Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets.

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.

PorA-specific differences in antibody avidity after vaccination with a hexavalent Men B outer membrane vesicle vaccine in toddlers and school children.

A clinical phase II trial with an experimental hexavalent outer membrane vesicle (OMV) vaccine (HexaMen) containing six different porin A (PorAs) was carried out in toddlers (2-3 years) and schoolchildren (7-8 years) in The Netherlands. HexaMen exists of two OMVs each containing three different PorA types. The serum bactericidal activity (SBA) after vaccination against the six PorAs was significantly different and was higher in toddlers than in schoolchildren. After vaccination the SBA against P1.5-2,10 was 4-6 times higher than against P1.7-2,4. The aim of this study was to test whether the differences in SBA could be explained by a difference in subtype-specific antibody avidity maturation. The avidity index (AI) of antibodies against three subtypes (PorA types P1.5-2,10; P1.12-1,13 and P1.7-2,4) was measured by ELISA and evaluated in relation to SBA. A significant avidity maturation for the 3 PorA subtypes was found. This maturation was most pronounced for P1.5-2,10 (mean AI = 72%), correlating with the highest SBA titres. Generally, the avidity titre correlated best with SBA. No differences in avidity indices against the three tested PorAs were found between toddlers and school children indicating that avidity maturation induced by this vaccine is not age-dependent.

Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Cross-reactivity of antibodies against PorA after vaccination with a meningococcal B outer membrane vesicle vaccine.

The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and PorA-identical patient isolates were compared as a target in SBA, to ensure that the vaccine strains are representative for patient isolates. Geometric mean titers (GMTs) in SBA against patient isolates with subtypes P1.5-2,10 and P1.5-1,2-2 after vaccination with HexaMen were generally lower than those against vaccine strains with the same subtype, although the percentage of vaccine responders (> or =4-fold increase in SBA after vaccination) was not affected. Using various P1.7-2,4 patient isolates, GMTs as well as the number of vaccine responders were higher than for the P1.7-2,4 vaccine strain, indicating that the use of the P1.7-2,4 vaccine strain may have underestimated the immunogenicity of this subtype in HexaMen. Secondly, the cross-reactivity of antibodies induced by MonoMen and HexaMen was studied using several patient isolates that differed from the vaccine subtypes by having minor antigenic variants of one variable region (VR), by having a completely different VR or by having a different combination of VRs. MonoMen induced P1.4-specific antibodies that were cross-reactive with P1.4 variants P1.4-1 and P1.4-3. HexaMen induced a broader cross-reactive antibody response against various patient isolates with one VR identical to a vaccine subtype or a combination of VRs included in HexaMen. Cross-reactivity, measured by a fourfold increase in SBA after vaccination, against these strains ranged from 23 to 92% depending on the subtype of the tested strain and was directed against both VR1 and VR2. The extended cross-reactivity of vaccinee sera induced by HexaMen against antigenic variants has important favorable implications for meningococcal B OMV vaccine coverage.

Antibody avidity and immunoglobulin G isotype distribution following immunization with a monovalent meningococcal B outer membrane vesicle vaccine.

The avidity maturation and immunoglobulin G (IgG) isotype distribution of antibodies after vaccination with a meningococcal B outer membrane vesicle (OMV) vaccine were evaluated as indicators of protective immunity. Pre- and postvaccination sera from 134 healthy toddlers (ages, 2 to 3 years) immunized with a monovalent meningococcal B OMV (serosubtype P1.7-2,4) vaccine adsorbed with AlPO(4) or Al(OH)(3) were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. The children were vaccinated three times with intervals of 3 to 6 weeks between vaccinations or twice with an interval of 6 to 10 weeks between vaccinations. A booster was given after 20 to 40 weeks. The avidity index (AI) of antibodies increased significantly during the primary series of vaccinations and after the booster was given. No differences in AIs were found when the results obtained with the two vaccination schedules or with the two adjuvants were compared. After vaccination, IgG1 was the predominant IgG isotype, followed by IgG3. No IgG2 or IgG4 was detected. There was a strong correlation between serum bactericidal activity (SBA) and ELISA titers (r = 0.85 [P < 0.0001] for total IgG, r = 0.83 for IgG1 [P < 0.0001], r = 0.82 for IgG3 [P < 0.0001], and r = 0.84 [P < 0.0001] for the avidity titer). When two subgroups with similar anti-OMV IgG levels were compared before and after the booster vaccination, the higher AI after the booster vaccination was associated with significantly increased SBA. We concluded that avidity maturation occurs after vaccination with a monovalent meningococcal B OMV vaccine, especially after boosting, as indicated by a significant increase in the AI. Vaccination with the monovalent OMV vaccine induced mainly IgG1 and IgG3 isotypes, which are considered to be most important for protection against meningococcal disease. An increase in the AI of antibodies is associated with increased SBA, independent of the level of specific IgG and the IgG isotype distribution. Measuring the AI and IgG isotype distribution of antibodies after vaccination can be a supplementary method for predicting protective immunity for evaluation in future phase III trials with meningococcal serogroup B vaccines.

Serum bactericidal activity and isotype distribution of antibodies in toddlers and schoolchildren after vaccination with RIVM hexavalent PorA vesicle vaccine.

A clinical phase II trial with the RIVM hexavalent OMV vaccine containing six different PorAs was carried out in toddlers (2-3 years) and schoolchildren (7-8 years) in The Netherlands. Children were vaccinated three times (0, 2, 8 months). Sera after two and three vaccinations were analysed for serum bactericidal activity (SBA) and isotype distribution in whole cell enzyme linked immunosorbent assay (ELISA). The SBA after vaccination against the six PorAs was significantly different. We investigated whether the age specific and PorA specific differences in SBA titers correlated with differences in PorA specific IgG isotype distribution. The SBA titers were higher in toddlers compared with schoolchildren. After vaccination, IgG1 antibodies dominated the response followed by IgG3 antibodies. IgG2 levels were low, whereas IgG4 was not detected. Irrespective of PorA, IgG total and isotype specific titers after two and three vaccinations were significantly higher in toddlers than in schoolchildren. A weak correlation was found between IgG total or IgG1 and SBA. Although the immunogenicity of the six PorAs is very different, the isotype distribution was similar for all six tested PorAs. We conclude that the RIVM hexavalent PorA vesicle vaccine induces bactericidal antibodies mainly of the IgG1 and IgG3 isotypes that are considered to be most important for protection against disease. The isotype distribution of the response is not age-dependent.

Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7-8 years of age.

To study the reactogenicity and immunogenicity of a hexavalent meningococcal outer-membrane-vesicle vaccine (OMV), two different dosages of this vaccine (7.5 and 15 microg of individual PorA proteins) consisting of vesicles expressing class 1 outer-membrane proteins (OMPs) of subtypes P1.7,16; P1.5,2; P1.19,15 and P1.5(c), 10; P1.12,13; P1.7(h),4 were administered to a group of 7-8 year (n=165) and a group of 2-3 year old children (n=172). Control groups of children with similar ages were vaccinated against hepatitis B. All participants received three injections. Pre- and postimmunisation sera were tested for bactericidal antibodies against six isogenic meningococcal vaccine strains expressing different PorA proteins. Antibody titres against OMP of the two different vesicles (PL16215 and PL10124) were measured by ELISA. The meningococcal hexavalent OMV vaccine was well tolerated. No statistically significant differences were seen between the high and low dose of hexavalent meningococcal OMV vaccine. The percentage of children showing a fourfold increase of bactericidal antibody titres against the specific serosubtype varied in toddlers from 28 to 98% and in older children from 16 to 100%. Both ELISA antibody titres and bactericidal activity showed the highest level in the youngest age-group.

A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols.

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.

Light microscopic and ultrastructural investigation of the dopaminergic innervation of the ventrolateral outgrowth of the rat inferior olive.

The ventrolateral outgrowth of the inferior olive is involved in the control of compensatory eye movement responses to optokinetic stimuli about the horizontal axis that is perpendicular to the ipsilateral anterior semicircular canal. Combining immunocytochemistry with retrograde tracing of WGA-BSA-gold, we demonstrated in the present study that this olivary subnucleus receives a substantial dopaminergic input, and that the prerubral parafascicular area and its surrounding regions form the sole source of this input. In addition, we investigated the postsynaptic distribution of the dopaminergic terminals in the inferior olive at the ultrastructural level. About a third (32%) of the dopaminergic terminals was found to make synaptic contacts in the olivary neuropil. The majority (81%) of these boutons terminated on cell bodies or extraglomerular dendrites, while the remaining terminals contacted dendritic spines inside glomeruli. In contrast, GABAergic terminals in the inferior olive formed more frequently (66%) synaptic contacts and they terminated more frequently (38%) in glomeruli. Thus, the ventrolateral outgrowth receives a dopaminergic input from the mesodiencephalic junction, and the postsynaptic distribution of this input reveals a characteristic pattern.

Human B- and T-cell responses after immunization with a hexavalent PorA meningococcal outer membrane vesicle vaccine.

The PorA protein from Neisseria meningitidis, a potential vaccine candidate, induces human bactericidal antibodies which are serosubtype specific. We developed a hexavalent PorA outer membrane vesicle vaccine based on reference strain H44/76. This vaccine contains the six most prevalent PorA serosubtypes as found in many countries. We previously reported on the immune responses of 20 adult volunteers after a single immunization with this vaccine. In this study, the B- and T-cell responses in three adult volunteers were studied after three consecutive immunizations (0, 2, and 11 months). The first immunization induced a strong B-cell response resulting in high immunoglobulin G levels in an outer membrane vesicle enzyme-linked immunosorbent assay. At least a fourfold increase in bactericidal activity was observed against the majority (four to six) of the vaccine antigens compared to prevaccination titers. Immunodominance was observed for one or two of the PorAs in the bactericidal assay with a set of six isogenic H44/76-derived PorA target strains. These strains carry the individual PorAs as present in the vaccine. The second and third immunizations did not induce a further increase in the immune responses. A decline in time with respect to PorA-specific antibodies was observed after each immunization. These observations were reflected by the T-cell proliferation responses. Two additional sets of isogenic H44/76-derived mutant strains were used to study the specificity and/or cross-reactivity of the induced bactericidal antibodies. These target strains differ only in expressing mutant family variants of either PorA P1.7,16 or P1.5,10, both present in the PorA vesicle vaccine. The bactericidal antibody responses found were directed predominantly against the P1.7 (loop 1 of P1.7,16) and the P1.10 (loop 4 of P1.5,10) epitopes. This indicates that different portions of PorA were involved in the induction of bactericidal antibodies depending upon the PorA serosubtype.

Distribution of dopamine immunoreactivity in the rat, cat and monkey spinal cord.

In the present study, the distribution of dopamine (DA) was identified light microscopically in all segments of the rat, cat, and monkey spinal cord by using immunocytochemistry with antibodies directed against dopamine. Only fibers and (presumed) terminals were found to be immunoreactive for DA. Strongest DA labeling was present in the sympathetic intermediolateral cell column (IML). Strong DA labeling, consisting of many varicose fibers, was found in all laminae of the dorsal horn, including the central canal area (region X), but with the exception of the substantia gelatinosa, which was only sparsely labeled, especially in rat and monkey. In the motoneuronal cell groups DA labeling was also strong and showed a fine granular appearance. The sexually dimorphic cremaster nucleus and Onuf's nucleus (or its homologue) showed a much stronger labeling than the surrounding somatic motoneurons. In the parasympathetic area at sacral levels, labeling was moderate. The remaining areas, like the intermediate zone (laminae VI-VIII), were only sparsely innervated. The dorsal nucleus (column of Clarke) showed the fewest DA fibers, as did the central cervical nucleus, suggesting that cerebellar projecting cells were avoided by the DA projection. In all species, the descending fibers were located mostly in the dorsolateral funiculus, but laminae I and III also contained many rostrocaudally oriented fibers. It is concluded that DA is widely distributed within the spinal cord, with few differences between species, emphasizing that DA plays an important role as one of the monoamines that influences sensory input as well as autonomic and motor output at the spinal level.

Specificity of human bactericidal antibodies against PorA P1.7,16 induced with a hexavalent meningococcal outer membrane vesicle vaccine.

A set of isogenic strains was constructed from the meningococcal reference strain H44/76 (B:15:P1.7,16) which differed only in their outer membrane protein (OMP) compositions. First, three isogenic strains lacking the expression of either class 3 (PorB) or class 4 (RmpM) OMP or both were obtained. Second, three isogenic class 1 OMP loop-deficient strains of H44/76 lacking the predicted loop 1 or 4 or both of class 1 OMP (PorA) were obtained. Third, three isogenic class 1 OMP strains which differed by point mutations in the predicted loop 4 of subtype P1.16 were constructed. Strains were constructed through transformation with gene constructs made in Escherichia coli and their homologous recombination into the meningococcal chromosome. This study describes the contribution of one of the six class 1 OMPs, PorA P1.7,16, in the development of bactericidal antibodies after a single immunization of adult volunteers with 50 or 100 micrograms of protein within a hexavalent PorA outer membrane vesicle vaccine. PorA-, PorB-, and RpmM-deficient isogenic strains were used to define the human immune response against PorA. The loop-deficient isogenic strains were used to define the contribution of loops 1 and 4 of PorA in the development of bactericidal anti-PorA antibodies. The isogenic strains carrying a point mutation in loop 4 were used to study the cross-reactivity of the induced bactericidal antibodies against target strains showing microheterogeneity. The results indicate that a single immunization with the hexavalent PorA vaccine induced a dose-dependent bactericidal immune response, which is directed mainly against PorA. The epitope specificity of antibodies is directed mostly against loop 1, although loop 4 and as-yet-unidentified epitopes of PorA P1.7,16 are also involved.

Ultrastructural localization of cholinergic muscarinic receptors in rat brain cortical capillaries.

Cholinergic innervation of the cerebrovasculature is known to regulate vascular tone, perfusion rate and permeability of the microvascular wall. Notably the cholinergic innervation of cerebral capillaries is of interest since these capillaries form the blood-brain barrier. Although there is a general consensus as to the presence of nicotinic and muscarinic receptors in the domain of the capillary wall, their precise anatomical position is unknown. The subcellular localization of muscarinic receptors in rat cortical capillaries was approached by way of immunocytochemistry at the ultrastructural level using monoclonal antibody M35 against muscarinic receptor protein. Binding of this antibody in the microvascular domain was found in 5% of the capillaries studied and was exclusively present in perivascular astroglia, and never in endothelium or pericytes. Combined with reported data on presynaptic cholinergic innervation the results indicate a cholinergic innervation pattern of non-directed presynaptic terminal structures in apposition to cholinoceptive perivascular astroglia with muscarinic receptor positive endfeet embracing the capillary basement membrane. The possible functional significance of such a cholinergic vascular innervation pattern is discussed with respect to capillary dynamics and barrier function.

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization.

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.

Short inescapable stress produces long-lasting changes in the brain-pituitary-adrenal axis of adult male rats.

Recently, we reported that rats exposed to a single and short session of inescapable footshocks showed alterations in behavioural response to environmental stimuli which developed progressively over a week and remained present for at least 28 days. The aim of the present study was to investigate whether these behavioural changes were accompanied by alterations in the brain-pituitary-adrenal axis. Male Wistar rats were subjected to 10 inescapable footshocks (S) of 6 s duration and 1 mA intensity during a period of 15 min. Control rats (C) were placed in the shock apparatus for 15 min without receiving shocks. The effects of these experimental procedures were studied 14 days later. Exposure to shocks did not affect basal plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT). However, the novelty-induced ACTH response was increased in S rats as compared to C rats whereas the CORT response did not differ between C and S rats. The ACTH content of the anterior pituitary gland and adrenal weight were not affected by exposure to inescapable footshocks 14 days earlier. Quantitative immunocytochemistry of vasopressin (AVP) and corticotropin-releasing factor (CRF) in the external zone of the median eminence showed that prior footshock exposure increased the AVPi stores to 167% as compared to C rats, whereas CRFi content was not changed. In addition, S rats showed increased mineralocorticoid (MR) and glucocorticoid (GR) receptor binding capacity in the hippocampus as compared to C rats, whereas affinities were not affected. We conclude that a single and short session of inescapable footshocks has long-lasting effects on brain-pituitary-adrenal functioning concomitant with behavioural alterations.

Characterization of stress-induced long-term behavioural changes in rats: evidence in favor of anxiety.

Recently, we reported that rats exposed to one brief session of inescapable footshocks showed a gradually developing and long-lasting decrease in behavioural activity and an increase in defecation in an open field. The aim of the present study was to further characterize the long-lasting changes in behavioural responsiveness to environmental stimuli. For this purpose, behavioural paradigms validated as tools in the preclinical study of the psychobiology of depression were used. Footshocked rats (S) showed a decreased response latency in an one-way avoidance-escape task and decreased immobility in a forced swim test as compared to nonshocked control rats (C) 14 days after shock exposure. These S rats showed decreased behavioural activity and increased defecation as compared to the C rats in an open field test carried out 28 days after footshock exposure. In addition, footshock exposure did not affect the preference for or consumption of a 0.05% saccharin solution on a long-term basis, although a decreased consumption of this solution was evident in S rats on day 1 postshock. These S rats showed an exaggerated immobility response to a sudden reduction in background noise level compared to C rats while placed in a novel environment on day 11 postshock. We conclude that the long-term effects of one short session of inescapable footshocks are not compatible with what is supposed to represent behavioural manifestations of depression in animals. It is argued that the common denominator of shock-induced long-lasting changes is increased behavioural defensiveness, which is more likely related to increased fear and/or anxiety.

Corticosteroid receptor plasticity in the central nervous system of various rat models.

The action of corticosteroids in the central nervous system (CNS) is mediated by two distinct corticosteroid receptors: the mineralocorticoid and glucocorticoid receptors (MR and GR respectively). Using an established in vitro binding assay system, MR and GR binding parameters were determined in the hippocampal, hypothalamic and pituitary cytosol of various rat models. In the (pharmaco-)genetically selected rat lines, the apomorphine susceptible (APO-SUS) rats showed a significant increase in the hippocampal and pituitary MR binding capacities (but not affinity) compared to those in the apomorphine-unsusceptible (APO-UNSUS) rats. In immunologically-altered Lewis (LEW/N) rats and in spontaneously hypertensive rats (SHR), increased hippocampal MR capacity (but not affinity) and hypothalamic MR capacity were observed compared to their respective control, Wistar (WIST) and Wistar Kyoto (WKY) rats. In addition, compared to WKY rats, SHR rats also showed a much greater pituitary but lesser hypothalamic GR binding capacity. In rats subjected to alteration in environmental conditions, the long-term effects of a short inescapable stress resulted in a significant increase in both hippocampal MR and GR while the pituitary and hypothalamic MR and GR do not differ in the stress and control groups. In rats subjected to a defeat test, a decrease in hippocampal MR and GR was observed 3 weeks (but not 1 week) later.

Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry.

A digoxigenin-labeled antisense 42-mer oligonucleotide was used for the localization of the dopamine D2 receptor mRNA in the rat brain. The digoxigenin label was identified with alkaline phosphatase conjugated sheep-anti-digoxigenin. In good analogy with the known terminal fields of the dopaminergic system, various nuclei throughout the brain were labeled. Positive in situ hybridization signals were also found in dopamine cell groups of the substantia nigra and ventral tegmental area and in regions where a dopaminergic innervation is controversial, like the cerebellar cortex and the hippocampus. The non-radioactive in situ hybridization procedure described, shows the localization of the dopamine D2 receptor mRNA with a very high contrast and an optimal histological resolution.

Inescapable footshocks induce progressive and long-lasting behavioural changes in male rats.

Long-term behavioural consequences of exposure to a brief (15 min) session of inescapable footshocks (10 x 6 s, 1 mA) were investigated in male rats. The time course of the effects of inescapable footshocks was assessed by studying the behaviour of groups of rats at different post-stress intervals. Footshocked rats (S) did not differ from control (C) rats (exposed to the shock box for 15 min) in their behavioural response to an open field whether tested 1 h or 4 h post-stress. However, one day after shocks, S rats showed less locomotion and rearing, and more immobility and attention as compared to C rats. At 7 days or 14 days post-stress, S rats exhibited decreased locomotion, rearing, sniffing, and grooming, and increased immobility, attention, and defecation relative to C rats. In a second experiment, we investigated whether footshocks affect the behavioural response to a sudden drop in background noise during exposure to a novel environment. At 21 days post-stress, S rats showed a markedly enhanced immobility response to this stimulus as compared to C rats. In order to investigate whether rats could be exposed repeatedly to the open field without affecting the differences in behaviour between the two treatment groups, C and S rats were tested in an open field for the first time at 7 days post-stress, which yielded the typical effects of footshocks. When these rats were exposed to a second open-field test one week later, the behavioural responses of C and S rats were not different.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of anxiolytic and antidepressant drugs on long-lasting behavioural deficits resulting from one short stress experience in male rats.

Exposure of male Wistar rats to one single session of ten inescapable footshocks induces changes in the behavioural responses to environmental stimuli as measured in the "noise test" 14 days later. Shocked (S) rats showed decreased locomotion and rearing during the first 3 min of exposure to a novel environment compared to control (C) rats. When the 85 dB background noise was switched off a marked immobility response emerged in S rats, concomitant with a further decrease in locomotion and rearing. In response to noise off, C rats showed hardly any immobility and a much smaller reduction in locomotion and rearing compared to S rats. These long-lasting changes in behaviour were not reversed by acute treatment with the antidepressants fluvoxamine (3.0-30.0 mg/kg) and desmethylimipramine (DMI, 2.5-10.0 mg/kg) injected IP 30 min before the noise test on day 14 following the shock session. Chronic treatment (day 1 to day 14) with fluvoxamine or DMI did not reverse the behavioural deficits induced by shock exposure. Diazepam (0.6-5.0 mg/kg) administered acutely only reversed the effects of shock on locomotion during the first 3 min of the noise test. Chronic treatment with diazepam normalized the shock-induced decrease in locomotion and attenuated the rearing decrease during the first 3 min of the test, and partially restored shock-induced changes in behavioural response to switching off the noise. The most potent drug in this study was the 5-HT1A receptor agonist flesinoxan (0.3-3.0 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)