Titanium Ions Play a Synergistic Role in the Activation of NLRP3 Inflammasome in Jurkat T Cells.

Release of titanium ions (Ti ions) frequently occurs around dental implants and, as a consequence, higher content of Ti is typically present in peri-implantitis tissue. Unlike chronic periodontitis, Ti ions may play a role in the development of peri-implantitis. Inflammasomes are multiprotein signal transduction complexes, involved in inflammation and immune response, which lead to the secretion of mature cytokines associated with the progression of peri-implantitis. It is well known that T lymphocytes dominate the immune response in peri-implantitis, but whether Ti ions can impact the assembly of functional inflammasomes in T cells still remains unclear. Here, we observed that the mRNA expression of NLRP3 and CASP1, as well as the secretion of IL-1ß, increased after 6-h incubation of Jurkat T cells with PHA and Ti ions. Moreover, measurement by confocal microscopy and flow cytometry assay indicates that Ti ions can promote the production of ROS, while NLRP3 expression and IL-1ß secretion are reduced after treatment of Jurkat cells with NAC (ROS scavenger). Taken together, we presently show that Ti ions can activate NLRP3 inflammasome and then promote IL-ß secretion in vitro, where ROS may play a mechanistic role in this activation process.

Development and reactivity of the immune system of Japanese quail lines divergently selected for the shape of the growth curve.

1. Selection strategies for broilers must balance rapid growth with the welfare and health of animals, strategies must deal with the trade-off with other vital functions.2. Divergent selection of Japanese quail for high (HG) and low (LG) relative body weight gain between 11 and 28 days of age has been conducted to accelerate linear phase growth without influencing the final adult body weight. Higher body growth rate is often connected with a weakened immune system. Therefore, the present study explored the immunological characterisation of quail from HG and LG lines, which differ substantially in their growth rate.3. The trial evaluated the maternal investment to immunologically active substances, cell-mediated immunity stimulated by phytohaemagglutinin (PHA) injection and the acute phase of the immune response to lipopolysaccharide (LPS) administration in three different phases of early postnatal growth.4. Except for higher lysozyme activity in the LG group when compared to the HG line, the maternal investment did not differ between the two lines. Plasma antibody concentrations responded quickly to any change in growth rate in both lines. Overall, it seems that initial rapid growth of the LG line had long-lasting effects on immune responsiveness, even after the growth rate of the HG line escalated during the linear phase of growth.5. The study indicated that changes in the growth rate caused by the selection for growth in meat-type Japanese quail can influence the acute phase of the immune response and development of the immune system.

Low interferon-gamma release in response to phytohemagglutinin predicts the high severity of diseases.

A clinically useful immune biomarker could potentially assist clinicians in their decision making. We stimulated T-cell proliferation to secret interferon gamma (IFN-γ) by phytohemagglutinin, and then measured the production of IFN-γ (mitogen value [M value]). We aimed to determine the relationship between the M value, clinical severity, and outcomes of diseases.In all, 484 patients admitted to intensive care units were enrolled in this retrospective study. The Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were collected within the first 24 hours. M value, C-reaction protein (CRP), procalcitonin (PCT), erythrocyte sedimentation rate (ESR), and routine blood tests were analyzed and collected during the study.When APACHE II scores were greater than 15 and M values were less than 6, the hospital mortality rose in a straight line. There was an inverse correlation between APACHE II score and M value (rs = -0.212, P < .001). There was a positive correlation between M value and lymphocyte numbers (b' = 0.249, P < .001); however, there was an inverse correlation between M value and WBC (b' = -0.230, P < .001), and ESR (b' = -0.100, P = .029). Neurological diseases had the greatest influence on APACHE II scores (b' = 10.356, P < .001), whereas respiratory diseases had the greatest influence on M value (b' = 1.933, P < .001). Furthermore, in the respiratory system, severe pneumonia had a greater influence on M value. Taking the APACHE II score as the gold standard, the area under the curve of M was 0.632 (95% confidence interval [CI] 0.575-0.690, P < .001), PCT was 0.647 (95% CI 0.589-0.705, P < .001), CRP was 0.570 (95% CI 0.511-0.629, P = .022), and ESR was 0.553 (95% CI 0.494-0.612, P = .078). Divided by M value = 5, the positive predictive value of the M value is 37.22% (115/309) and negative predictive value is 75.43% (132/175).The results show that the M values, PCT, and CRP were better than ESR to predict the severity of diseases. The number and proportion of lymphocytes also affected the result of the M value. To a certain extent, the M value may be a clinically useful immune biomarker, which may help clinicians objectively evaluate the severity of diseases, especially in the respiratory system.

Assessment of Trade-Offs between Simultaneous Immune Challenges in a Slow-Living Subterranean Rodent.

The coexistence of two or more infectious agents in the same host is common in nature. Given this, the study of trade-offs within the immune system itself is key to understanding how immune defenses act in wild species in their natural environment. Here we assessed the possible trade-off between an inflammatory response (induced by phytohemagglutinin [PHA]; involving innate and adaptive responses in the study species) and an antibody response (induced by sheep red blood cells [SRBC]; adaptive response) in a slow-living subterranean rodent, the Talas tuco-tuco (Ctenomys talarum Thomas, 1898). According to life-history theory, slow-living species should rely more heavily on adaptive immunity, which develops more slowly than an innate response but is beneficial against repeated infections. Individual physiological condition (estimated by measuring levels of infection and immune, nutritional, and stress parameters) was analyzed during immune challenges. Contrary to what was expected, we found that the magnitude and energetic costs of both immune responses were similar when stimulated alone or simultaneously. Variation in natural antibodies, neutrophils, basophils, total leukocytes, and the ratio of neutrophils to lymphocytes in relation to the different treatments was also detected. In particular, natural antibodies were negatively affected by the induction of both immune challenges simultaneously and an increase of neutrophil counts was detected in all animals with the exception of those challenged with SRBC, while the pattern of variation of basophils, total leukocytes, and ratio of neutrophils to lymphocytes was not clearly associated with any triggered immune response. In general, our results suggest the absence of an energetic or resource-based trade-off between the immune responses triggered by PHA and SRBC in C. talarum.

Early effects on the intestinal barrier and pancreatic function after enteral stimulation with protease or kidney bean lectin in neonatal rats.

Gut maturation naturally accelerates at weaning in altricial mammalian species, such as the rat. Mimicking this, gut development can also be induced precociously, 3-4 d earlier than it would occur naturally, by enteral exposure to phytohaemagglutinin (PHA), or various proteases. We investigated the early effects of gut provocation on intestinal barrier and pancreatic functions, to get a better understanding of the mechanisms that initiate gut maturation. The effects of oral administration of protease (trypsin) or PHA to 14-d-old suckling rats were studied during 24 h in comparison with water-fed controls. Intestinal in vivo permeability was assessed by oral administration of different-sized marker molecules and measuring their passage into the blood or urine 3 h later. A period of 24 h following oral administration, both PHA and protease provocation stimulated small intestinal (SI) growth and pancreatic secretion, as indicated by decreased pancreatic trypsin and increased luminal enzyme content. Within 1 h of oral administration, both treatments prevented the absorption of macromolecules to blood that was observed in controls. PHA treatment hindered the passage of fluorescein isothiocyanate-dextran (FD) 4 to blood, whereas protease treatment temporarily increased plasma levels of FD4, and the urine lactulose:mannitol ratio, indicating increased intestinal leakiness. Following protease treatment, fluorescence microscopy showed decreased vesicular uptake of FD70 in the proximal SI and increased epithelial fluorescence in the distal SI. In conclusion, PHA and protease differed in their early effects on the intestinal barrier; both exerted a blocking effect on epithelial endocytosis, whereas protease treatment alone temporarily increased epithelial leakiness, which seemed to be confined to the distal SI.

Impact of dietary induced precocious gut maturation on cecal microbiota and its relation to the blood-brain barrier during the postnatal period in rats.

BACKGROUND: Precocious maturation of the gastrointestinal barrier (GIB) in newborn mammals can be induced by dietary provocation, but how this affects the gut microbiota and the gut-brain axis remains unknown. The objective of this study was to investigate effects of induced GIB maturation on gut microbiota composition and blood-brain barrier (BBB) permeability. METHODS: Suckling rats were studied at 72 h after gavage with phytohemagglutinin (PHA) or microbial protease (PT) to induce maturation of GIB. For comparison, untreated suckling and weaned rats were included (n = 10). Human serum albumin (HSA) was administered orally and analyzed in blood to assess permeability of the GIB, while intraperitoneally injected bovine serum albumin (BSA) was measured in the brain tissue for BBB permeability. The cecal microbial composition, plasma lipopolysaccharide-binding protein (LBP) levels and short-chain fatty acids in serum and brain were analyzed. KEY RESULTS: Cessation of HSA passage to blood after PHA or PT treatment was similar to that seen in weaned rats. Interestingly, concomitant increases in cecal Bacteroidetes and plasma LBP levels were observed after both PHA and PT treatments. The BBB passage of BSA was surprisingly elevated after weaning, coinciding with lower plasma LBP levels and specific microbial taxa and increased acetate uptake into the brain. CONCLUSIONS & INFERENCES: This study provides evidence that the gut microbiota alteration following induced precocious GIB maturation may induce low-grade systemic inflammation and alter SCFAs utilization in the brain which may also play a potential role in GIB-BBB dysfunction disorders in neonates.

Phaseolus vulgaris lectins: A systematic review of characteristics and health implications.

Legume lectins are carbohydrate-binding proteins of non-immune origin. Significant amounts of lectins have been found in Phaseolus vulgaris beans as far back as in the last century; however, many questions about their potential biological roles still remain obscure. Studies have shown that lectins are anti-nutritional factors that can cause intestinal disorders. Owing to their ability to act as toxic allergens and hemagglutinins, the Phaseolus vulgaris lectins are of grave concern for human health and safety. Nonetheless, their potential beneficial health effects, such as anti-cancer, anti-human immunodeficiency virus (anti-HIV), anti-microbial infection, preventing mucosal atrophy, reducing type 2 diabetes and obesity, promoting nutrients absorption and targeting drugs, are of immense interest. The significance of Phaseolus vulgaris lectins in biological researches and the potential biomedical applications have placed tremendous emphasis on the development of purification strategies to obtain the protein in pure and stable forms. These purification strategies entail considerations such as effects of proteolysis, heating, gamma radiation, and high-hydrostatic-pressure that can have crucial outcomes in either eliminating or improving bioactivities of the lectins. Thus, up-to-date research findings of Phaseolus vulgaris lectins on different aspects such as anti-nutritional and health impacts, purification strategies and novel processing trends, are systematically reviewed.

Acute hypoxia activates hypothalamic paraventricular nucleus-projecting catecholaminergic neurons in the C1 region.

Catecholaminergic C1 cells reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM) and can be activated by hypoxia. These neurons regulate the hypothalamic pituitary axis via direct projections to the hypothalamic paraventricular nucleus (PVH) and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. Based on the various effects attributed to the C1 cells and what is currently known of their synaptic inputs, our hypothesis is that acute hypoxia (AH) activates RVLM projecting catecholaminergic neurons to PVH. Anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) was unilaterally injected into the RVLM and a retrograde tracer Cholera toxin b (CTb) was unilaterally injected into the PVH region. After ten days, male Wistar rats that received CTb injection into the PVH were subjected to AH (8% O2, balanced with N2) or normoxia (21% O2) for 3h. Acute hypoxia significantly increased Fos immunoreactivity in the C1 region (68±2 neurons), and half of the RVLM cells activated are catecholaminergic (35±2 neurons). We observed that 23±4% of the RVLM projecting PVH cells that were activated by AH were also C1 cells. The presence of varicosities containing PHA-L in PVH region was also observed. The present results suggest that catecholaminergic C1-PVH projection is hypoxia-sensitive and the pathway between these two important brain areas can be one more piece in the complex puzzle of neural control of autonomic regulation during hypoxia.

An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L).

A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50-200µm in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specialization, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be visualized in the same tissue sections as PHA-L localized with immunofluorescence techniques. © 1984. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms.

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.

Reference levels for corticosterone and immune function in farmed saltwater crocodiles (Crocodylus porosus) hatchlings using current Code of Practice guidelines.

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice.

PHA-induced inflammation is not energetically costly in the subterranean rodent Ctenomys talarum (tuco-tucos).

Immune activity has been proposed to be associated with substantial costs, due to trade-offs with other functions or activities that share common resources and contribute to an animal's fitness. However, direct estimates of the cost of mounting an immune response are few and have been performed mainly in birds. Thus, further work is needed to clarify the relative costs of different components of the immune system and the role of environmental and life-history traits in modulating the costs of resistance. Within the components of immunity, inflammation is considered to be associated with a larger energetic expenditure. Here, we evaluated the energetic cost of the inflammatory response to phytohemagglutinin (PHA) in a wild population of a subterranean rodent, Ctenomys talarum, and the trade-offs between immune activity and reproduction. C. talarum develops an inflammatory response to PHA, but contrary to our predictions, this response was not associated with an increase in oxygen consumption regardless of reproductive status or sex. Our study shows that an immune challenge may not always result in a detectable energetic cost. We discuss the possibility that other currencies could be underlying the cost, such as micro-or macronutrients requirements, autoimmunity or oxidative stress.

Micronutrient supplementation and T cell-mediated immune responses in patients with tuberculosis in Tanzania.

Limited studies exist regarding whether incorporating micronutrient supplements during tuberculosis (TB) treatment may improve cell-mediated immune response. We examined the effect of micronutrient supplementation on lymphocyte proliferation response to mycobacteria or T-cell mitogens in a randomized trial conducted on 423 patients with pulmonary TB. Eligible participants were randomly assigned to receive a daily dose of micronutrients (vitamins A, B-complex, C, E, and selenium) or placebo at the time of initiation of TB treatment. We found no overall effect of micronutrient supplements on lymphocyte proliferative responses to phytohaemagglutinin or purified protein derivatives in HIV-negative and HIV-positive TB patients. Of HIV-negative TB patients, the micronutrient group tended to show higher proliferative responses to concanavalin A than the placebo group, although the clinical relevance of this finding is not readily notable. The role of nutritional intervention in this vulnerable population remains an important area of future research.

T-cell responses in oiled guillemots and swans in a rehabilitation setting.

Aquatic birds are commonly affected by oil spills. Despite rehabilitation efforts, the majority of rehabilitated common guillemots (Uria aalge) do not survive, whereas mute swans (Cygnus olor) tend to have higher postrelease survival. Polyaromatic hydrocarbons (PAHs) present in crude oil and diesel are immunotoxic in birds affecting cell-mediated responses to immunogens. Because it is a target of PAH toxicity, T-lymphocyte response to controlled mitogen administration (phytohemagglutinnin test) was investigated in a scoping study as a potentially useful minimally invasive in vivo test of cell-mediated immunity. The test was performed on 69 mute swans and 31 common guillemots stranded on the Norfolk and Lincolnshire coastline and inland waterways in England (UK) either due to injury or to contamination with crude or diesel oil. T-lymphocyte response was significantly decreased in swans with greater oil scores. T-lymphocyte responses were also decreased in guillemots, but this finding was not statistically significant.

On lateral septum-like characteristics of outputs from the accumbal hedonic "hotspot" of Peciña and Berridge with commentary on the transitional nature of basal forebrain "boundaries".

Peciña and Berridge (2005; J Neurosci 25:11777-11786) observed that an injection of the µ-opioid receptor agonist DAMGO (D-ala(2) -N-Me-Phe(4) -Glycol(5) -enkephalin) into the rostrodorsal part of the accumbens shell (rdAcbSh) enhances expression of hedonic "liking" responses to the taste of an appetitive sucrose solution. Insofar as the connections of this hedonic "hotspot" were not singled out for special attention in the earlier neuroanatomical literature, we undertook to examine them. We observed that the patterns of inputs and outputs of the rdAcbSh are not qualitatively different from those of the rest of the Acb, except that outputs from the rdAcbSh to the lateral preoptic area and anterior and lateral hypothalamic areas are anomalously robust and overlap extensively with those of the lateral septum. We also detected reciprocal interconnections between the rdAcbSh and lateral septum. Whether and how these connections subserve hedonic impact remains to be learned, but these observations lead us to hypothesize that the rdAcbSh represents a basal forebrain transition area, in the sense that it is invaded by neurons of the lateral septum, or possibly transitional neuronal forms sharing properties of both structures. We note that the proposed transition zone between lateral septum and rdAcbSh would be but one of many in the basal forebrain and conclude by reiterating the longstanding argument that the transitional nature of such boundary areas has functional importance, of which the precise nature will remain elusive until the neurophysiological and neuropharmacological implications of such zones of transition are more generally acknowledged and better addressed.

Connections of the lateral hypothalamic area juxtadorsomedial region in the male rat.

The connections of the lateral hypothalamic area juxtadorsomedial region (LHAjd) were investigated in a series of pathway-tracing experiments involving iontophoretic co-injection of the tracers Phaseolus vulgaris-leucoagglutinin (PHA-L; for outputs) and cholera toxin B subunit (CTB; for inputs). Results revealed that the LHAjd has connections with some 318 distinct gray matter regions encompassing all four subsystems-motor, sensory, cognitive, and behavioral state-included in a basic structure-function network model of the nervous system. Integration of these subsystems is necessary for the coordination and control of emotion and behavior, and in that regard the connections of the LHAjd indicate that it may have a prominent role. Furthermore, the LHAjd connections, together with the connections of other LHA differentiations studied similarly to date, indicate a distinct topographic organization that suggests each LHA differentiation has specifically differing degrees of involvement in the control of multiple behaviors. For the LHAjd, its involvement to a high degree in the control of defensive behavior, and to a lesser degree in the control of other behaviors, including ingestive and reproductive, is suggested. Moreover, the connections of the LHAjd suggest that its possible role in the control of these behaviors may be very broad in scope because they involve the somatic, neuroendocrine, and autonomic divisions of the nervous system. In addition, we suggest that connections between LHA differentiations may provide, at the level of the hypothalamus, a neuronal substrate for the coordinated control of multiple themes in the behavioral repertoire.

Experimentally increased in ovo testosterone leads to increased plasma bactericidal activity and decreased cutaneous immune response in nestling house wrens.

Maternally derived testosterone in the eggs of birds may benefit nestlings by increasing various aspects of their growth, condition and behavioral development, but these benefits may come at a cost, including suppression of immune responsiveness. Experiments on a variety of species in which in ovo levels of testosterone have been experimentally increased have produced mixed results; some have found increased growth and suppressed immune function of nestlings whereas others have found the opposite. In an attempt to clarify the relationship between in ovo testosterone and nestling size, mass, health state and immune responsiveness, we experimentally increased levels of testosterone in the eggs of house wrens (Troglodytes aedon). We simultaneously determined the size, mass, hematocrit (a measure of health state), cutaneous immune response to phytohaemagglutinin and plasma bactericidal activity of nestlings near the time of fledging. We predicted that nestlings hatching from testosterone-injected eggs would exhibit lower immune responsiveness, but achieve greater mass, size and condition, than nestlings hatching from vehicle-injected control eggs. Instead, we found that nestlings hatching from testosterone-injected eggs had a weaker cutaneous immune response but greater bactericidal activity than those hatching from control eggs. They did not, however, differ significantly in mass, size or hematocrit from controls. These results suggest that experimentally increased in ovo testosterone induced a trade-off between bactericidal activity and the cutaneous immune response. The opposite responses by two different measures of immune function to experimentally increased in ovo testosterone underscore the importance of including multiple immune assays when investigating the potential for trade-offs with the immune system and other physiological functions.

Basal superoxide as a sex-specific immune constraint.

There is increasing evidence that reactive oxygen species (ROS), a group of unstable and highly reactive chemical molecules, play a key role in regulating and maintaining life-history trade-offs. Upregulation of ROS in association with immune activation is costly because it may result in an imbalance between pro- and antioxidants and, hence, oxidative damage. Previous research aimed at quantifying this cost has mostly focused on changes in the pro-/antioxidant balance subsequent to an immune response. Here, we test the hypothesis that systemic ROS may constrain immune activation. We show that systemic, pre-challenge superoxide (SO) levels are negatively related to the strength of the subsequent immune response towards the mitogen phytohaemagglutinin in male, but not female painted dragon lizards (Ctenophorus pictus). We therefore suggest that systemic SO constrains immune activation in painted dragon males. We speculate that this may be due to sex-specific selection pressures on immune investment.

Brain circuits mediating baroreflex bradycardia inhibition in rats: an anatomical and functional link between the cuneiform nucleus and the periaqueductal grey.

Defence responses triggered experimentally in rats by stimulation of the dorsomedial nucleus of the hypothalamus (DMH) and the dorsolateral periaqueductal grey matter (PAG) inhibit the cardiac baroreflex response (i.e. bradycardia). It has also been proposed that the midbrain cuneiform nucleus (CnF) is involved in active responses. Our aim was to identify the neurocircuitry involved in defence-induced baroreflex inhibition, with a particular focus on the link between DMH, CnF and dorsolateral PAG. Microinjection of the anterograde tracer Phaseolus vulgaris leucoaggutinin into the CnF revealed a dense projection to the dorsolateral PAG. Moreover, activation of neurons in the CnF induced increased expression of Fos protein in the dorsolateral PAG. Inhibition of neurons of the CnF or dorsolateral PAG prevented the inhibition of baroreflex bradycardia induced by DMH or CnF stimulation, respectively. These results provide a detailed description of the brain circuitry underlying acute baroreflex modulation by neurons of the DMH. Our data have shown for the first time that the CnF plays a key role in defence reaction-associated cardiovascular changes; its stimulation, from the DMH, activates the dorsolateral PAG, which, in turn, inhibits baroreflex bradycardia.

Microcapillary specifically designed for pressure microinjections of very low volumes.

Tracer micronjections are very widely used in brain mapping research. While administration of small quantities/volumes of tracers can easily be achieved through iontophoresis, in the case of pressure injections the volume of the substances injected are much more difficult to control. Instead of using different variables like hydrodynamic conductance, pressure pulses, pressure-temperature-dependent protocols to quantify very small volumes in the nanoliter range out of a higher volume, within the confines of the present study a novel microcapillary design is presented. This microcapillary contains exactly the volume of tracer one intends to inject, therefore the danger of flooding large brain areas or the risk of tracer leakage to neighbouring nuclei are completely eliminated, and in the same time this design assures that very small and circumscribed areas can be labeled and their connections mapped, thus making the experiments more specific. In combination with high precision stereotaxic measurements these small volumes of tracers can yield well targeted and very discrete injection sites that make possible the mapping of individual nuclear subdivisions or delicate nuclei in the brain.