Brainstem catecholaminergic neurons play key roles in the autonomic, neuroendocrine, and behavioral responses to glucoprivation, yet the functions of the individual groups are not fully understood. Adrenergic C3 neurons project widely throughout the brain, including densely to sympathetic preganglionic neurons in the spinal cord, yet their function is completely unknown. Here we demonstrate in rats that optogenetic stimulation of C3 neurons induces sympathoexcitatory, cardiovasomotor functions. These neurons are activated by glucoprivation, but unlike the C1 cell group, not by hypotension. The cardiovascular activation induced by C3 neurons is less than that induced by optogenetic stimulation of C1 neurons; however, combined stimulation produces additive sympathoexcitatory and cardiovascular effects. The varicose axons of C3 neurons largely overlap with those of C1 neurons in the region of sympathetic preganglionic neurons in the spinal cord; however, regional differences point to effects on different sympathetic outflows. These studies definitively demonstrate the first known function of C3 neurons as unique cardiovasomotor stimulatory cells, embedded in the brainstem networks regulating cardiorespiratory activity and the response to glucoprivation.
Increased activity of the sympathetic nervous system is thought to play a role in the development and progression of cardiovascular disease. Recent work has shown that physical inactivity versus activity alters neuronal structure in brain regions associated with cardiovascular regulation. Our physiological studies suggest that neurons in the rostral ventrolateral medulla (RVLM) are more responsive to excitation in sedentary versus physically active animals. We hypothesized that enhanced functional responses in the RVLM may be due, in part, to changes in the structure of RVLM neurons that control sympathetic activity. We used retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) to identify bulbospinal catecholaminergic (C1) neurons in sedentary and active rats after chronic voluntary wheel-running exercise. We then digitally reconstructed their cell bodies and dendrites at different rostrocaudal levels. The dendritic arbors of spinally projecting TH neurons from sedentary rats were more branched than those of physically active rats (P?0.05). In sedentary rats, dendritic branching was greater in more rostral versus more caudal bulbospinal C1 neurons, whereas, in physically active rats, dendritic branching was consistent throughout the RVLM. In contrast, cell body size and the number of primary dendrites did not differ between active and inactive animals. We suggest that these structural changes provide an anatomical underpinning for the functional differences observed in our in vivo studies. These inactivity-related structural and functional changes may enhance the overall sensitivity of RVLM neurons to excitatory stimuli and contribute to an increased risk of cardiovascular disease in sedentary individuals.
Visualization is an integral aspect of genomics data analysis. Algorithmic-statistical analysis and interactive visualization are most effective when used iteratively. Epiviz (http://epiviz.cbcb.umd.edu/), a web-based genome browser, and the Epivizr Bioconductor package allow interactive, extensible and reproducible visualization within a state-of-the-art data-analysis platform.
Bulbospinal neurons in the ventral medulla play important roles in the regulation of sympathetic outflow. Physiological evidence suggests that these neurons are activated by N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors. In this study, we examined bulbospinal neurons in the ventral medulla for the presence of immunoreactivity for the NMDA NR1 subunit, which is essential for NMDA receptor function. Rats received bilateral injections of cholera toxin B into the tenth thoracic spinal segment to label bulbospinal neurons. Triple immunofluorescent labeling was used to detect cholera toxin B with a blue fluorophore, NR1 with a red fluorophore, and either tyrosine hydroxylase or tryptophan hydroxylase with a green fluorophore. In the rostral ventrolateral medulla, NR1 occurred in all bulbospinal tyrosine hydroxylase-positive neurons and 96% of bulbospinal tyrosine hydroxylase-negative neurons, which were more common in sections containing the facial nucleus. In the raphe pallidus, the parapyramidal region, and the marginal layer, 98% of bulbospinal tryptophan hydroxylase-positive neurons contained NR1 immunoreactivity. NR1 was also present in all of the bulbospinal tryptophan hydroxylase-negative neurons, which comprised 20% of bulbospinal neurons in raphe pallidus and the parapyramidal region. These results show that virtually all bulbospinal tyrosine hydroxylase and non-tyrosine hydroxylase neurons in the rostral ventrolateral medulla and virtually all bulbospinal serotonin and non-serotonin neurons in raphe pallidus and the parapyramidal region express NR1, the obligatory subunit of the NMDA receptor. NMDA receptors on bulbospinal neurons in the rostral ventral medulla likely influence sympathoexcitation in normal and pathological conditions.
Worldwide, lung cancer in never-smokers is ranked the seventh most common cause of cancer death; however, the etiology of lung cancer in never-smokers is unclear. We investigated associations for body mass index (BMI) at various ages, waist circumference, hip circumference, and physical activity with lung cancer in 158,415 never-smokers of the NIH-AARP Diet and Health Study. Multivariable hazard ratios (HR) and 95% confidence intervals (CI) were estimated from Cox proportional hazards models. Over 11 years of follow-up, 532 lung cancer cases occurred. The risk estimate for obese (BMI ? 30 kg/m(2)) participants at baseline was 1.21 (95%CI = 0.95-1.53) relative to those with a normal BMI between 18.5 ? BMI<25.0. Overweight (25.0 ? BMI<30.0) at age 18 (HR(overweight-vs-normal) = 1.51;95%CI = 1.01-2.26) and time spent sitting (HR(? 3 hrs-vs-<3 hrs) = 1.32;95%CI = 1.00-1.73) was each associated with lung cancer after adjustment for baseline BMI, as was waist (HR(Q4-vs-Q1) = 1.75;95%CI = 1.09-2.79) and hip circumference (HRQ4-vs-Q1 = 0.62;95%CI = 0.39-0.99), after mutual adjustment for each other and baseline BMI. No associations were observed for vigorous activity or television watching. In summary, using a large prospective cohort study, we found no evidence that BMI at baseline or middle age was associated with decreased lung cancer risk in never smokers. If anything, we observed some evidence for positive associations with a larger BMI or waist circumference.
Oxytocin (OXT) has been implicated in reproduction and social interactions and in the control of digestion and blood pressure. OXT-immunoreactive axons occur in the dorsal vagal complex (DVC; nucleus tractus solitarius, NTS, dorsal motor nucleus of the vagus, DMV, and area postrema, AP), which contains neurons that regulate autonomic homeostasis. The aim of the present work is to provide a systematic investigation of the OXT-immunoreactive innervation of dorsal motor nucleus of the vagus (DMV) neurons involved in the control of gastrointestinal (GI) function.
Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality.
Glucagon-like peptide 1 (GLP-1) and its analogue exendin-4 inhibit food intake, reduce blood glucose levels and increase blood pressure and heart rate by acting on GLP-1 receptors in many brain regions. Within the CNS, GLP-1 is produced only by preproglucagon (PPG) neurons. We suggest that PPG neurons mediate the central effects of GLP-1 by modulating sympathetic and vagal outflow. We therefore analysed the projections of PPG neurons to brain sites involved in autonomic control. In transgenic mice expressing yellow fluorescent protein (YFP) under the control of the PPG promoter, we assessed YFP-immunoreactive innervation using an anti-GFP antiserum and avidin-biotin-peroxidase. PPG neurons were intensely YFP-immunoreactive and axons could be easily discriminated from dendrites. YFP-immunoreactive cell bodies occurred primarily within the caudal nucleus tractus solitarius (NTS) with additional somata ventral to the hypoglossal nucleus, in raphé obscurus and in the intermediate reticular nucleus. The caudal NTS contained a dense network of dendrites, some of which extended into the area postrema. Immunoreactive axons were widespread throughout NTS, dorsal vagal nucleus and reticular nucleus with few in the hypoglossal nucleus and pyramids. The dorsomedial and paraventricular hypothalamic nuclei, ventrolateral periaqueductal grey and thalamic paraventricular nucleus exhibited heavy innervation. The area postrema, rostral ventrolateral medulla, pontine central grey, locus coeruleus/Barringtons nucleus, arcuate nucleus and the vascular organ of the lamina terminalis were moderately innervated. Only a few axons occurred in the amygdala and subfornical organ. Our results demonstrate that PPG neurons innervate primarily brain regions involved in autonomic control. Thus, central PPG neurons are ideally situated to modulate sympathetic and parasympathetic outflow through input at a variety of central sites. Our data also highlight that immunohistochemistry improves detection of neurons expressing YFP. Hence, animals in which specific populations of neurons have been genetically-modified to express fluorescent proteins are likely to prove ideal for anatomical studies.
The innervation of the nonpregnant rat uterus has been studied in histological sections, which contain only small samples of nerves and are unlikely to afford a complete picture of uterine innervation. Here we used whole-mount preparations of entire full-thickness uterine horns from nonpregnant rats in estrus to visualize autonomic or sensory nerves with peroxidase immunohistochemistry. Immunoreactivity was studied for tyrosine hydroxylase (TH)-labeled sympathetic nerves; vesicular acetylcholine transporter (VAChT), parasympathetic nerves; and substance P (SP) and calcitonin gene-related peptide (CGRP), sensory nerves. Neuropeptide Y (NPY) and nitric oxide synthase (NOS) identified more than one of these functionally distinct nerve types. Axons of all neurochemical classes entered the uterus at the mesometrium and innervated the uterine smooth muscle. The linea uteri, a dense band of longitudinal muscle opposite the mesometrium, contained more TH-, NPY-, CGRP-, and VAChT-immunoreactive axons than the remaining smooth muscle. Axons immunoreactive for NPY, SP, NOS, and VAChT formed a plexus near the circular muscle-endometrium interface. Rare TH- and NPY-immunoreactive axons and occasional CGRP-immunoreactive axons occurred close to uterine glands. Blood vessels had dense perivascular plexuses of TH- and NPY-containing axons and less dense NOS-, SP-, CGRP-, and VAChT-positive plexuses. The circular muscle plexus and glands were absent opposite the mesometrium. Uterine arterioles formed an interconnected network throughout the uterus. This article provides the first comprehensive description of the autonomic and sensory innervation of the nonpregnant rat uterus and will be a foundation for future studies on changes in uterine innervation caused by normal physiological or pathophysiological challenges.
Immunofluorescently stained whole mounts have proved useful for defining the innervation of the gut and large blood vessels. Nerves supplying other hollow organs are usually studied in sections, which provide much less information. Aiming to describe the entire innervation of rat uterus, we developed a method for immunoperoxidase staining of full-thickness whole mounts that allowed us to visualize all immunoreactive axons. Uterine horns were dissected out, slit open, stretched, pinned flat and fixed. Entire horns were treated with methanol/peroxide, buffered Triton X-100 and normal serum and then incubated in primary antibodies, biotinylated secondary antibodies and avidin-horseradish peroxidase (HRP), each for at least 3 days. Peroxidase reactions revealed immunoreactivity. Immunostained horns were dehydrated, infiltrated with epoxy resin, mounted on slides under Aclar coverslips and polymerized. We treated bladders, gut, major pelvic ganglia and thick sections of perfused medulla oblongata similarly to assess the applicability of the method. Using this method, we could map the entire uterine innervation provided by axons immunoreactive for a variety of antigens. We could also assess the entire tyrosine hydroxylase-immunoreactive innervation in all layers of bladder, gut and ganglia whole mounts and throughout 300 ?m sections of medulla. These observations show that this method for immunoperoxidase staining reliably reveals the complete innervation of full-thickness whole mounts of hollow organs and thick sections of central nervous tissue. The method has several advantages. The resin-embedded tissue does not degrade; the immunostaining is non-fading and permanent and neurochemically defined features can be mapped at large scale without confocal microscopy.
The promise, status and challenges of developing fusion power are outlined. The key physics and engineering principles are described and recent progress quantified. As the successful demonstration of 16 MW of fusion in 1997 in the Joint European Torus showed, fusion works. The central issue is therefore to make it work reliably and economically on the scale of a power station. We argue that to meet this challenge in 30 years we must follow the aggressive programme known as the Fast Track to Fusion. This programme is described in some detail.
Sympathetic preganglionic neurons (SPN) are critical links in the sympathetic neural circuitry that controls every organ in the body. All sympathetic outflow to the periphery comes from SPN, which send their axons from thoracic and upper lumbar spinal segments to innervate post-ganglionic neurons in sympathetic ganglia and chromaffin cells in the adrenal medulla. Despite over 30 years of study, we still do not have a sufficiently detailed understanding of the synaptic circuits through which these important neurons receive information from other central sites. We know that there is direct synaptic input to SPN from both supraspinal and intraspinal neurons, but not sensory neurons. Ultrastructural studies support functional evidence that amino acids are the primary fast-acting transmitters controlling SPN activity and indicate that an amino acid transmitter occurs in every synaptic input to an SPN. In addition, axons that synapse on SPN contain neuropeptides and monoamines, which would co-exist with and be released with the amino acids. Receptors and transporters for transmitters have also been localized in SPN inputs. Light and electron microscopic observations suggest that there are qualitative and/or quantitative differences in the neurochemical types and origins of axons, which provide synaptic input to SPN that supply different targets or have different functions. However, more research is required before it can be confirmed that SPN receive projection- or function-specific patterns of innervation. This information is likely to be important if we are to understand how the central nervous system differentially regulates sympathetic outflow to different target tissues.
The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague-Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1-L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS-positive and NOS-negative/ChAT-positive lateral horn neurons in SD with those in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS-positive and NOS-negative/ChAT-positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG-projecting, 54.8% +/- 0.7%; SCG-projecting, 75.3% +/- 1.2%; MPG-projecting, 89% +/- 1.1% and AM-projecting, 98.6% +/- 0.2%). Within each retrogradely labelled group of SPN, the NOS-positive proportion also varied with subnuclear location (e.g., 25.5% +/- 4.0% of CG-projecting SPN in the CAA vs. 82.7% +/- 7.6% of CG-projecting SPN in the dorsolateral funiculus). The numbers of NOS-positive and NOS-negative/ChAT-positive neurons in T9-T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide-synthesizing SPN in normotensive SD and hypertensive SHR.
Plant reproduction depends on pollen dispersal. For anemophilous (wind-pollinated) species, such as grasses and many trees, shedding pollen from the anther must be accomplished by physical mechanisms. The unknown nature of this process has led to its description as the paradox of pollen liberation. A simple scaling analysis, supported by experimental measurements on typical wind-pollinated plant species, is used to estimate the suitability of previous resolutions of this paradox based on wind-gust aerodynamic models of fungal-spore liberation. According to this scaling analysis, the steady Stokes drag force is found to be large enough to liberate anemophilous pollen grains, and unsteady boundary-layer forces produced by wind gusts are found to be mostly ineffective since the ratio of the characteristic viscous time scale to the inertial time scale of acceleration of the wind stream is a small parameter for typical anemophilous species. A hypothetical model of a stochastic aeroelastic mechanism, initiated by the atmospheric turbulence typical of the micrometeorological conditions in the vicinity of the plant, is proposed to contribute to wind pollination.
Exposure to chronic intermittent hypoxia (CIH) leads to significant autonomic and respiratory changes, similar to those observed in obstructive sleep apnea. The hypertension associated with CIH is due to sympathoexcitation triggered by long-term exposure to intermittent hypoxia. However, the mechanisms underlying these effects are unknown. Changes in central regulation of sympathetic activity may underlie CIH-induced hypertension. Since NO appears to be mainly sympathoinhibitory in the nucleus of the solitary tract (NTS), we hypothesized that CIH augments sympathetic activity, in part by reducing neuronal nitric oxide synthase (nNOS) expression and consequently nitric oxide (NO) production in this brain region. To test our hypothesis, juvenile male Wistar rats were exposed to CIH for 8 h/day for 10 days and sections of perfused brainstem were either stained to reveal nNOS-immunoreactivity or loaded with DAF 2-DA to label neurons containing NO. CIH rats showed a significant increase in mean arterial pressure and heart rate compared to controls. However, there was no significant difference in the distribution, staining intensity or numbers of nNOS-immunoreactive neurons in the NTS between experimental and control rats. We also found no significant change in NO content in the DAF 2-DA-loaded sections of NTS from CIH rats. Our data show that NO is not altered in the NTS of juvenile CIH rats, suggesting that nitrergic mechanisms, at least in the NTS, are unlikely to be involved in the sympathetic excitation that generates the hypertension observed after 10 days of CIH.
Preproglucagon (PPG) neurons produce glucagon-like peptide-1 (GLP-1) and occur primarily in the nucleus tractus solitarius (NTS). GLP-1 affects a variety of central autonomic circuits, including those controlling the cardiovascular system, thermogenesis, and most notably energy balance. Our immunohistochemical studies in transgenic mice expressing YFP under the control of the PPG promoter showed that PPG neurons project widely to central autonomic regions, including brainstem nuclei. Functional studies have highlighted the importance of hindbrain receptors for the anorexic effects of GLP-1. In this study, we assessed YFP innervation of neurochemically identified brainstem neurons in transgenic YFP-PPG mice. Immunoreactivity for YFP plus choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and/or serotonin (5-HT) was visualised with two- or three-colour immunoperoxidase labelling using black (YFP), brown and blue-grey reaction products. In the dorsal motor nucleus of the vagus (DMV), terminals from fine YFP-immunoreactive axons closely apposed a small proportion of ChAT-positive and rare TH-positive/ChAT-positive motor neurons, mostly ventral to AP. YFP-immunoreactive innervation was virtually absent from the compact and loose formations of the nucleus ambiguus. In the NTS, some TH-immunoreactive neurons were closely apposed by YFP-containing axons. In the A1/C1 column in the ventrolateral medulla, close appositions on TH-positive neurons were more common, particularly in the caudal portion of the column. A single YFP-immunoreactive axon usually provided 1-3 close appositions on individual ChAT- or TH-positive neurons. Serotonin-immunoreactive neurons were most heavily innervated, with the majority of raphé pallidus, raphé obscurus and parapyramidal neurons receiving several close appositions from large varicosities of YFP-immunoreactive axons. These results indicate that GLP-1 neurons innervate various populations of brainstem autonomic neurons. These include vagal efferent neurons and catecholamine neurons in areas linked with cardiovascular control. Our data also indicate a synaptic connection between GLP-1 neurons and 5-HT neurons, some of which might contribute to the regulation of appetite.
Although obesity has been directly linked to the development of many cancers, many epidemiological studies have found that body mass index (BMI)--a surrogate marker of obesity--is inversely associated with the risk of lung cancer. These studies are difficult to interpret because of potential confounding by cigarette smoking, a major risk factor for lung cancer that is associated with lower BMI.
Increasing numbers of cellular pathways are now recognized to be regulated via proteolytic processing events. The rhomboid family of serine proteases plays a pivotal role in a diverse range of pathways, activating and releasing proteins via regulated intramembrane proteolysis. The prototype rhomboid protease, rhomboid-1 in Drosophila, is the key activator of epidermal growth factor (EGF) receptor pathway signalling in the fly and thus affects multiple aspects of development. The role of the rhomboid family in plants is explored and another developmental phenotype, this time in a mutant of an Arabidopsis chloroplast-localized rhomboid, is reported here. It is confirmed by GFP-protein fusion that this protease is located in the envelope of chloroplasts and of chlorophyll-free plastids elsewhere in the plant. Mutant plants lacking this organellar rhomboid demonstrate reduced fertility, as documented previously with KOM-the one other Arabidopsis rhomboid mutant that has been reported in the literature-along with aberrant floral morphology.
The Large Hadron Collider (LHC) machine and detectors are now working superbly. There are good reasons to hope and expect that the new domain that the LHC is already exploring, operating at 7 TeV with a luminosity of 10(33) cm(-2) s(-1), or the much bigger domain that will be opened up as the luminosity increases to over 10(34) and the energy to 14 TeV, will provide clues that will usher in a new era in particle physics. The arguments that new phenomena will be found in the energy range that will be explored by the LHC have become stronger since they were first seriously analysed in 1984, although their essence has changed little. I will review the evolution of these arguments in a historical context, the development of the LHC project since 1984, and the outlook in the light of reports on the performance of the machine and detectors presented at this meeting.
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