volatile specie generation variables were carried out using two level full factorial design (2(3)) with 3 replicates of the central point. Considering a sample consumption of 25mL, an enrichment factor of 13-fold was obtained. The detection limit (3sigma) was 10ngL(-1) and the precision (relative standard deviation) was 3.1% (n=10) at the 5microgL(-1) level. The calibration curve using the preconcentration system for mercury was linear with a correlation coefficient of 0.9995 at levels near the detection limit up to at least 1000microgL(-1). Satisfactory results were obtained for the analysis of mercury in tap water and hair samples.

Preconcentration and speciation of chromium in drinking water samples by coupling of on-line sorption on activated carbon to ETAAS determination.

An on-line flow injection (FI) preconcentration-electrothermal atomic absorption spectrometry (ETAAS) method is developed for trace determination of chromium in drinking water samples by sorption on a conical minicolumn packed with activated carbon (AC) at pH 5.0. The chromium was removed from the minicolumn with 1.0% (v/v) nitric acid. An enrichment factor (EF) of 35-fold for a sample volume of 10ml was obtained. The detection limit (DL) value for the preconcentration method proposed was 3.0ngl(-1). The precision for 10 replicate determinations at the 0.5mugl(-1) Cr level was 4.0% relative standard deviation (R.S.D.), calculate with the peak heights obtained. The calibration graph using the preconcentration system for chromium was linear with a correlation coefficient of 0.9992 at levels near the detection limits up to at least 50mugl(-1). The method was successfully applied to the determination of Cr(III) and Cr(VI) in drinking water samples.

On-line preconcentration/determination of lead in Ilex paraguariensis samples (mate tea) using polyurethane foam as filter and USN-ICP-OES.

The present paper proposes an on-line preconcentration procedure for lead determination in Ilex paraguariensis (St. Hilaire) samples by ultrasonic nebulization associated to inductively coupled plasma optical emission spectrometry (USN-ICP-OES). It is based on the precipitation of lead(II) ion on a minicolumn packed with polyurethane foam using 2-(5-bromo-2-pyridilazo)-5-diethylaminophenol (5-Br-PADAP) as precipitating reagent. The collected analyte precipitate was quantitatively eluted from the minicolumn with 20% (v/v) nitric acid. An enhancement factor of 225-fold was obtained (15 for USN and 15 for preconcentration). The detection limit (DL) value for the preconcentration of 10.0 ml of sample was 40.0 ng/l. The relative standard deviation (R.S.D.) was 3.0% for a Pb concentration of 1 microg/l, calculated from the peak heights obtained. The calibration graph using the preconcentration system for lead was linear with a correlation coefficient of 0.9997, at levels near the detection limits up to at least 100 microg/l. The preconcentration procedure was successfully applied to the determination of lead in mate tea samples.

On-line preconcentration/determination of copper in parenteral solutions using activated carbon by inductively coupled plasma optical emission spectrometry.

A method for the on-line preconcentration of copper using a minicolumn packed with activated carbon and its subsequent determination by inductively coupled plasma optical emission spectrometry (ICP-OES) coupled with flow injection (FI) was studied. In order to determinate the copper concentration present in parenteral solutions; it was retained on activated carbon (AC) at pH 9.5. A sensitivity enrichment factor of 30-fold was obtained with respect to the copper determination by ICP-OES without preconcentration. The detection limit for the preconcentration of 25 ml of sample was 0.1 microgl(-1). The precision for the ten replicate determinations at the 2.5 microgl(-1) Cu level was 3.0% relative standard deviation (R.S.D.), calculated with the peak heights. The calibration graph using the preconcentration method for cooper species was linear with a correlation coefficient of 0.9996 at levels near the detection limits up to at least 200 microgl(-1). The method was successfully applied to the determination of copper in parenteral solutions.

Time-Varying Analysis of the Heart Rate Variability during Arousals from Sleep.

The goal of this work was to apply a time-varying spectral approach to study the dynamic changes in Heart Rate Variability (HRV) produced by the arousal from sleep in obese patients with and without Obstructive Sleep Apnea (OSA). We studies 10 subjects, group I without OSA and group II with OSA diagnostic. RR intervals series was obtained from ECG records. HRV spectral indexes (HF & LF component) were computed from a time variant AR model. In general, group II has more variability than group I. Results are in agreement with literature for group I. However, group II results don't show statistical differences. Our preliminary results are comparable to others reported. Therefore, this technique allows characterizing transitory phenomena like cardiovascular response to arousals.

Trace element determination of Argentine wines using ETAAS and USN-ICP-OES.

The objective of this work was to develop a method to determine the metal content in wine samples from the province of Mendoza in Argentina. Ten samples of white wine and 10 samples of red wine available in the supermarket were analyzed for the metals aluminium, cadmium, calcium, chromium, copper, iron, nickel, lead and zinc by electrothermal atomic spectrometry (ETAAS) and ultrasonic nebulization was coupled to inductively coupled plasma optical emission spectrometry (USN-ICP-OES). The aluminium, cadmium, calcium, copper, iron, lead, zinc, chromium concentrations were between 17.0-18.0 microg l(-1), 1.0-4.7microg l(-1), 10.0-15.0 mg l(-1), 23.0-28.0 microg l(-1), 480-790 microg l(-1), 50-90 microg l(-1), 24-130 microg l(-1), and <0.2-6.25 microg l(-1), respectively. The levels compare well with those reported for similar wines from some other parts of the world. A significant aspect in this paper is the samples mineralization step, which allowed the direct determination of the metals. Concerning to the Cd determination, a refluxing digestion system was used for the pretreatment of the samples.

Variations of Celiac Artery in Dogs: Anatomic Study for Experimental, Surgical and Radiological Practice / Variaciones de la Arteria Celiaca en Perros: Estudio Anatómico para Prácticas Experimental, Quirúrgica y Radiológica

The purpose of this study is gain a better understanding of the anatomical variations of celiac artery and its branches in dogs. The celiac artery supply was analyzed in thirty dogs.The animals were injected with solution of Petrolatex S65 and dissected to evidence the celiac artery origin and its proximal ramifications. The celiac artery and its proximal branches were dissected and lengthen. The celiac artery more frequently emerged ahead of the first lumbar vertebra in one way form. The medium length of the celiac artery was of 0.98 cm. The gastrolienal trunk was the predominant morphological arrangement with medium length of 0.19 cm. The lienal artery emerged in one way form emitting more frequently 2 main branches. A branch emerging of the celiac artery irrigating the left adrenal occurred in 4 animals. The left gastric artery presented 1 branch in 17 animals (56.7%), 2 branches in 11 animals (36.6%) and 3 branches in 3 animals (10%). The left pancreatic lobe received a main branch from the lienal artery in 24 animals (80.0%), two branches in 4 animals (13.4%), and a branch emerging of the gastrolienal trunk in 2 animals (6.6%). The present data should therefore provide important information for devising experiments and interpreting results when using the mongred dogs as a model for experimental surgery and radiolocal practice of the celiac artery specially when making comparisons to human anatomy.

El propósito de este estudio es comprender mejor las variaciones anatómicas de la arteria celiaca y de sus ramas en los perros. Estudiamos la arteria celiaca en 30 perros (Canis familiaris). La parte abdominal de la aorta y el tronco celiaco fueron inyectados con una solución de Petrolatex S65, disecándose, desde su origen, la arteria celiaca y sus ramificaciones proximales. La arteria celiaca se originaba, de manera más frecuente, a nivel de la primera vértebra lumbar. La longitud media de la arteria celiaca fue de 0.98 cm. El tronco gastrolienal era el elemento morfológico predominante, con una longitud promedio de 0.19 cm. La arteria lienal se originaba de un tronco y emitía, frecuentemente, dos ramas principales. En 4 animales, se originaba una rama de la arteria celiaca, la cual irrigaba la glándula adrenal izquierda. La arteria gástrica izquierda emitía una rama en 17 animales (56.7%), dos ramas en 11 animales (36.6%) y 3 ramas en 3 animales (10%). El lóbulo pancreático izquierdo recibía una rama principal de la arteria lienal en 24 animales (80.0%), 2 ramas en 4 animales (13.4%), y una rama que se originaba del tronco gastrolienal en 2 animales (6.6%). Los resultados obtenidos se deben tener presentes cuando son utilizados perros como modelo en cirugía experimental y práctica radiolocal, especialmente, cuando se hacen comparaciones con la anatomía humana.

Optimization of the preconcentration system of cadmium with 1(2-thiazolylazo)-p-cresol using a knotted reactor and flame atomic absorption spectrometric detection.

The present paper proposes an on-line preconcentration procedure for cadmium determination in drinking water samples. It is based on the precipitation of cadmium(II) ions on a knotted reactor (KR) using 1(2-thiazolylazo)-p-cresol (TAC) as complexing reagent. The optimization step was performed using a full factorial design involving the variables: pH, eluent concentration (nitric acid) and TAC concentration. The results of this experiment demonstrated that these variables at chosen levels are not statistically significant. Under optimized experimental established conditions, analytical parameters for the preconcentration method were: a detection limit of 40.0 ng/l, precision as relative standard deviation (RSD) of 1.2 and 1.0%, for cadmium concentration of 2.5 and 20.0 microg/l, respectively. The preconcentration factor considering the slopes of the analytical curves with and without preconcentration is 23 for a sample volume of 10 ml. This system shows a sampling frequency of 25 h(-1). In order to check the accuracy, the standard reference material, NIST SRM 1643d trace elements in water was analyzed. A comparison, using t-test demonstrates that there is not significant difference among the achieved results with proposed method and the certified values. The addition/recovery experiments in the samples analyzed demonstrated the accuracy and applicability of the system developed for cadmium determination in water samples.

Factorial design for multivariate optimization of an on-line preconcentration system for platinum determination by ultrasonic nebulization coupled to inductively coupled plasma optical emission spectrometry.

A system for on-line preconcentration and determination of platinum by ultrasonic nebulization (USN) coupled to inductively coupled plasma optical emission spectrometry (ICP-OES) was studied. It is based on the chemical sorption of platinum on a column packed with polyurethane foam loaded with thiocyanate reagent. The optimization step was carried out using two level full factorial design. Three variables (pH, loading flow rate (LFR) and eluent concentration) were regarded as factors in the optimization. Results of the two level factorial design 2(3) with three replicates of the central point for platinum preconcentration, based on the variance analysis (ANOVA), demonstrated that the factors and their interactions are not statistically significant. The proposed procedure allowed the determination of platinum with a detection limit of 0.28mugl(-1). The precision for 10 replicate determinations at 10.0mugl(-1) Pt level was 3.8% relative standard deviation (R.S.D.), calculated from the peak heights obtained. A total enhancement factor of 100 was obtained with respect to ICP-OES using pneumatic nebulization (10 for USN and 10 for preconcentration). A sampling frequency of 50 samples per hour was obtained. The effect of other ions in concentrations agreeing with water samples was studied. The addition/recovery experiments in the samples analyzed demonstrated the accuracy and applicability of the system developed for platinum determination in spiked water samples.

Operant discriminative learning and evidence of subtelencephalic plastic changes after long-term detelencephalation in pigeons.

We analyzed operant discrimination in detelencephalated pigeons and neuroanatomical substrates after long-term detelencephalation. In Experiment I, experimental pigeons with massive telencephalic ablation and control pigeons were conditioned to key peck for food. Successive discrimination was made under alternating red (variable-ratio reinforcement) and yellow (extinction) lights in one key of the chamber. These relations were interchanged during reversal discrimination. The sessions were run until steady-state rates were achieved. Experiment II analyzed the morphology of the nucleus rotundus and optic tectum in long-term detelencephalated and control birds, using a Klüver-Barrera staining and image analyzer system. Detelencephalated birds had more training sessions for response shaping and steady-state behavior (p < 0.001), higher red key peck rates during discrimination (p < 0.01), and reversal discrimination indexes around 0.50. Morphometric analysis revealed a decreased number of neurons and increased vascularity, associated with increases in the perimeter (p < 0.001) in the nucleus rotundus. In the optic tectum, increases in the perimeter (p < 0.05) associated with disorganization in the layers arrangement were seen. The data indicate that telencephalic systems might have an essential function in reversal operant discrimination learning. The structural characteristics of subtelencephalic systems after long-term detelencephalation evidence plastic changes that might be related to functional mechanisms of learning and neural plasticity in pigeons.

On-line preconcentration and determination of nickel in natural water samples by flow injection-inductively coupled plasma optical emission spectrometry (FI-ICP-OES).

An on-line nickel preconcentration and determination system implemented with inductively coupled plasma optical emission spectrometry (ICP-OES) associated to flow injection (FI) was studied. Trace amounts of nickel were preconcentrated by sorption on a conical minicolumn packed with activated carbon (AC) at pH 5.0. The nickel was removed from the minicolumn with 20% nitric acid. An enrichment factor of 80-fold for a sample volume of 50 ml was obtained. The detection limit (DL) value for the preconcentration method proposed was 82 ng l(-1). The precision for ten replicate determinations at the 0.5 microg l(-1) Ni level was 3.0% relative standard deviation (R.S.D.), calculated from the peak heights obtained. The calibration graph preconcentration method for nickel was linear with a correlation coefficient of 0.9997 at levels near the detection limits (DL) up to at least 100 microg l(-1). The method was successfully applied to the determination of nickel in natural water samples.

S100 immunoreactivity is increased in reactive astrocytes of the visual pathways following a mechanical lesion of the rat occipital cortex.

After demonstration of the paracrine action of glial neurotrophic factors, gliosis has also been considered to be related to neuronal trophism and plasticity rather than solely a repair event following brain injury. S100 is a Ca2+ binding protein, present mainly in astrocytes, that exerts paracrine trophic effects on several neuronal populations. This study analyses the presence of S100 protein by means of immunohistochemistry combined with stereology in the reactive glial cells of the rat visual pathways following a lesion of the visual cortex. Adult male Wistar rats were submitted to a unilateral aspiration of the occipital cortex or to a sham operation. One week later the rats were killed and their brain processed for immunochemistry. Single antibody immunohistochemistry was performed for the visualization of glial fibrillary acidic protein (GFAP, a marker for astrocytes), OX-42 (a marker for microglia) and S100 protein. Double immunofluorescence procedures were applied for co-localization of the S100/GFAP and S100/OX-42. An optical dissector, point interceptors and rotators were used to quantify the degree of glial activation and the changes in the S100 immunoreactivity. We observed an intense microglial and astroglial reaction in addition to an increased S100 immunoreactivity in the occipital cerebral cortex, geniculate nucleus and hippocampus ipsilateral to the lesion. In the ipsilateral superior colliculus, an intense astroglial activation was accompanied by an up-regulation of S100 immunoreactivity. Double-immunofluoresence revealed an increased S100 immunoreactivity in reactive astrocytes, but not in the reactive microglia. Evidence has therefore been obtained that after mechanical trauma, the astroglial S100 protein participates in the trophism and plasticity of the injured visual pathways.

The functional value of sound and exploratory behaviour in detelencephalated pigeons.

Exploratory behaviour in response to a sound previously paired to a shock was analyzed before and after massive telencephalic lesions. Six pigeons were submitted to sound shock associations, habituation tests, telencephalic lesion and habituation retesting. Eighteen birds divided into three sham-lesioned groups received sound shock pairing (Control 1), pre-exposure to sound stimulation (Control 2) or pre-exposure to electric shocks (Control 3) before the behavioural tests. The pre-surgery tests consisted of 25 1 s pulses of sound A (1000 Hz, 83 dB) or B (500 Hz, 85 dB), every 30 s. Retests took place 10 days after surgery using the same sequence of procedures. The behaviour exhibited immediately after each sound stimulation was recorded manually and on video during the sessions. Analysis of variance showed an effect of group {F(3, 20)= 13.37, p < 0.0001) suggesting that pre-exposure to tone-shock association affected the exploration in response to the sound presented in another context. The Experimental and Control 1 birds showed no robust decrease in responses during the pre-surgery sessions. Post-surgery exploration data for detelencephalated birds showed a lower level of responding (p < 0.05). These data suggest a retardation in the typical reduction of responses to recurrent stimulation and support other evidences of the potentiating effect of sound shock association on responding. They are also indicative of telencephalic modulation of exploratory behaviour and strengthen the arguments for the subtelencephalic storage of associative information concerning the functional value of acoustic stimulation.

Operant discrimination learning in detelencephalated pigeons (Columba livia).

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into three groups: non-lesioned (N = 6), sham-lesioned (N = 5) and telencephalon lesioned (N = 10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80% of the ad libitum weight. Successive discrimination was programmed by the alteration of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with variable-ratio reinforcement; the yellow key was correlated with extinction. Session were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning by detelencephalated birds. Response shaping and steady-state rates required a larger number of sessions for lesioned pigeons (P < 0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components as compared to the non-lesioned (red: 16.51 +/- 2.0; yellow: 2.02 +/- 0.64) and sham-lesioned (red: 22.84 +/- 1.77; yellow: 4.72 +/- 1.99) groups (P < 0.05). These data show that telencephalic systems are not essential for operant discrimination learning but play a role in the modulation of discriminative behavior. Subtelencephalic systems appear to be functionally important for the organization and storage of learning.

Operant discrimination learning in detelencephalated pigeons (Columba livia)

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into the three groups: non-lesioned (N=6), sham-lesioned (N=5) and telencephalon lesioned (N=10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80 percent of the ad libitum weight. Successive discrimination was programmed by the alternation of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with extinction. Sessions were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning required a larger number of sessions for lesioned pigeons (P<0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components...