Disinhibition of the mediodorsal thalamus induces fos-like immunoreactivity in both pyramidal and GABA-containing neurons in the medial prefrontal cortex of rats, but does not affect prefrontal extracellular GABA levels.

Stimulation of the mediodorsal and midline thalamic nuclei excites cortical neurons and induces c-fos expression in the prefrontal cortex. Data in the literature data suggest that pyramidal neurons are the most likely cellular targets. In order to determine whether cortical interneurons are also impacted by activation of mediodorsal/midline thalamic nuclei, we studied the effects of thalamic stimulation on (1) Fos protein expression in gamma-aminobutyric acid (GABA)-immunoreactive neurons and on (2) extracellular GABA levels in the prefrontal cortex of rats. Perfusion of the GABA-A receptor antagonist bicuculline for 20 minutes through a dialysis probe implanted into the mediodorsal thalamus induced Fos-like immunoreactivity (IR) approximately 1 hour later in the thalamus and in the medial prefrontal cortex of freely moving rats. Immunohistochemical double-labeling for Fos-like IR and GABA-like IR showed that about 8% of Fos-like IR nuclei in the prelimbic and infralimbic areas were located in GABA-like IR neurons. Fos-like IR was detected in three major subsets of GABAergic neurons defined by calbindin, parvalbumin, or vasoactive intestinal peptide (VIP)-like IR. Dual probe dialysis showed that the extracellular levels of GABA in the prefrontal cortex did not change in response to thalamic stimulation. These data indicate that activation of thalamocortical neurons indeed affects the activity of GABAergic neurons as shown by the induction of Fos-like IR but that these metabolic changes are not reflected in changes of extracellular GABA levels that are sampled by microdialysis.

Heart defects in connexin43-deficient mice.

Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.

Modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial prefrontal cortex induced by disinhibition of the mediodorsal thalamus: a study employing microdialysis and immunohistochemistry.

We studied the interaction of catecholaminergic and thalamic afferents of the medial prefrontal cortex (PFC) by analyzing the effects of catecholamine depletion on thalamus-induced c-fos expression in the PFC of freely moving rats. Thalamic projections to the PFC were pharmacologically activated by perfusing the GABA-A receptor antagonist bicuculline (0.03 mM or 0.1 mM) through a dialysis probe implanted into the mediodorsal thalamic nucleus. Bicuculline perfusion induced Fos-like immunoreactivity in the thalamic projection areas, including the PFC, and in the thalamic nuclei surrounding the dialysis probe. 6-Hydroxydopamine lesions of the ventral tegmental area causing a 70-80% depletion of catecholamines in the PFC did not influence the increase in the number of Fos-like immunoreactive nuclei in the prefrontal cortex in response to thalamic stimulation. However, densitometric image analysis revealed that the intensity of Fos-like immunoreactivity in the PFC of lesioned rats perfused with 0.1 mM bicuculline was higher than in correspondingly treated controls. The behavioral activity to bicuculline perfusion, an increase of non-ambulatory activity (0.03 mM) followed by locomotion and rearing (0.1 mM), was not changed in 6-hydroxydopamine-lesioned rats. It is suggested that the thalamically induced c-fos response is directly mediated by excitatory, presumably glutamatergic, transmission and not indirectly by an activation of catecholaminergic afferents of the PFC. The increase in the intensity of Fos-like immunostaining in strongly stimulated, catecholamine-depleted rats suggests that catecholamines modulate the degree to which thalamic activity can activate the PFC of awake animals.

C-Fos expression in the rat brain after pharmacological stimulation of the rat "mediodorsal" thalamus by means of microdialysis.

In order to visualize target cells of thalamic projections in the rat brain we examined the induction of c-fos messenger RNA and Fos-like immunoreactivity following stimulation of the "mediodorsal" thalamus (midline, mediodorsal and intralaminar nuclei) in freely moving rats. The thalamic neurons were activated through disinhibition by perfusion of the GABAA antagonist bicuculline-methyl chloride via a microdialysis cannula placed in the mediodorsal nucleus of the thalamus. The rats were allowed a recovery period of at least 20 h after surgery before being coupled to the perfusion pump. Cannula implantation with or without 4 h of Ringer perfusion caused hardly any detectable c-fos expression in the brain, but 20 min of bicuculline (0.1 mM) perfusion induced high levels of c-fos messenger RNA and Fos protein expression in the area adjacent to the dialysis membrane, indicating activated thalamic neurons. In situ hybridization as well as immunohistochemical analysis of the frontal cortical areas and limbic structures showed a rapid, specific and transient c-fos expression in the medial and lateral prefrontal cortex, nucleus accumbens, mediodorsal striatum, claustrum, nucleus reticularis of the thalamus and amygdala. The overall spatial distribution of the c-fos response was comparable to the innervation patterns of thalamic efferents known from anatomical tracing experiments. The rats were perfused with Ringer while asleep, but they woke up during treatment with bicuculline and displayed an increase in general behavioural activity, which could be correlated to the amount of bicuculline measurable in the dialysate. Pathological behaviours, such as epilepsy, were not noticeable during bicuculline treatment. These results show that it is possible to selectively activate defined anatomical pathways by pharmacological application of drugs using microdialysis in unanesthetized unrestrained animals and to visualize the transsynaptically activated target neurons of these projections. We conclude that this novel experimental approach is indeed suitable for studying functional anatomical pathways.

Developmental changes in rat brain monoamine metabolism and beta-adrenoceptor subtypes after chronic prenatal exposure to propranolol.

The aim of this study was to investigate whether a chronic prenatal beta-blockade can alter the maturation of the noradrenergic system in the rat brain. Pregnant female and adult male rats were treated for 10 days with the beta-antagonist propranolol dissolved in the drinking water (40-50 mg/kg/day). Direct and long-term effects on beta-adrenoceptors and monoamine metabolism in various rat brain regions were determined. After the prenatal treatment the propranolol level in the foetal brain was 0.9 micrograms/g, while in the adult brain 2.0 micrograms/g was present. The foetal beta 1-receptors were significantly up-regulated by propranolol (200%), whereas the beta 2-receptor number remained unaltered. On postnatal days 4 and 21 the number of both beta-subtypes was the same as that of controls. Noradrenaline, its metabolite 3-methoxy-4-hydroxyphenylglycol and their ratio were unaltered directly after the prenatal treatment. In the PN 21 offspring, however, the metabolite level had increased in the frontal cortex (+ 17%) and hippocampus (+ 32%), and the ratio in the hippocampus (37%) and medulla pons (+ 34%). Prenatal treatment also induced a significant increase of the 5-hydroxyindoleacetic acid.5-hydroxytryptamine ratio (+ 15%) in the medulla pons at GD 21. No direct or lasting effects were found on dopamine metabolism. Propranolol treatment of adult rats gave no direct changes in monoamine metabolism. We concluded that chronic prenatal propranolol exposure (a) reversibly up-regulates foetal beta 1-adrenoceptors, and (b) increases the NA activity in the brain in later life.

Determination of propranolol, labetalol and clenbuterol in rat brain by high-performance liquid chromatography.

A rapid and simple HPLC method for the measurement of adrenergic drugs (propranolol, labetalol and clenbuterol) in rat brain is described. This method was applied to establish if these drugs can pass the blood-brain barrier in prenatal or early post-natal life. The chromatography was performed using a C18 column and a phosphate buffer (pH 3)-acetonitrile (65:35, v/v) mixture. After homogenization of the brain tissue in perchloric acid, the supernatant was buffered at pH 9 and extracted with diethyl ether, followed by back-extraction in sulphuric acid. Recoveries of between 80 and 100% were achieved. The method was found to be accurate (100%) and precise (coefficient of variation around 10%). All three drugs were readily detected in the brain of neonatal rats after peripheral administration. In addition, we demonstrated the presence of propranolol in the fetal brain after maternal administration.

Postnatal exposure to the beta-adrenoceptor agonist clenbuterol has regionally selective direct and long-term effects on rat brain beta-adrenoceptors and monoamine metabolism.

The effects of chronic postnatal beta 2-adrenoceptor activation on the maturation of the rat brain noradrenergic system have been studied. For that purpose, rat pups have been treated twice daily during the first 10 days of life with the beta 2-agonist clenbuterol-HCl (2.5 mg/kg s.c.), and the effects on the beta-adrenoceptor number and monoamine metabolism have been determined directly after the treatment and in adulthood. On postnatal day 10, 90 min after the last clenbuterol injection 4.5 micrograms/g of the drug was present in the brain. At the end of the treatment the beta-receptor binding had decreased in the cerebellum (35%), but not in the frontal cortex or mesolimbic system. Clenbuterol significantly increased the steady-state brain levels of noradrenaline (NA) in the striatum 90 min after the last injection, whereas the levels in the frontal cortex, meso-limbic system, medulla pons and cerebellum were unaffected. The NA metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), had significantly increased in the frontal cortex and striatum. The serotonergic (5-HT) and dopaminergic (DA) system were not altered. In general, no long-lasting effects on beta-adrenoceptor number and affinity or monoamine metabolism were measurable, except for the frontal cortex which showed a sustained increase of MHPG, a decrease of 5-HT and an increase of 5-HIAA/5-HT on PN 60. In conclusion, chronic postnatal activation of beta 2-adrenoceptors by clenbuterol treatment selectively causes changes in the setting of the neurochemical parameters investigated in the frontal cortex.

Changes in adrenoceptors and monoamine metabolism in neonatal and adult rat brain after postnatal exposure to the antihypertensive labetalol.

1. The purpose of the present study was to investigate the acute (single injection), direct (chronic treatment) and the long-lasting effects after exposure to the alpha 1/beta-adrenoceptor antagonist labetalol during rat brain development on adrenoceptors and monoamine metabolism. 2. In 10-day-old rat pups, subcutaneously administered labetalol (10 mg kg-1) passed the blood-brain barrier, reaching a level of 2.1 micrograms g-1 tissue in the brain 90 min after injection. 3. Chronic labetalol treatment (10 mg kg-1, s.c., twice daily) during the first 10 days of life significantly increased alpha 1-adrenoceptor binding in the hypothalamus (+39%), but not in the occipital cortex. 4. This chronic postnatal labetalol treatment did not result in long-lasting changes in alpha 1- and beta-receptors measured on day 60. 5. A single labetalol injection (10 mg kg-1, s.c.) on postnatal day 10 significantly increased noradrenaline (NA) metabolism in all brain regions tested (+25 to 105%), but had no effects on 5-hydroxytryptamine (5-HT) or dopamine metabolism. 6. Chronic labetalol treatment between postnatal (PN) days 1 and 10 also increased NA metabolism on PN 10 (3-methoxy-4-hydroxyphenylglycol (MHPG)/NA, +20 to 100%), suggesting that tolerance to the acute effect of labetalol did not occur. A slight increase in 5-HT metabolism (20%) was induced by the chronic labetalol treatment in the hippocampus and meso-limbic system. 7. In general, long-lasting effects on NA metabolism could not be detected on day 60 more than one month after the treatment. However, 5-HT metabolism was significantly increased in all four brain regions measured (+20 to 70%). 8. We conclude that chronic labetalol exposure during early postnatal rat brain development does not cause long-lasting changes in beta-receptor number or NA metabolism, but appears to be critical for the rate of 5-HT metabolism in later life.

Chronic propranolol treatment in developing rats: acute and lasting effects on monoamines and beta-adrenergic receptors in the rat brain.

During early postnatal development rat pups were treated twice daily with the beta-adrenergic antagonist propranolol (15 mg/kg) in order to study the acute and long-lasting effects of early blockade of noradrenergic beta-mediated neurotransmission. Treatments from postnatal days 1-10 or days 11-20 did not induce alterations in the number of beta-adrenergic receptors as measured three days after the last injection, nor could lasting effects be shown at 60 days of age. The day 1-10 treatment, however, had a significant effect on the regional brain levels of noradrenaline (NA) and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), measured 90 min after the last injection. The metabolite had increased by 40% in all brain regions examined. On day 60, the MHPG concentrations were still increased when compared to postnatally saline-treated animals. Propranolol treatment from day 11-20 only marginally increased MHPG on day 20 and induced no lasting differences. These results suggest that propranolol treatment during the first ten days of life produces a long-lasting increase in NA metabolism, possibly reflecting an increased neuronal NA turnover.

Nonionic block polymer surfactants modulate the humoral immune response against Streptococcus pneumoniae-derived hexasaccharide-protein conjugates.

Nonionic block polymer surfactants (NBPs) were tested for the capacity to stimulate the antibody response against hexasaccharide (HS), derived from Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS), which was conjugated to proteins. The immune response was evaluated in the (CBA/N x BALB/c)F1 progeny, in which female mice are phenotypically normal whereas male mice carry an X-chromosome-linked immunodeficiency. NBPs L101, L121, 1101, and 1501 were able to increase anti-HS immunoglobulin M (IgM) and IgG levels in both normal and X-chromosome-linked immunodeficient mice (with up to 74-fold stimulation of antibody titers). Distribution of S3PS-specific antibodies over the various IgG isotypes was restricted after immunization with either HS-bovine serum albumin or HS-keyhole limpet hemocyanin (HS-KLH). Addition of NBPs (in particular 1501) resulted in a more diverse immune response with either antigen as judged by isotype distribution. Isoelectric focusing of individual sera and subsequent detection of S3PS-binding antibodies in these sera by immunochemical staining revealed a restricted number of different spectrotypes in the course of the immune response. Upon immunization of mice with HS-KLH, spectra of secreted antibodies were slightly more complex and more densely stained than after immunization with HS-bovine serum albumin. Furthermore, NBPs 1101 and 1501 appeared to be able to stimulate the secretion of antibodies, which were secreted only in small amounts without the use of NBPs. Different explanations for increased spectrotype diversity after immunization with KLH as the carrier and after administration of NBPs as the adjuvant are discussed.

Nonionic block polymer surfactants enhance immunogenicity of pneumococcal hexasaccharide-protein vaccines.

Incorporated in oil-in-water emulsions, nonionic block polymer surfactants change the kinetics of generated antibody responses against pneumococcal hexasaccharide-protein conjugates: prolonged immunoglobulin M and immunoglobulin G responses are realized. Nonionic block polymer surfactants favor the immunogenicity of hexasaccharide-protein conjugates in young mice in such a way that a single injection yields long-lasting protection.

Measurement of the humoral immune response against Streptococcus pneumoniae type 3 capsular polysaccharide and oligosaccharide containing antigens by ELISA and ELISPOT techniques.

A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.