Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats

The behavioral effects of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), a metabotropic glutamate receptor (mGluR) agonist, or 0.9% (w/v) saline, injected into the dorsal periaqueductal gray (DPAG), was investigated. Male Wistar rats showed defense reactions characterized by jumps toward the top edges of the cages (saline = 0 vs t-ACPD = 6.0, medians P<0.05) and gallops (saline = 0 vs t-ACPD = 10.0, medians P<0.05) during the 60-s period after the beginning of the injection. In another experiment animals were placed inside an open arena for 5 min immediately after injection. Their behavior was recorded by a video camera and a computer program analyzed the videotapes. Eleven of fifteen rats injected with t-ACPD showed a short-lasting (about 1 min) flight reaction. No saline-treated animal showed this reaction (P<0.0005, chi-square test). The drug induced an increase in turning behavior (P = 0.002, MANOVA) and a decrease in the number of rearings (P<0.001, MANOVA) and grooming episodes (P<0.001, MANOVA). These results suggest that mGluRs play a role in the control of defense reactions in the DPAG.

Infusions of AP5 into the basolateral amygdala impair the formation, but not the expression, of step-down inhibitory avoidance

We evaluated the effects of infusions of the NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) on the formation and expression of memory for inhibitory avoidance. Adult male Wistar rats (215-300 g) were implanted under thionembutal anesthesia (30 mg/kg, ip) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Bilateral infusions of AP5 (5.0 µg) were given 10 min prior to training, immediately after training, or 10 min prior to testing in a step-down inhibitory avoidance task (0.3 mA footshock, 24-h interval between training and the retention test session). Both pre- and post-training infusions of AP5 blocked retention test performance. When given prior to the test, AP5 did not affect retention. AP5 did not affect training performance, and a control experiment showed that the impairing effects were not due to alterations in footshock sensitivity. The results suggest that NMDA receptor activation in the BLA is involved in the formation, but not the expression, of memory for inhibitory avoidance in rats. However, the results do not necessarily imply that the role of NMDA receptors in the BLA is to mediate long-term storage of fear-motivated memory within the amygdala.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Incorporation of dietary trans monounsaturated fatty acids into tissues of Walker 256 tumor-bearing rats

The correlation between dietary trans fatty acids and neoplasia was examined in the present study. Walker 256 tumor-bearing and control rats were fed a trans monounsaturated fatty acid (MUFA)-rich diet for 8 weeks and the incorporation of trans fatty acids by tumor tissue was examined. Also, the effect of tumor growth on trans fatty acid composition of plasma and liver, and the content of thiobarbituric acid-reactive substances (TBARS) was determined. Walker 256 tumor cells presented both trans and cis MUFAs given in the diet. The equivalent diet proportions were 0.66 for trans and 1.14 for cis. Taking into consideration the proportion of trans MUFAs in plasma (11.47%), the tumor incorporated these fatty acids in a more efficient manner (18.27%) than the liver (9.34%). Therefore, the dietary trans fatty acids present in the diet are actively incorporated by the tumor. Tumor growth itself caused marked changes in the proportion of polyunsaturated fatty acids in the plasma and liver but provoked only slight modifications in both trans and cis MUFAs. Tumor growth also reduced the unsaturation index in both plasma and liver, from 97.79 to 86.83 and from 77.51 to 69.64, respectively. This effect was partially related to an increase in the occurrence of the lipid oxidation/peroxidation process of TBARS content which was increased in both plasma (from 0.428 to 0.505) and liver (from 9.425 to 127.792) due to tumor growth.

Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Cardiovascular and respiratory responses to microinjection of L-glutamate into the caudal pressor area in conscious and anesthetized rats

The role of the caudal pressor area (CPA) in the maintenance of vasomotor tonus in anesthetized and decerebrate animals has been clearly established. In conscious animals, however, the participation of CPA in the cardiovascular control remains to be fully elucidated. In the present study, unilateral L-glutamate (L-Glu) (10 and/or 20 nmol/70 nl) microinjection into CPA, in conscious male Wistar rats (250-280 g) caused a significant increase in mean arterial blood pressure (MAP; control: 112 ± 1.9 mmHg; after 20 nmol L-Glu: 139 ± 4.5 mmHg, N = 12, P<0.05) and respiratory rate (control: 81 ± 3.5 breaths/min; after 10 nmol L-Glu: 92 ± 3 breaths/min, P<0.05; after 20 nmol L-Glu: 104 ± 5 breaths/min, N = 6, P<0.05). The subsequent anesthesia with urethane caused a significant increase in basal respiratory frequency (conscious: 81 ± 3.5 breaths/min; under urethane: 107 ± 1.3 breaths/min, N = 6, P<0.05). Anesthesia also significantly attenuated L-Glu-evoked pressor (conscious: deltaMAP = +27 mmHg; anesthetized: deltaMAP = +18 mmHg, P<0.05) and respiratory responses. These results suggest that glutamatergic receptors in the CPA are involved in cardiovascular and respiratory modulation in conscious rats.

Excitotoxic median raphe lesions aggravate working memory storage performance deficits caused by scopolamine infusion into the dentate gyrus of the hippocampus in the inhibitory avoidance task in rats

The interactions between the median raphe nucleus (MRN) serotonergic system and the septohippocampal muscarinic cholinergic system in the modulation of immediate working memory storage performance were investigated. Rats with sham or ibotenic acid lesions of the MRN were bilaterally implanted with cannulae in the dentate gyrus of the hippocampus and tested in a light/dark step-through inhibitory avoidance task in which response latency to enter the dark compartment immediately after the shock served as a measure of immediate working memory storage. MRN lesion per se did not alter response latency. Post-training intrahippocampal scopolamine infusion (2 and 4 µg/side) produced a more marked reduction in response latencies in the lesioned animals compared to the sham-lesioned rats. Results suggest that the immediate working memory storage performance is modulated by synergistic interactions between serotonergic projections of the MRN and the muscarinic cholinergic system of the hippocampus.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 ± 3.6 to 110 ± 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 ± 8.9 to 76 ± 8.8 mmHg, N = 7) or pentobarbital (from 118 ± 4.5 to 57 ± 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 ± 4.1 to 92 ± 5.9 mmHg, N = 7) and anesthetized rats (from 123 ± 6.9 to 87 ± 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition.

Photophysical studies of zinc phthalocyanine and chloroaluminum phthalocyanine incorporated into liposomes in the presence of additives

The photophysical properties of zinc phthalocyanine (ZnPC) and chloroaluminum phthalocyanine (AlPHCl) incorporated into liposomes of dimyristoyl phosphatidylcholine in the presence and absence of additives such as cholesterol or cardiolipin were studied by time-resolved fluorescence, laser flash photolysis and steady-state techniques. The absorbance of the drugs changed linearly with drug concentration, at least up to 5.0 µM in homogeneous and heterogeneous media, indicating that aggregation did not occur in these media within this concentration range. The incorporation of the drugs into liposomes increases the dimerization constant by one order of magnitude (for ZnPC, 3.6 x 10(4) to 1.0 x 10(5) M-1 and for AlPHCl, 3.7 x 10(4) to 1.5 x 10(5) M-1), but this feature dose does not rule out the use of this carrier, since the incorporation of these hydrophobic drugs into liposomes permits their systemic administration. Probe location in biological membranes and predominant positions of the phthalocyanines in liposomes were inferred on the basis of their fluorescence and triplet state properties. Both phthalocyanines are preferentially distributed in the internal regions of the liposome bilayer. The additives affect the distribution of these drugs within the liposomes, a fact that controls their delivery when both are used in a biological medium, retarding their release. The addition of the additives to the liposomes increases the internalization of phthalocyanines. The interaction of the drugs with a plasma protein, bovine serum albumin, was examined quantitatively by the fluorescence technique. The results show that when the drugs were incorporated into small unilamellar liposomes, the association with albumin was enhanced when compared with organic media, a fact that should increase the selectivity of tumor targeting by these phthalocyanines (for ZnPC, 0.71 x 10(6) to 1.30 x 10(7) M-1 and for AlPHCl, 4.86 x 10(7) to 3.10 x 10(8) M-1).

Translation into Brazilian Portuguese, cultural adaptation and evaluation of the reliability of the Disabilities of the Arm, Shoulder and Hand Questionnaire

The objective of the present study was to translate, adapt and validate a Brazilian Portuguese version of the Disabilities of the Arm, Shoulder and Hand (DASH) Questionnaire. The study was carried out in two steps. The first was to translate the DASH into Portuguese and to perform cultural adaptation and the second involved the determination of the reliability and validity of the DASH for the Brazilian population. For this purpose, 65 rheumatoid arthritis patients of either sex (according to the classification criteria of the American College of Rheumatology), ranging in age from 18 to 60 years and presenting no other diseases involving the upper limbs, were interviewed. The patients were selected consecutively at the rheumatology outpatient clinic of UNIFESP. The following results were obtained: in the first step (translation and cultural adaptation), all patients answered the questions. In the second step, Spearman's correlation coefficients for interobserver evaluation ranged from 0.762 to 0.995, values considered to be highly reliable. In addition, intraclass correlation coefficients ranged from 0.97 to 0.99, also highly reliable values. Spearman's correlation coefficients and the intraclass correlation coefficients obtained during intra-observer evaluation ranged from 0.731 to 0.937 and from 0.90 to 0.96, respectively, being highly reliable values. The Ritchie Index showed a weak correlation with Brazilian DASH scores, while the visual analog scale of pain showed a good correlation with DASH score. We conclude that the Portuguese version of the DASH is a reliable instrument.

Maternal aggression in Wistar rats: effect of 5-HT2A/2C receptor agonist and antagonist microinjected into the dorsal periaqueductal gray matter and medial septum

The objective of the present study was to assess the role of the 5-HT2A/2C receptor at two specific brain sites, i.e., the dorsal periaqueductal gray matter (DPAG) and the medial septal (MS) area, in maternal aggressive behavior after the microinjection of either a 5-HT2A/2C receptor agonist or antagonist. Female Wistar rats were microinjected on the 7th postpartum day with the selective agonist alpha-methyl-5-hydroxytryptamine maleate (5-HT2A/2C) or the antagonist 5-HT2A/2C, ketanserin. The agonist was injected into the DPAG at 0.2 (N = 9), 0.5 (N = 10), and 1.0 µg/0.2 µl (N = 9), and the antagonist was injected at 1.0 µg/0.2 µl (N = 9). The agonist was injected into the medial septal area (MS) at 0.2 (N = 9), 0.5 (N = 7), and 1.0 µg/0.2 µl (N = 6) and the antagonist was injected at 1.0 µg/0.2 µl (N = 5). For the control, saline was injected into the DPAG (N = 7) and the MS (N = 12). Both areas are related to aggressive behavior and contain a high density of 5-HT receptors. Non-aggressive behaviors such as horizontal locomotion (walking) and social investigation and aggressive behaviors such as lateral threat (aggressive posture), attacks (frontal and lateral), and biting the intruder were analyzed when a male intruder was placed into the female resident's cage. For each brain area studied, the frequency of the behaviors was compared among the various treatments by analysis of variance. The results showed a decrease in maternal aggressive behavior (number of bites directed at the intruder) after microinjection of the agonist at 0.2 and 1.0 µg/0.2 µl (1.6 ± 0.7 and 0.9 ± 0.3) into the DPAG compared to the saline group (5.5 ± 1.1). There was no dose-response relationship with the agonist. The present findings suggest that the 5-HT2A/2C receptor agonist has an inhibitory effect on maternal aggressive behavior when microinjected into the DPAG and no effect when microinjected into the MS. Ketanserin (1.0 µg/0.2 µl) decreased locomotion when microinjected into the DPAG and MS, but did not affect aggressive behavior. We interpret these findings as evidence for a specific role of 5-HT2A/2C receptors in the DPAG in the inhibition of female aggressive behavior, dissociated from those on motor activity.

Validation of the Social Phobia and Anxiety Inventory for Children (SPAI-C) in a sample of Brazilian children

The purpose of the present study was to examine the factor structure and psychometric properties of the Social Phobia and Anxiety Inventory for Children (SPAI-C), an instrument developed in the United States and applied to a sample of Brazilian schoolchildren. The process included the translation of the original material from English into Portuguese by two bilingual psychiatrists and a back translation by a bilingual physician. Both the front and back translations were revised by a bilingual child psychiatrist. The study was performed using a cross-sectional design and the Portuguese version of the SPAI-C was applied to a sample of 1954 children enrolled in 3rd to 8th grade attending 2 private and 11 public schools. Eighty-one subjects were excluded due to an incomplete questionnaire and 2 children refused to participate. The final sample consisted of 1871 children, 938 girls (50.1%) and 933 boys (49.8%), ranging in age from 9 to 14 years. The majority of the students were Caucasian (89.0%) and the remainder were African-Brazilian (11.0%). The Pearson product-moment correlation showed that the two-week test-retest reliability coefficient was r = 0.780 and Cronbach's alpha was 0.946. The factor structure was almost similar to that reported in previous studies. The results regarding the internal consistency, the test-retest reliability and the factor structure were similar to the findings obtained in studies performed on English speaking children. The present study showed that the Portuguese language version of SPAI-C is a reliable and valid measure of social anxiety for Brazilian children.