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Adenosine 5'-triphosphate (ATP) has been shown to induce release of cytokines implicated in fever, including interleukin(IL)-1beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha). The role of ATP-mediated purinergic signalling in fever and cytokine release during systemic inflammation was investigated by studying the effects of P2 receptor antagonists suramin, pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and Brilliant Blue G (BBG) on changes in body temperature and the increases in plasma levels of IL-1beta, IL-6, and TNFalpha induced by bacterial lipopolysaccharide (LPS) in rats. LPS (Escherichia coli; 50 microg kg(-1))-induced febrile response was attenuated by suramin (25 mg kg(-1) and 100 mg kg(-1)), PPADS (25 mg kg(-1)), and a more selective P2X(7) receptor antagonist BBG (100 mg kg(-1)) injected intraperitoneally before the induction of fever. The increase in plasma concentrations of IL-1beta and IL-6, measured 1 h after LPS treatment, was reduced by PPADS (25 mg kg(-1)) and BBG (100 mg kg(-1)). LPS-induced increase in plasma TNF-alpha concentration was also markedly attenuated by BBG (100 mg kg(-1)), but not by PPADS (25 mg kg(-1)). These data indicate that purinergic signalling plays an important role in the mechanisms responsible for the LPS-induced febrile response and increases in the levels of circulating cytokines. We suggest that ATP acting via P2X(7) receptors induces release of pyrogenic cytokines to mediate fever during systemic inflammation.

The pathogenesis of hepatic encephalopathy (HE) in cirrhosis is multifactorial and ammonia is thought to play a key role. Astroglial dysfunction is known to be present in HE. Astrocytes are extensively connected by gap junctions formed of connexins, which also exist as functional hemichannels allowing exchange of molecules between the cytoplasm and the extracellular milieu. The astrocyte-neuron lactate shuttle hypothesis suggests that neuronal activity is fuelled (at least in part) by lactate provided by neighbouring astrocytes. We hypothesised that in HE, astroglial dysfunction could impair metabolic communication between astrocytes and neurons. In this study we determined whether hyperammonemia leads to hemichannel dysfunction and impairs lactate transport in the cerebral cortex using rat models of HE (bile duct ligation [BDL] and induced-hyperammonemia [HA]) and also evaluated the effect of ammonia-lowering treatment (ornithine phenylacetate, OP). Plasma ammonia concentration in BDL rats was indeed significantly reduced by OP treatment. Biosensor recordings demonstrated that HE is associated with a significant reduction in both tonic and hypoxia-induced lactate release in the cerebral cortex, which was normalized by OP treatment. Cortical dye loading experiments revealed hemichannel dysfunction in HE with improvement following OP treatment, while the expression of key connexins was unaffected. Conclusion. The results of the present study demonstrate that HE is associated with CNS hemichannel dysfunction, with ammonia playing a key role. The data provide evidence of a potential neuronal energy deficit due to impaired hemichannel-mediated lactate transport between astrocytes and neurons as a possible mechanism underlying pathogenesis of HE. This article is protected by copyright. All rights reserved.

Recent evidence suggests that ATP is a mediator of central (within the ventral surface of the medulla) and peripheral (within the carotid body) chemosensory transduction. This short review discusses the data obtained in experiments in vivo and in vitro supporting this hypothesis. P2 receptors for ATP are expressed within the ventrolateral medulla as well as by the peripheral chemosensory afferent neurones. Blockade of P2 receptors in the ventrolateral medulla attenuates the CO2-induced increase in respiration while blockade of purinergic signalling impairs carotid body function and diminishes the ventilatory response to hypoxia. Furthermore, ATP is released from the ventral surface of the medulla during hypercapnia and from the carotid body during hypoxia. Finally, exogenous ATP applied on the ventral surface of the medulla evokes rapid increase in phrenic nerve activity, while ATP applied to the carotid body evokes marked excitation of the carotid sinus nerve afferents. We suggest that in the ventrolateral medulla ATP is produced following CO2/H+-induced activation of central chemosensory elements (neuronal and/or glial) and acts within the respiratory network to produce physiologically relevant changes in ventilation. In the carotid body, ATP contributes in a significant manner to the transmission of the sensitivity of the carotid body to changes in arterialPand may be considered as a key transmitter released by chemoreceptor cells to activate endings of the sinus nerve afferent fibres.

Supplemental Digital Content is available in the text., Systemic arterial hypertension has been previously suggested to develop as a compensatory condition when central nervous perfusion/oxygenation is compromised. Principal sympathoexcitatory C1 neurons of the rostral ventrolateral medulla oblongata (whose activation increases sympathetic drive and the arterial blood pressure) are highly sensitive to hypoxia, but the mechanisms of this O2 sensitivity remain unknown. Here, we investigated potential mechanisms linking brainstem hypoxia and high systemic arterial blood pressure in the spontaneously hypertensive rat. Brainstem parenchymal PO2 in the spontaneously hypertensive rat was found to be ≈15 mm Hg lower than in the normotensive Wistar rat at the same level of arterial oxygenation and systemic arterial blood pressure. Hypoxia-induced activation of rostral ventrolateral medulla oblongata neurons was suppressed in the presence of either an ATP receptor antagonist MRS2179 or a glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol, suggesting that sensitivity of these neurons to low PO2 is mediated by actions of extracellular ATP and lactate. Brainstem hypoxia triggers release of lactate and ATP which produce excitation of C1 neurons in vitro and increases sympathetic nerve activity and arterial blood pressure in vivo. Facilitated breakdown of extracellular ATP in the rostral ventrolateral medulla oblongata by virally-driven overexpression of a potent ectonucleotidase transmembrane prostatic acid phosphatase results in a significant reduction in the arterial blood pressure in the spontaneously hypertensive rats (but not in normotensive animals). These results suggest that in the spontaneously hypertensive rat, lower PO2 of brainstem parenchyma may be associated with higher levels of ambient ATP and l-lactate within the presympathetic circuits, leading to increased central sympathetic drive and concomitant sustained increases in systemic arterial blood pressure.

Respiratory depression remains an important clinical problem that limits the use of opiate analgesia. Activation of AMPA glutamate receptors has been shown to reverse fentanyl-induced respiratory changes. Here, we explored whether tianeptine, a drug known for its ability to phosphorylate AMPA receptors, can be used to prevent opiate-induced respiratory depression. A model of respiratory depression in conscious rats was produced by administration of morphine (10 mg/kg, i.p.). Rats were pre-treated with test compounds or control solutions 5 min prior to administration of morphine. Respiratory activity was measured using whole-body plethysmography. In conscious animals, tianeptine (2 and 10 mg/kg, ip) and DP-201 (2-(4-((3-chloro-6-methyl-5,5-dioxido-6,11-dihydrodibenzo[c,f][1,2] thiazepin-11-yl)amino)butoxy)acetic acid; tianeptine analogue; 2 mg/kg, ip) triggered significant (~30%) increases in baseline respiratory activity and prevented morphine-induced respiratory depression. These effects were similar to those produced by an ampakine CX-546 (15 mg/kg, ip). The antinociceptive effect of morphine (hot plate test) was unaffected by tianeptine pre-treatment. In conclusion, the results of the experiments conducted in conscious rats demonstrate that systemic administration of tianeptine increases respiratory output and prevents morphine-induced respiratory depression without interfering with the antinociceptive effect of opiates.

The time course of adenosine release in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) during acute systemic hypoxia was investigated in the anaesthetised rat by means of amperometric enzymatic sensors. It was found that acute hypoxia induced a significant delayed increase in adenosine level (reaching levels as high as 5 microM) in the NTS and that hypoxia-induced release of adenosine was similar at various regions of the NTS along its rostro-caudal axis. Significantly smaller or no increases in adenosine levels at all in response to hypoxia were observed in the VLM. The increase in adenosine level in the NTS occurred during reoxygenation after the termination of the hypoxic challenge and was accompanied by a smaller increase in inosine concentration. At the dorsal surface of the brainstem, only release of inosine was detected following acute hypoxia. Addition of the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP (200 microM) to the dorsal surface of the brainstem completely abolished the signal evoked by hypoxia, suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. This study indicates that following systemic hypoxia, adenosine levels in the NTS increase to a significantly greater extent than in the VLM. However, the increase in adenosine concentration in the NTS occurs too late to be responsible for the hypoxia-induced depression of the respiratory activity.

1 P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the regulation of body temperature (T-b). The role of ATP acting on these P2 receptors in thermoregulation was investigated by studying the effects of the stable ATP analogue alpha,beta-methyleneATP (alpha,beta-meATP) and P2 receptor antagonists suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on T-b when injected intracerebroventricularly (i.c.v.) via a pre-implanted cannula in conscious rats at various ambient temperatures and during lipopolysaccharide (LPS)-induced fever.2 Depending on ambient temperature, alpha,beta-meATP (0.2 mumol, i.c.v.) induced a fall in T-b (-3.3degreesC, P < 0.05), no changes in T-b when compared to pre-injection levels, or an increase in T-b (similar to1.0degreesC), in rats maintained at 10degreesC, 25degreesC and 30degreesC ambient temperature, respectively.3 Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in T-b (on average 1.2degreesC. P

Astrocytes occupy a strategic position in the brain where they can act as an interface between neurones and blood vessels, and neurones and the cerebro-spinal fluid. This location is ideal for functioning as interoceptors, as they may sense changes in brain microenvironment and contribute to the adaptive homeostatic responses coordinated by neuronal networks. Here we briefly review some of the recent evidence, which implicates the involvement of astrocytes in the central nervous control of breathing, sympathetic tone and blood glucose levels. L-lactate appears a potentially crucial signaling molecule in the communication between astrocytes and neurones. Based on the available evidence, we conclude that astrocytes contribute to the homeostasis by playing a significant role in the brain's interoceptive mechanisms.

The central nervous origins of functional parasympathetic innervation of cardiac ventricles remain controversial.This study aimed to identify a population of vagal preganglionic neurons that contribute to the control of ventricular excitability. An animal model of synuclein pathology relevant to Parkinson's disease was used to determine whether age-related loss of the activity of the identified group of neurons is associated with changes in ventricular electrophysiology.In vivo cardiac electrophysiology was performed in anesthetized rats in conditions of selective inhibition of the dorsal vagal motor nucleus (DVMN) neurons by pharmacogenetic approach and in mice with global genetic deletion of all family members of the synuclein protein.In rats anesthetized with urethane (in conditions of systemic beta-adrenoceptor blockade), muscarinic and neuronal nitric oxide synthase blockade confirmed the existence of a tonic parasympathetic control of cardiac excitability mediated by the actions of acetylcholine and nitric oxide. Acute DVMN silencing led to shortening of the ventricular effective refractory period (vERP), a lowering of the threshold for triggered ventricular tachycardia, and prolongation of the corrected QT (QTc) interval. Lower resting activity of the DVMN neurons in aging synuclein-deficient mice was found to be associated with vERP shortening and QTc interval prolongation.Activity of the DVMN vagal preganglionic neurons is responsible for tonic parasympathetic control of ventricular excitability, likely to be mediated by nitric oxide. These findings provide the first insight into the central nervous substrate that underlies functional parasympathetic innervation of the ventricles and highlight its vulnerability in neurodegenerative diseases.

Nature Communications 5: Article number: 3284 (2014); Published: 11 February 2014; Updated: 28 May 2014. While this Article was under consideration, Bozzo et al. published their findings on the inhibitory effects of L-lactate on cortical neuron activity. This paper should have been cited in the Discussion as follows

This review highlights the importance of neural mechanisms capable of protecting the heart against lethal ischemia/reperfusion injury. Increased parasympathetic (vagal) activity limits myocardial infarction, and recent data suggest that activation of autonomic reflex pathways contributes to powerful innate mechanisms of cardioprotection underlying the remote ischemic conditioning phenomena.

This study tested the hypothesis that during hypercapnia partial removal of a tonic GABA-mediated inhibition contributes to the increase in activity of the ventrolateral medulla (VLM) inspiratory neurones. Extracellular recordings were taken from 22 inspiratory neurones in the VLM of rats anaesthetised with pentobarbitone and artificially ventilated. It was found that during hypercapnia, changes in the discharge pattern (i.e. an increase in the discharge frequency during the neurone's normally active phase) and firing frequency of the VLM inspiratory neurones were similar to those evoked by GABA(A) receptor antagonist bicuculline methiodide (BMI, 10 mM, 20 nA), applied ionophoretically in conditions of normocapnia. During hypercapnia BMI (20 nA) failed to evoke a further increase in firing of these neurones. This suggests that CO2-evoked activation of VLM inspiratory neurones may involve a withdrawal in part of a tonic GABA(A) receptor-mediated inhibition. This disinhibition may play a role in the hypercapnia-induced increase in ventilatory activity.

Reperfusion following myocardial ischaemia induces a rapid change in endothelial function. Endotheliumdependent vasodilatation is impaired due to attenuated production of nitric oxide (NO) and/or enhanced inactivation of NO by superoxide. In addition, production of the vasoconstrictor peptide endothelin is increased. This results in a change from a state of vasorelaxation in the normal situation to an increased vasoconstrictor tone following reperfusion. There is a marked infiltration of neutrophils into the jeopardized myocardium during early reperfusion, which contributes to cell death. The adhesion of neutrophils to the endothelium is mediated by cell adhesion molecules which are expressed on endothelial cells, vascular smooth muscle cells and neutrophils during reperfusion. The reduction in NO bioavailability during early reperfusion contributes to several events in the reperfusion injury. Therefore, maintenance of NO levels is important for protection against reperfusion injury. Administration of the NO substrate l-arginine or NO donors in connection with reperfusion attenuates neutrophil accumulation and reduces infarct size. Furthermore, blockade of adhesions molecules reduces the extent of reperfusion injury. Endothelin also seems to be of importance for the development of reperfusion injury. Endothelin receptor antagonists improve endothelial and ventricular function and reduce infarct size following ischaemia and reperfusion. The mechanism behind the cardioprotective eect of endothelin receptor antagonists seems to involve inhibition of neutrophil-mediated injury and NO production. The endothelium plays an important role during the development of reperfusion injury. The reperfusion injury is reduced by enhancing NO levels, blocking endothelin receptors and inhibiting neutrophil adhesion.

The influence of bombesin (1 μg/0.1 ml artificial cerebrospinal fluid) on neuronal thermosensitivity of the preoptic area of the anterior hypothalamus in brain slices of 5- ( n =7 neurons) and 10-day-old ( n =36 neurons) Muscovy ducks ( Cairina moschata ) was investigated. Similar to adult mammals, most of the neurons investigated increased the firing rate (FR) after bombesin application. Changes in FR were not related to changes in thermal coefficient (TC). The neurons react to bombesin also under synaptic blockade. The bombesin-induced effect on TC (increase or decrease in nearly the same number of neurons, e.g. nine neurons increased and ten decreased TC in 10-day-old ducklings) in the postnatal bird neurons investigated was different from the results described in adult mammals, where the main reaction to bombesin was an increase of TC in warm-sensitive and temperature-insensitive-neurons and a transformation of temperature-insensitive-neurons into warm-sensitive ones. This may be related to the assumption that during early ontogeny, body functions react to exogenous and endogenous factors nonspecifically. It is to speculate, that later, probably at the end of embryonic development or during the early postnatal period, the reactivity of these functions changes qualitatively, so that the reaction of an individual function to different factors becomes specific (ultimately adaptive).

Tumor necrosis factor double receptor-knockout (TNFR KO) mice lacking functional genes for the TNF p55 and p75 receptors show exacerbated febrile responses to endotoxin. TNFR KO mice and wild-type (WT) control animals were intraperitoneally treated with 2.5 mg/kg of lipopolysaccharide (LPS), and the effect of this treatment on plasma levels of TNF, interleukin (IL)-6, IL-10 and IL-1 type II (decoy) receptor was studied. TNFR KO mice showed markedly higher levels of TNF 1.5 and 8 h after LPS compared to WT controls. There was no difference in circulatory levels of IL-6 and IL-1 type II receptor between TNFR KO and WT mice 1.5 and 8 h after LPS challenge. There was no difference in plasma IL-10 levels 1.5 h after LPS treatment between TNFR KO and WT mice. However, 8 h after LPS injection TNFR KO mice showed significantly lower levels of IL-10 in plasma than control animals (13.2±2.7 pg/ml vs. 47.6±11.4 pg/ml, P=0.001). These results indicate that under certain conditions the antipyretic action of TNF may be mediated via IL-10 which is considered a major antiinflammatory and antipyretic cytokine. The data obtained also suggest that the exacerbated fever in TNFR KO mice is probably not due to alterations of IL-6 or IL-1 type II (decoy) receptor concentrations in the plasma.

Interleukin (IL)-11 is a member of the gp130 family of cytokines. In contrast to IL-6 (another gp130 cytokine), IL-11 does not induce fever in humans. In the present study, the effect of recombinant human IL-11 (hrIL-11) injected intracerebroventricularly on body temperature of afebrile and febrile rats was studied. Results showed that: (i) hrIL-11 in doses of 5, 50 and 500 ng injected into the cerebral ventricles does not alter body temperature in rats; (ii) febrile response induced by intraperitoneal injection of E. coli endotoxin (50 µg/kg) was initiated more rapidly in rats injected with 500 ng of hrIL-11 in the cerebral ventricles, and (iii) the enhancement of the initial phase of fever induced by hrIL-11 was not accompanied by changes in plasma concentrations of IL-6 and tumor necrosis factor (TNF). These results indicate that hrIL-11 is not pyrogenic when administered into the brain ventricles. The data obtained also demonstrate that central application of hrIL-11 alters body temperature in conditions of pyrogenic stimulation, but that this effect is not due to the alterations in plasma concentrations of IL-6 or TNF. These data suggest that during the development of the systemic inflammatory response, activation of gp130 subunit becomes effective in altering body temperature.

New experimental evidence supports theidea that future therapeutic strategies fortreating cardiovascular disease could tar-get sites within the brain. Our recentstudy demonstrates the existence of previ-ously unrecognized central nervousβ-adrenoceptor (β-AR) mechanisms,blockade of which has a beneficial effecton the failing heart. It has been assumedthat β-AR antagonists (β-blockers) have adirect favorable influence on the heart,but our results challenge this view, sug-gesting that β-blockers may also actdirectly in the brain and that this actioncould contribute to their clinical effect-iveness in heart failure. These data add toan increasing body of evidence support-ing the idea that novel approaches mightbe found to treat cardiovascular disease byaiming at sites within the CNS.Heart failure (HF) is one of the mostimportant public health problems. Mor-bidity and mortality associated with HFremain high, despite considerable achieve-ments in its treatment. Analysis of recentclinical trials indicates similar, or possiblysuperior, beneficial effects of β-AR block-ade in comparison to angiotensin-convert-ing enzyme (ACE) inhibition. In fact, thechoice of ACE inhibitors as first-line ther-apy in patients with HF has recently beenquestioned

The study by Stavrakis et al. [(1)][1] is an interesting contribution to the emerging area of human neuromodulation. The complexity of interaction between the parasympathetic nervous system and the immune system [(2)][2], as well as the heart [(3)][3], is increasingly well recognized. As the

We thank Prof Andrea Porzionato1 for her comment and support of our review2 that raises the issue of the role of the carotid body in obesity-related sympathoactivation. In our review,2 we summarized the intriguing association of raised peripheral chemosensitivity in several disease states, including hypertension, heart failure, and sleep apnea. On the basis of our recent preclinical data,3 we emphasized that in addition to increased peripheral chemoreflex sensitivity, the carotid body develops substantial afferent tone in hypertensive (but not normotensive) rats, and that by severing its connection to the central nervous system, arterial pressure falls substantially. This was associated with a reduction in sympathetic vasomotor …

We read with interest the Letter by Drs Giblett and Hoole in response to our research article ‘Glucagon-Like Peptide-1 (GLP-1) Mediates Cardioprotection by Remote Ischaemic Conditioning’.1 We thank Drs Giblett and Hoole for their interest in our work and provide our responses to the critical comments raised. First, we believe that it is not entirely appropriate to compare the effects of exogenous application of native GLP-1 or GLP-1 receptor (GLP-1R) agonists with the effects induced by remote ischaemic conditioning (RIc). The efficacy of RIc in protecting against left ventricular dysfunction and myocardial stunning may be compromised by the study design and/or inability of a significant proportion of patients to recruit vagal activity, which appears to be critically important for RIc cardioprotection.2,3 We reported that in rats vagotomy blocks RIc cardioprotection, …

Astrocytes might function as brain interoceptors capable of detecting different (chemo)sensory modalities and transmitting sensory information to the relevant neural networks controlling vital functions. For example, astrocytes that reside near the ventral surface of the brainstem (central respiratory chemosensitive area) respond to physiological decreases in pH with vigorous elevations in intracellular Ca2+and release of ATP. ATP transmits astroglial excitation to the brainstem respiratory network and contributes to adaptive changes in lung ventilation. Here we show that in terms of pH-sensitivity, ventral brainstem astrocytes are clearly distinct from astrocytes residing in the cerebral cortex. We monitored vesicular fusion in cultured rat brainstem astrocytes using total internal reflection fluorescence microscopy and found that ∼35% of them respond to acidification with an increased rate of exocytosis of ATP-containing vesicular compartments. These fusion events require intracellular Ca2+signaling and are independent of autocrine ATP actions. In contrast, the rate of vesicular fusion in cultured cortical astrocytes is not affected by changes in pH. Compared to cortical astrocytes, ventral brainstem astrocytes display higher levels of expression of genes encoding proteins associated with ATP vesicular transport and fusion, including vesicle-associated membrane protein-3 and vesicular nucleotide transporter. These results suggest that astrocytes residing in different parts of the rat brain are functionally specialized. In contrast to cortical astrocytes, astrocytes of the brainstem chemosensitive area(s) possess signaling properties that are functionally relevant—they are able to sense changes in pH and respond to acidification with enhanced vesicular release of ATP.

Inorganic polyphosphate is known to be present in the mammalian brain at micromolar concentrations. Here we show that polyphosphate may act as a gliotransmitter, mediating communication between astrocytes. It is released by astrocytes in a calcium-dependent manner and signals to neighbouring astrocytes through P2Y1 purinergic receptors, activation of phospholipase C and release of calcium from the intracellular stores. In primary neuroglial cultures, application of polyP triggers release of endogenous polyphosphate from astrocytes while neurons take it up. In vivo, central actions of polyphosphate at the level of the brainstem include profound increases in key homeostatic physiological activities, such as breathing, central sympathetic outflow and the arterial blood pressure. Together, these results suggest a role for polyphosphate as a mediator of astroglial signal transmission in the mammalian brain., Inorganic polyphosphates have been identified in the central nervous system. Holmström and colleagues examine neuroglial cultures in vitro and cardiorespiratory responses in vivo, and find that inorganic polyphosphates trigger calcium-dependent activation of astrocytes and increase cardiorespiratory activity.

The combination of optogenetics and functional magnetic resonance imaging (fMRI) is referred to as opto-fMRI. Optogenetics utilises genetic engineering to introduce light sensitive actuator proteins into cells. Functional MRI (fMRI) is a specialist form of magnetic resonance imaging concerned with imaging changes in blood flow and oxygenation, linked to regional variation in metabolic activity, in the brain. This study describes a methodological concern regarding the effects of light delivery into the brain for the purposes of opto-fMRI. We show that blue light delivery to the naive rat brain causes profound fMRI responses, despite the absence of optogenetic activation. We demonstrate that these fMRI responses are dependent upon laser power and show that the laser causes significant heating. We identify how heating impacts upon the MR signal causing NMR frequency shifts, and T1 and T2* changes. This study brings attention to a possible confounder which must be taken into account when opto-fMRI experiments are designed.

The peripheral chemoreflex is known to be enhanced in individuals with hypertension. In pre-hypertensive (PH) and adult spontaneously hypertensive rats (SHRs) carotid body type I (glomus) cells exhibit hypersensitivity to chemosensory stimuli and elevated sympathoexcitatory responses to peripheral chemoreceptor stimulation. Herein, we eliminated carotid body inputs in both PH-SHRs and SHRs to test the hypothesis that heightened peripheral chemoreceptor activity contributes to both the development and maintenance of hypertension. The carotid sinus nerves were surgically denervated under general anaesthesia in 4- and 12-week-old SHRs. Control groups comprised sham-operated SHRs and aged-matched sham-operated and carotid sinus nerve denervated Wistar rats. Arterial blood pressure was recorded chronically in conscious, freely moving animals. Successful carotid sinus nerve denervation (CSD) was confirmed by testing respiratory responses to hypoxia (10% O(2)) or cardiovascular responses to i.v. injection of sodium cyanide. In the SHR, CSD reduced both the development of hypertension and its maintenance (P0.05) and was associated with a reduction in sympathetic vasomotor tone (as revealed by frequency domain analysis and reduced arterial pressure responses to administration of hexamethonium; P0.05 vs. sham-operated SHR) and an improvement in baroreflex sensitivity. No effect on blood pressure was observed in sham-operated SHRs or Wistar rats. In conclusion, carotid sinus nerve inputs from the carotid body are, in part, responsible for elevated sympathetic tone and critical for the genesis of hypertension in the developing SHR and its maintenance in later life.

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO2/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Usingin vivo(conscious and anesthetized rats) andin situ(arterially perfused rat brainstem–spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO2-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupledDrosophilaallatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and thein siturat preparations. Postinspiratory activity was also reducedin situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO2-evoked abdominal expiratory activity in anesthetized rats andin situpreparations. Inspiratory responses evoked by rising levels of CO2in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO2-evoked increases of inspiratory activity.

An increase in PCO(2) in the arterial blood triggers immediate release of ATP from the ventral chemosensory site(s) on the surface of the medulla oblongata. Systemic hypoxia in anesthetized rats was also associated with increased ATP release on the ventral medullary surface. During both hypoxia and hypercapnia, ATP and possibly other gliotransmitters released in the ventral medulla seemed to enhance cardiorespiratory responses to these stressors, and some of this ATP was proposed to be derived from astrocytes. Astrocytes also play a vital role controlling local blood flow. Astrocytes are activated by neurotransmitter release - especially glutamate and ATP. The astrocytic activation is manifest as a rise in intracellular Ca(2+) that is closely coupled to the metabolic activity of neurons in the active area. The activation of astrocytes spreads as a wave from astrocyte to astrocyte and causes release of ATP, adenosine, and other gliotransmitters that may alter neuronal function in the region of astrocytic activation. In addition, ATP, adenosine and other vasoactive substances, when released at the endfeet of astrocytes, interact with vascular receptors that may either dilate or constrict the vessels in the region closely adjacent to the site of neuronal activity. Thus, astrocytes seem to integrate neuronal metabolic needs by responding to the level of neuronal activity to regulate local blood flow and cardiorespiratory responses to hypoxia and hypercapnia to match substrate need (oxygen and glucose) with substrate availability and with the removal of CO(2). In so doing, astrocytes assume a larger role in information processing and in the regulation of neuronal activity and homeostasis of the entire organism than has been ascribed to them in the past.

Dopamine is one of the key neurotransmitters in the central nervous system and plays an important role in physiological processes, as well as in the development of many diseases. Here we report a receptor-independent signalling pathway induced by dopamine in CNS neurons. In cultured neurons from midbrain, cortex and hippocampus, dopamine uptake via dopamine or monoamine transporters induces plasmalemmal membrane depolarization, leading to opening of voltage gated calcium channels and a cytosolic calcium signal. This dopamine-induced calcium signal is unaffected by inhibition of the known dopamine receptors. In anaesthetized rats, application of dopamine in the presence of dopamine receptor antagonists to brainstem structures controlling cardiovascular activity results in an increase in heart rate, arterial blood pressure and sympathetic nerve activity. These data identify a novel dopamine-induced signalling pathway in CNS neurons which may have an important functional role in the central mechanisms controlling complex behaviours.

Cardiorespiratory activity is controlled by a network of neurons located within the lower brainstem. The basic rhythm of breathing is generated by neuronal circuits within the medullary pre-Bötzinger complex, modulated by pontine and other inputs from cell groups within the medulla oblongata and then transmitted to bulbospinal pre-motor neurons that relay the respiratory pattern to cranial and spinal motor neurons controlling respiratory muscles. Cardiovascular sympathetic and vagal activities have characteristic discharges that are patterned by respiratory activity. This patterning ensures ventilation–perfusion matching for optimal respiratory gas exchange within the lungs. Peripheral arterial chemoreceptors and central respiratory chemoreceptors are crucial for the maintenance of cardiorespiratory homeostasis. Inputs from these receptors ensure adaptive changes in the respiratory and cardiovascular motor outputs in various environmental and physiological conditions. Many of the connections in the reflex pathway that mediates the peripheral arterial chemoreceptor input have been established. The nucleus tractus solitarii, the ventral respiratory network, pre-sympathetic circuitry and vagal pre-ganglionic neurons at the level of the medulla oblongata are integral components, although supramedullary structures also play a role in patterning autonomic outflows according to behavioural requirements. These medullary structures mediate cardiorespiratory reflexes that are initiated by the carotid and aortic bodies in response to acute changes in PO 2 , PCO 2 and pH in the arterial blood. The level of arterial PCO 2 is the primary factor in determining respiratory drive and although there is a significant role of the arterial chemoreceptors, the principal sensor is located either at or in close proximity to the ventral surface of the medulla. The cellular and molecular mechanisms of central chemosensitivity as well as the neural basis for the integration of central and peripheral chemosensory inputs within the medulla remain challenging issues, but ones that have some emerging answers.

to the editor: Amniote vertebrates adjust the acid-base status of the blood through ventilatory alterations of PaCO2 and, to some degree, by renal modulation of bicarbonate concentration. Receptor systems for the ventilatory acid-base regulation has been studied mainly in mammals, in which the

Current knowledge of the central nervous system distribution of the beta(1)-adrenergic receptors (beta(1)-AR) is incomplete. Here we present a general map of the beta(1)-AR distribution in the rat brain. beta(1)-AR-immunoreactivity was detected throughout the entire rat brain, but particularly dense staining was observed in the cerebellar cortex and basal ganglia. Brainstem areas displaying significant beta(1)-AR-immunoreactivity include the ventrolateral medulla, nucleus ambiguus and the nucleus of the solitary tract. Within the hypothalamus, only the paraventricular nucleus and the median eminence (ME) showed beta(1)-AR immunostaining. Numerous beta(1)-AR-immunoreactive cells were also found in the hippocampus, basal ganglia and cerebral cortex. These results extend our knowledge of the expression profile of beta(1)-AR in the central nervous system. The identification of several distinct beta(1)-AR immunoreactive substrates linked with neuropathophysiological roles in cardiovascular disease supports the hypothesis that the therapeutic benefit of beta(1)-AR blockade may be conferred at least in part through central nervous system mechanisms.

Intercellular purinergic signalling, which utilizes ATP as a transmitter, is fundamental for the operation of the autonomic nervous system. ATP is released together with 'classical' transmitters from sympathetic and parasympathetic nerves supplying various peripheral targets, modulates neurotransmission in autonomic ganglia, has an important role in local enteric neural control and coordination of intestinal secretion and motility, and acts as a common mediator for several distinct sensory modalities. Recently, the role of ATP-mediated signalling in the central nervous control of autonomic function has been addressed. Emerging data demonstrate that in the brain ATP is involved in the operation of several key cardiorespiratory reflexes, contributes to central processing of viscerosensory information, mediates central CO(2) chemosensory transduction and triggers adaptive changes in breathing, and modulates the activities of the brainstem vagal preganglionic, presympathetic and respiratory neural networks.

Respiratory activity is controlled by a network of neurons located within the lower brain stem. The basic rhythm of breathing is generated by a population of neurons within the so-called pre-Botzinger complex of the medulla oblongata and is then subsequently shaped, modified, and transmitted to bulbospinal premotor neurons, which relay the respiratory pattern to spinal motor neurons controlling respiratory muscles. The respiratory network receives peripheral chemosensory and mechanosensory inputs and modulatory inputs from the other parts of the brain. These inputs are essential for adaptive changes in the respiratory motor output, ensuring appropriate ventilation of the lungs in variable environmental and physiological conditions.

We have developed an amperometric microbiosensor for real time monitoring l -glutamate release in neural tissue, based on enzymatic oxidation catalyzed by the l -glutamate oxidase. By means of a sol–gel coating method, l -glutamate oxidase was entrapped in a biocompatible gel layer that provided a benign environment and retained enzyme activity on the surface of Pt microelectrode. Prior to gel layer formation, a modification on the surface of Pt microelectrode with poly(phenylene diamine) enabled the microbiosensor screen majority of common potential interfering substances existing in physiological samples. The miniaturized biosensor achieved a steady state response to l -glutamate within 10 s and exhibited a linear dependence on the concentration of l -glutamate from 0.5 to 100 μmol L−1 with a high sensitivity of 279.4 ± 2.0 μA (mmol L−1)−1 cm−2 (n = 4, R.S.D. = 2.8%). The microbiosensor also exhibited excellent long-term stability in dry storage. We have successfully used the microbiosensor for real time measuring of l -glutamate in vivo.

Acid-sensitive K+ channels of the tandem P-domain K +-channel family (TASK-1 and TASK-3) have been implicated in peripheral and central respiratory chemosensitivity; however, because of the lack of decisive pharmacological agents, the final proof of the role of the TASK channel in the chemosensory control of breathing has been missing. In the mouse, TASK-1 and TASK-3 channels are dispensable for central respiratory chemosensitivity (Mulkey et al., 2007). Here, we have used knock-out animals to determine whether TASK-1 and TASK-3 channels play a role in the carotid body function and chemosensory control of breathing exerted by the carotid body chemoreceptors. Ventilatory responses to hypoxia (10% O2 in inspired air) and moderate normoxic hypercapnia (3-6% CO2 in inspired air) were significantly reduced in TASK-1 knock-out mice. In contrast, TASK-3-deficient mice showed responses to both stimuli that were similar to those developed by their wild-type counterparts. TASK-1 channel deficiency resulted in a marked reduction of the hypoxia (by 49%)-and CO2 (by 68%)-evoked increases in the carotid sinus nerve chemoafferent discharge recorded in the in vitro superfused carotid body/carotid sinus nerve preparations. Deficiency in both TASK-1 and TASK-3 channels increased baseline chemoafferent activity but did not cause a further reduction of the carotid body chemosensory responses. These observations provide direct evidence that TASK-1 channels contribute significantly to the increases in the carotid body chemoafferent discharge in response to a decrease in arterial PO2 or an increase in PCO2 /[H+]. TASK-1 channels therefore play a key role in the control of ventilation by peripheral chemoreceptors. Copyright © 2008 Society for Neuroscience., This work was supported by the Medical Research Council (S.T.), The Wellcome Trust (A.V.G.), and the J. Ernest Tait Estate (W.W.).

The Breuer-Hering inflation reflex is initiated by activation of the slowly adapting pulmonary stretch receptor afferents (SARs), which monosynaptically activate second-order relay neurones in the dorsal medullary nucleus of the solitary tract (NTS). Here we demonstrate that during lung inflation SARs release both ATP and glutamate from their central terminals to activate these NTS neurones. In anaesthetized and artificially ventilated rats, ATP- and glutamate-selective microelectrode biosensors placed in the NTS detected rhythmic release of both transmitters phase-locked to lung inflation. This release of ATP and glutamate was independent of the centrally generated respiratory rhythm and could be reversibly abolished during the blockade of the afferent transmission in the vagus nerve by topical application of local anaesthetic. Microionophoretic application of ATP increased the activity of all tested NTS second-order relay neurones which receive monosynaptic inputs from the SARs. Unilateral microinjection of ATP into the NTS site where pulmonary stretch receptor afferents terminate produced central apnoea, mimicking the effect of lung inflation. Application of P2 and glutamate receptor antagonists (pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid, suramin and kynurenic acid) significantly decreased baseline lung inflation-induced firing of the second-order relay neurones. These data demonstrate that ATP and glutamate are released in the NTS from the central terminals of the lung stretch receptor afferents, activate the second-order relay neurones and hence mediate the key respiratory reflex - the Breuer-Hering inflation reflex.

Receptors for extracellular ATP (both ionotropic and metabotropic) are widely expressed in the CNS both in neurones and glia. ATP can modulate neuronal activity in many parts of the brain and contributes to the central nervous control of several physiological functions. Here we show that during the systemic inflammatory response the extracellular concentrations of ATP increase in the anterior hypothalamus and this has a profound effect on the development of the thermoregulatory febrile response. In conscious rabbits we measured ATP release in real time with novel amperometric biosensors and monitored a marked increase in the concentration of ATP (4.0 +/- 0.7 microm) in the anterior hypothalamus in response to intravenous injection of bacterial endotoxin - lipopolysaccharide (LPS). No ATP release was observed in the posterior hypothalamus. The release of ATP coincided with the development of the initial phase of the febrile response, starting 18 +/- 2 min and reaching its peak 45 +/- 2 min after LPS injection. Application of the ATP receptor antagonists pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid, Brilliant Blue G or periodate oxidized ATP dialdehyde to the site of ATP release in the anterior hypothalamus markedly augmented and prolonged the febrile response. These data indicate that during the development of the systemic inflammation, ATP is released in the anterior hypothalamus to limit the magnitude and duration of fever. This release may also have a profound effect on the hypothalamic control of other physiological functions in which ATP and related purines have been implicated to play modulatory roles, such as food intake, hormone secretion, cardiovascular activity and sleep.

should also target sinusoidal cells. However, there are no in vitro research tools that correctly mimic the unique features of the sinusoid: adequate spatial distribution of cells, biomechanical stimulation of the endothelial lineage and free paracrine interactions. Our aim was to design, fabricate and validate a three-dimensional co-culture chamber with microfluidics that mimics the hepatic sinusoid. Methods: A transparent PMMA chamber composed by 3 growing areas of 9.7 cm arranged at different heights and separated by 0.5mm was manufactured. The higher level integrated a microfluidic system allowing the application of homogeneous shear stress on a reinforced porous membrane where the endothelium is grown. The middle level had a second culture membrane, and on the lower level, the growing area was the base of the camera. Functional validation: Endothelial cells stimulated with shear stress (5 dyn/cm), HSC, and hepatocytes were cultured for 24h in the chamber. Morphology, viability and endothelial nitric oxide production was analyzed. Translational Validation: The applicability of the chamber in the field of cirrhosis was assessed co-culturing activated HSC and capillarized LSEC under shear stress, or in static conditions. Furthermore, the effects of adding the vasoprotective agent simvastatin were analyzed and compared with traditional culture methods. Results: Functional validation: Cells grown in the chamber showed excellent viability. Endothelial cells were aligned in the direction of shear stress, and increased their production of nitric oxide. Translational Validation: Dysfunctional LSEC cultured in the device showed a marked improvement in their phenotype (activation of the KLF2 pathway and decrease in endothelin-1) that was not observed using ordinary culture methods. The shear stress-derived improvement in LSEC led to a beneficial paracrine effect on HSC (reduced collagen I and alpha-SMA). Simvastatin addition produced a strong protective effect on both cell types, which was significantly higher than that obtained using traditional “transwell” methods. Conclusions: We herein describe a novel, versatile, easy to operate and highly reproducible device that can be applied in different fields of vascular biomedical research, including hepatology.

Extracellular signalling by the purine nucleotide ATP has long been associated with sensory function. In the periphery, ATP mediates nociception, mechanosensitivity, thermal sensitivity and O2 chemosensitivity. These processes share a common mechanism that involves the release of ATP to excite afferent fibres via activation of ionotropic P2X and/or metabotropic P2Y receptors. Chemosensors located in the brainstem are crucial for the maintenance of physiological levels of blood gases through the regulation of breathing. Here we show that an increase in pCO2 in the arterial blood triggers the immediate release of ATP from three chemosensitive regions located on the ventral surface of the medulla oblongata. Blockade of ATP receptors at these sites diminishes the chemosensory control of breathing, suggesting that ATP release constitutes a key step in central chemosensory transduction. These new data suggest that ATP, a phylogenetically ancient, unique and simple molecule, has been widely used in the evolution of afferent systems to mediate distinct forms of sensory transduction not only in the periphery but also within the central nervous system.

P2X2receptor subunits of the ATP-gated ion channels are expressed by physiologically identified respiratory neurons in the ventral respiratory column, implicating ATP in the control of respiratory activity. We now show that, during hypoxia, release of ATP in the ventrolateral medulla (VLM) plays an important role in the hypoxic ventilatory response in rats. By measuring ATP release in real time at the ventral surface of the medulla with novel amperometric biosensors, we found that hypoxia (10% O2; 5 min) induced a marked increase in the concentration of ATP (∼3 μm). This ATP release occurred after the initiation of enhanced respiratory activity but coincided with the later hypoxia-induced slowing of the respiratory rhythm. ATP was also released at the ventral surface of the medulla during hypoxia in peripherally chemodenervated animals (vagi, aortic, and carotid sinus nerve sectioned). By using horizontal slices of the rat medulla, we found that, during hypoxia, ATP is produced throughout the VLM in the locations corresponding to the ventral respiratory column. Blockade of ATP receptors in the VLM (microinjection of P2 receptor antagonist pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid; 100 μm) augmented the hypoxia-induced secondary slowing of the respiratory rhythm. Our findings suggest that ATP released within the ventral respiratory column is involved in maintenance of the respiratory activity in conditions when hypoxia-induced slowing of respiration occurs. These data illustrate a new functional role for ATP-mediated purinergic signaling in the medullary mechanisms controlling respiratory activity.

Recent evidence suggests that ATP is a mediator of central (within the ventral surface of the medulla) and peripheral (within the carotid body) chemosensory transduction. This short review discusses the data obtained in experiments in vivo and in vitro supporting this hypothesis. P2 receptors for ATP are expressed within the ventrolateral medulla as well as by the peripheral chemosensory afferent neurones. Blockade of P2 receptors in the ventrolateral medulla attenuates the CO2-induced increase in respiration while blockade of purinergic signalling impairs carotid body function and diminishes the ventilatory response to hypoxia. Furthermore, ATP is released from the ventral surface of the medulla during hypercapnia and from the carotid body during hypoxia. Finally, exogenous ATP applied on the ventral surface of the medulla evokes rapid increase in phrenic nerve activity, while ATP applied to the carotid body evokes marked excitation of the carotid sinus nerve afferents. We suggest that in the ventrolateral medulla ATP is produced following CO2/H(+)-induced activation of central chemosensory elements (neuronal and/or glial) and acts within the respiratory network to produce physiologically relevant changes in ventilation. In the carotid body, ATP contributes in a significant manner to the transmission of the sensitivity of the carotid body to changes in arterial PO2 and may be considered as a key transmitter released by chemoreceptor cells to activate endings of the sinus nerve afferent fibres.

Extracellular purine nucleotide and nucleoside signalling molecules, such as ATP and adenosine, acting through specific receptors (P2 and P1, respectively) play significant roles in the mechanisms underlying the febrile response. A variety of P2 and P1 receptor subunits have been identified in the hypothalamus, the area of the brain that orchestrates the febrile response. Importantly, both ATP and adenosine have been shown to modulate release and/or action of cytokines that are implicated in fever, as well as to be involved in the central mechanisms of cardiovascular and respiratory control. Our data indicate that at the level of the anterior hypothalamus extracellular ATP is involved in the control of the development of fever. A population of warm-sensitive neurones in the anterior hypothalamus is likely to be the site of action of ATP on body temperature. ATP-induced cytokine release does not appear to play a significant role in the hypothalamic mechanisms leading to the development of the febrile response. However, the blockade of fever by P2 receptor antagonists given systemically suggests that ATP-mediated signalling may play a role in the release of pyrogenic cytokines in the periphery. At the level of the anterior hypothalamus adenosine appears to be released tonically, and acts to maintain body temperature under afebrile conditions. There is also evidence that adenosine-mediated signalling may play a role in the hypothalamic mechanisms controlling the degree of body temperature increase during fever. Our investigations have identified possible mechanisms by which purines modulate the febrile response. The actions of purines on body temperature during fever are most likely "site specific" (brain vs. periphery), may or may not involve their effect on cytokine release and/or action, and are likely to involve P2 and P1 receptors of different subtypes. Further extensive studies are needed to elucidate these mechanisms in greater detail and may lead to the development of new approaches for modifying febrile, cytokine and acute-phase responses to infection.