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Supplementary figure 4. Cell viability data based to generate the synergy plots in Figures 2 and 3.

Supplementary figure 1A. Sequencing tracks of a part of the Zwilch gene targeted by a first unique sgRNA, with the wildtype sequence (top) and sgRNA treated sequence (bottom) shown. B. Tide analysis showing editing efficiency of the first Zwilch targeting sgRNA

Supplementary figure 1C. Sequencing tracks of a part of the Zwilch gene targered by a second unique sgRNA, with the wildtype sequence (top) and sgRNA treated sequence (bottom) shown. D. Tide analysis showing editing efficiency of the second Zwilch targeting sgRNA

Small cell lung cancer (SCLC) is generally regarded as very difficult to treat, mostly due to the development of metastases early in the disease and a quick relapse with resistant disease. SCLC patients initially show a good response to treatment with the DNA damaging agents cisplatin and etoposide. This is, however, quickly followed by the development of resistant disease, which urges the development of novel therapies for this type of cancer. In this study, we set out to compile a comprehensive overview of the vulnerabilities of SCLC. A functional genome-wide screen where all individual genes were knocked out was performed to identify novel vulnerabilities of SCLC. By analysis of the knockouts that were lethal to these cancer cells, we identified several processes to be synthetic vulnerabilities in SCLC. We were able to validate the vulnerability to inhibition of the replication stress response machinery by use of Chk1 and ATR inhibitors. Strikingly, SCLC cells were more sensitive to these inhibitors than nontransformed cells. In addition, these inhibitors work synergistically with either etoposide and cisplatin, where the interaction is largest with the latter. ATR inhibition by VE-822 treatment in combination with cisplatin also outperforms the combination of cisplatin with etoposide in vivo. Altogether, our study uncovered a critical dependence of SCLC on the replication stress response and urges the validation of ATR inhibitors in combination with cisplatin in a clinical setting.

Supplementary figure 2. Graphs showing the correlation between all three replicates of the genome�wide CRISPR�Cas9 screen at the start of the experiment (t=0) and at the final time point (t=14).

Supplementary figure 3A and B. Graph representing the mRNA levels of MYC and MYCL1 in a panel of 63 human SCLC cell lines. Cut-offs for the analysis of low and high expressing cells were set as indicated. C. Graphs showing the 2log(IC50) for a selection of CHK1 and ATR inhibitors of a panel of 63 human SCLC cell lines screened by Polley at al. (21). Cell lines were grouped on the basis of absence or presence of either MYC or MYCL1 expression. D. p-values for testing between the MYC/MYCL1 low and either high group were calculated using a Mann- Whitney U test and were corrected for multiple testing using Benjamini-Hochberg.

Small cell lung cancer (SCLC) is generally regarded as very difficult to treat, mostly due to the development of metastases early in the disease and a quick relapse with resistant disease. SCLC patients initially show a good response to treatment with the DNA damaging agents cisplatin and etoposide. This is, however, quickly followed by the development of resistant disease, which urges the development of novel therapies for this type of cancer. In this study, we set out to compile a comprehensive overview of the vulnerabilities of SCLC. A functional genome-wide screen where all individual genes were knocked out was performed to identify novel vulnerabilities of SCLC. By analysis of the knockouts that were lethal to these cancer cells, we identified several processes to be synthetic vulnerabilities in SCLC. We were able to validate the vulnerability to inhibition of the replication stress response machinery by use of Chk1 and ATR inhibitors. Strikingly, SCLC cells were more sensitive to these inhibitors than nontransformed cells. In addition, these inhibitors work synergistically with either etoposide and cisplatin, where the interaction is largest with the latter. ATR inhibition by VE-822 treatment in combination with cisplatin also outperforms the combination of cisplatin with etoposide in vivo. Altogether, our study uncovered a critical dependence of SCLC on the replication stress response and urges the validation of ATR inhibitors in combination with cisplatin in a clinical setting.

Badhai et al. describe a mouse model of mesothelioma with combined deletion of Bap1, Nf2, and Cdkn2ab that shows rapid onset and recapitulates human mesothelioma including its response to the standard treatment. This autochthonous model is well suited for testing cancer immunotherapies., We have generated mouse models of malignant mesothelioma (MM) based upon disruption of the Bap1, Nf2, and Cdkn2ab tumor suppressor loci in various combinations as also frequently observed in human MM. Inactivation of all three loci in the mesothelial lining of the thoracic cavity leads to a highly aggressive MM that recapitulates the histological features and gene expression profile observed in human patients. The tumors also show a similar inflammatory phenotype. Bap1 deletion alone does not cause MM but dramatically accelerates MM development when combined with Nf2 and Cdkn2ab (hereafter BNC) disruption. The accelerated tumor development is accompanied by increased Polycomb repression and EZH2-mediated redistribution of H3K27me3 toward promoter sites with concomitant activation of PI3K and MAPK pathways. Treatment of BNC tumor–bearing mice with cisplatin and pemetrexed, the current frontline treatment, prolongs survival. This makes the autochthonous mouse model described here very well suited to explore the pathogenesis of MM and validate new treatment regimens for MM, including immunotherapy.

Summary Fibroblast growth factor receptor 1 (FGFR1) is frequently amplified in human small-cell lung cancer (SCLC), but its contribution to SCLC and other lung tumors has remained elusive. Here, we assess the tumorigenic capacity of constitutive-active FGFR1 (FGFR1K656E) with concomitant RB and P53 depletion in mouse lung. Our results reveal a context-dependent effect of FGFR1K656E: it impairs SCLC development from CGRPPOS neuroendocrine (NE) cells, which are considered the major cell of origin of SCLC, whereas it promotes SCLC and low-grade NE bronchial lesions from tracheobronchial-basal cells. Moreover, FGFR1K656E induces lung adenocarcinoma (LADC) from most lung cell compartments. However, its expression is not sustained in LADC originating from CGRPPOS cells. Therefore, cell context and tumor stage should be taken into account when considering FGFR1 inhibition as a therapeutic option., Graphical Abstract, Highlights • FGRF signaling impairs SCLC development initiated from CGRPPOS NE cells • FGRF signaling promotes development of NE tumors initiated from K14POS cells • Rb;p53;Fgfr1 mice develop ADCs that retain signatures of their cell of origin • Low expression of Fgfr1 in progressed tumors suggests a role in tumor initiation, Ferone et al. show that inducing FGFR signaling in lung neuroendocrine (NE) cells of Rb;p53 mice results in impairment of SCLC and enhancement of peripheral NE lesions. The location and tumor characteristics depend on the targeted cell type. Rb;p53;Fgfr1 mice also develop adenocarcinomas with the incidence depending on the targeted cell type.

Summary Small-cell lung cancer is the most aggressive type of lung cancer, characterized by a remarkable response to chemotherapy followed by development of resistance. Here, we describe SCLC subtypes in Mycl- and Nfib-driven GEMM that include CDH1-high peripheral primary tumor lesions and CDH1-negative, aggressive intrapulmonary metastases. Cisplatin treatment preferentially eliminates the latter, thus revealing a striking differential response. Using a combined transcriptomic and proteomic approach, we find a marked reduction in proliferation and metabolic rewiring following cisplatin treatment and present evidence for a distinctive metabolic and structural profile defining intrinsically resistant populations. This offers perspectives for effective combination therapies that might also hold promise for treating human SCLC, given the very similar response of both mouse and human SCLC to cisplatin., Graphical Abstract, Highlights • Heterogeneity and plasticity of SCLC underlies differential sensitivity to cisplatin • Cisplatin selectively eradicates the CDH1-negative, NFIB-positive SCLC compartment • Identification of intrinsically cisplatin-resistant CDH1-high alveolar lesions • Multi-omics analysis identifies distinctive metabolic circuitry in resistant populations, Böttger et al. show the presence of histologically and molecularly distinct sub-populations of small-cell lung cancer (SCLC) in advanced mouse models. They demonstrate that the heterogeneity of SCLC is responsible for the differential sensitivity to cisplatin and identify metabolic circuitries that likely contribute to cisplatin resistance.

Tertiary lymphoid tissue (TLT) is lymphoid tissue that forms in adult life as a result of chronic inflammation in a tissue or organ. TLT has been shown to form in a variety of chronic inflammatory diseases, though it is not clear if and how TLT develops in the inflamed colon during inflammatory bowel disease. Here, we show that TLT develops as newly formed lymphoid tissue in the colon following dextran sulphate sodium induced colitis in C57BL/6 mice, where it can be distinguished from the preexisting colonic patches and solitary intestinal lymphoid tissue. TLT in the inflamed colon develops following the expression of lymphoid tissue-inducing chemokines and adhesion molecules, such as CXCL13 and VCAM-1, respectively, which are produced by stromal organizer cells. Surprisingly, this process of TLT formation was independent of the lymphotoxin signaling pathway, but rather under neuronal control, as we demonstrate that selective surgical ablation of vagus nerve innervation inhibits CXCL13 expression and abrogates TLT formation without affecting colitis. Sympathetic neuron denervation does not affect TLT formation. Hence, we reveal that inflammation in the colon induces the formation of TLT, which is controlled by innervation through the vagus nerve.

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH–SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

The brain plays a prominent role in the regulation of inflammation. Immune cells are under control of the so-called cholinergic anti-inflammatory reflex, mainly acting via autonomic innervation of the spleen. Activation of this reflex inhibits the secretion of proinflammatory cytokines and may reduce the development of atherosclerosis. Therefore, the aim of this study was to evaluate the effects of selective parasympathetic (Px) and sympathetic (Sx) denervation of the spleen on inflammatory status and atherosclerotic lesion development. Female APOE*3-Leiden.CETP mice, a well-established model for human-like lipid metabolism and atherosclerosis, were fed a cholesterol-containing Western-type diet for 4 wk after which they were subdivided into three groups receiving either splenic Px, splenic Sx, or sham surgery. The mice were subsequently challenged with the same diet for an additional 15 wk. Selective Px increased leukocyte counts (i.e., dendritic cells, B cells, and T cells) in the spleen and increased gene expression of proinflammatory cytokines in the liver and peritoneal leukocytes compared with Sx and sham surgery. Both Px and Sx increased circulating proinflammatory cytokines IL-1β and IL-6. However, the increased proinflammatory status in denervated mice did not affect atherosclerotic lesion size or lesion composition. Conclusion: Predominantly selective Px of the spleen enhances the inflammatory status, which, however, does not aggravate diet-induced atherosclerotic lesion development.

The hypothalamus uses hormones and the autonomic nervous system to balance energy fluxes in the body. Here we show that the autonomic nervous system has a distinct organization in different body compartments. The same neurons control intraabdominal organs (intraabdominal fat, liver, and pancreas), whereas sc adipose tissue located outside the abdominal compartment receives input from another set of autonomic neurons. This differentiation persists up to preautonomic neurons in the hypothalamus, including the biological clock, that have a distinct organization depending on the body compartment they command. Moreover, we demonstrate a neuronal feedback from adipose tissue that reaches the brainstem. We propose that this compartment-specific organization offers a neuroanatomical perspective for the regional malfunction of organs in type 2 diabetes, where increased insulin secretion by the pancreas and disturbed glucose metabolism in the liver coincide with an augmented metabolic activity of visceral compared with sc adipose tissue.

The arcuate nucleus (ARC) is crucial for the maintenance of energy homeostasis as an integrator of long- and short-term hunger and satiety signals. The expression of receptors for metabolic hormones, such as insulin, leptin, and ghrelin, allows ARC to sense information from the periphery and signal it to the central nervous system. The ventromedial ARC (vmARC) mainly comprises orexigenic neuropeptide agouti-related peptide and neuropeptide Y neurons, which are sensitive to circulating signals. To investigate neural connections of vmARC within the central nervous system, we injected the neuronal tracer cholera toxin B into vmARC. Due to variation of injection sites, tracer was also injected into the subependymal layer of the median eminence (seME), which showed similar projection patterns as the vmARC. We propose that the vmARC forms a complex with the seME, their reciprocal connections with viscerosensory areas in brain stem, and other circumventricular organs, suggesting the exchange of metabolic and circulating information. For the first time, the vmARC-seME was shown to have reciprocal interaction with the suprachiasmatic nucleus (SCN). Activation of vmARC neurons by systemic administration of the ghrelin mimetic GH-releasing peptide-6 combined with SCN tracing showed vmARC neurons to transmit feeding related signals to the SCN. The functionality of this pathway was demonstrated by systemic injection of GH-releasing peptide-6, which induced Fos in the vmARC and resulted in a reduction of about 40% of early daytime Fos immunoreactivity in the SCN. This observation suggests an anatomical and functional pathway for peripheral hormonal feedback to the hypothalamus, which may serve to modulate the activity of the SCN.

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day- and night-time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N-acetyltransferase (AA-NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h-pattern of melatonin secretion in SCN-lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN-lesioned and ganglionectomised rats, AA-NAT levels and pineal melatonin content were low (i.e. 12% of night-time control levels) for both day- and night-time periods. In SCN-lesioned rats, AA-NAT levels were intermediate (i.e. 30% of night-time control levels) and the 48-h secretion of melatonin presented constant levels not exceeding 20% of night-time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day-length (i.e. adaptation to seasons).

Background: Postoperative ileus is characterized by a transient impairment of the gastrointestinal motility after abdominal surgery. The intestinal inflammation, triggered by handling of the intestine, is the main factor responsible for the prolonged dysmotility of the gastrointestinal tract. Secondary lymphoid organs of the intestine were identified as essential components in the dissemination of inflammation to the entire gastrointestinal tract also called field effect. The involvement of the spleen, however, remains unclear. Aim: In this study, we investigated whether the spleen responds to manipulation of the intestine and participates in the intestinal inflammation underlying postoperative ileus. Methods: Mice underwent Laparotomy (L) or Laparotomy followed by Intestinal Manipulation (IM). Twenty-four hours later, intestinal and colonic inflammation was assessed by QPCR and measurement of the intestinal transit was performed. Analysis of homeostatic chemokines in the spleen was performed by QPCR and splenic cell populations analysed by Flow Cytometry. Blockade of the egress of cells from the spleen was performed by administration of the Sphingosine-1phosphate receptor 1 (S1P1) agonist CYM-5442 10 h after L/IM. Results: A significant decrease in splenic weight and cellularity was observed in IM mice 24 h post-surgery, a phenomenon associated with a decreased splenic expression level of the homeostatic chemokine CCL19. Splenic denervation restored the expression of CCL19 and partially prevented the reduction of splenocytes in IM mice. Treatment with CYM-5442 prevented the egress of splenocytes but did not ameliorate the intestinal inflammation underlying postoperative ileus. Conclusions: Intestinal manipulation results in two distinct phenomena: local intestinal inflammation and a decrease in splenic cellularity. The splenic response relies on an alteration of cell trafficking in the spleen and is partially regulated by the splenic nerve. The spleen however does not participate in the intestinal inflammation during POI.

In the present study we investigated how the suprachiasmatic nucleus (SCN) controls the E(2)-induced PRL surge in female rats. First, the role of vasopressin (VP), a SCN transmitter present in medial preoptic area (MPO) projections and rhythmically released by SCN neurons, as a circadian signal for the E(2)-induced PRL surge was investigated. Using a reverse microdialysis technique, VP was administered in the MPO during the PRL surge, resulting in a suppression of the surge. VP administration before the surge did not affect PRL secretion. Also, administration of a V1a receptor antagonist before the surge was ineffective. Second, lesions of the SCN were made that resulted in constant basal PRL levels, suggesting that with removal of the SCN a stimulatory factor for PRL secretion disappeared. Indeed, the PRL secretory response to blockade of pituitary dopamine receptors was significantly reduced in SCN-lesioned animals. These data suggest that the afternoon decrease of VP release in the MPO by SCN terminals enables the PRL surge to occur, and may thus be a circadian signal for the PRL surge. Simultaneously the SCN is involved in the regulation of the secretory capacity of the pituitary, possibly via specific PRL-releasing factors.

Nutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response; therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 min post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and α-bungarotoxin and vagal intestinal denervation. Accordingly, release of β-hexosaminidase by MC/9 mast cells following LPS or IgE-ovalbumin complexes was dose dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR α7, in bone marrow-derived mast cells from nAChR β2−/− and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR α7 and is independent of the nAChR β2. Together, the present study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex.

Background The biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), controls the daily rhythms in physiology and behavior. Early studies demonstrated that light exposure not only affects the phase of the SCN but also the functional activity of peripheral organs. More recently it was shown that the same light stimulus induces immediate changes in clock gene expression in the pineal and adrenal, suggesting a role of peripheral clocks in the organ-specific output. In the present study, we further investigated the immediate effect of nocturnal light exposure on clock genes and metabolism-related genes in different organs of the rat. In addition, we investigated the role of the autonomic nervous system as a possible output pathway of the SCN to modify the activity of the liver after light exposure. Methodology and Principal Findings First, we demonstrated that light, applied at different circadian times, affects clock gene expression in a different manner, depending on the time of day and the organ. However, the changes in clock gene expression did not correlate in a consistent manner with those of the output genes (i.e., genes involved in the functional output of an organ). Then, by selectively removing the autonomic innervation to the liver, we demonstrated that light affects liver gene expression not only via the hormonal pathway but also via the autonomic input. Conclusion Nocturnal light immediately affects peripheral clock gene expression but without a clear correlation with organ-specific output genes, raising the question whether the peripheral clock plays a “decisive” role in the immediate (functional) response of an organ to nocturnal light exposure. Interestingly, the autonomic innervation of the liver is essential to transmit the light information from the SCN, indicating that the autonomic nervous system is an important gateway for the SCN to cause an immediate resetting of peripheral physiology after phase-shift inducing light exposures.

BACKGROUND: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output. METHODOLOGY/PRINCIPAL FINDINGS: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA) does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS), activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA. CONCLUSIONS/SIGNIFICANCE: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

International audience; Daily variations in plasma glucose concentrations are controlled by the biological clock, located in the suprachiasmatic nucleus. Our previous studies indicated an important role for the sympathetic innervation of the liver in the generation of the daily glucose rhythm. In the present study, we investigated further the role of the autonomic nervous system (ANS) in the genesis of the plasma glucose rhythm. First, we showed that complete removal of the autonomic inputs to the liver did not impair the plasma glucose rhythm or the daily expression of the gluco-regulatory enzymes in the liver. Consequently, we studied whether the daily glucose rhythm is driven by the daily feeding activity in denervated animals. Surprisingly, also complete denervation combined with a non-circadian feeding schedule did not abolish the 24h-profile in plasma glucose, nor all daily rhythms in the gene expression of liver enzymes. These results demonstrate that the mechanisms used by the SCN to control the rhythmic expression of glucose metabolizing enzymes and the 24h-rhythm in plasma glucose concentrations are highly versatile and the glucose rhythm can be maintained in absence of hepatic ANS input and/or a day/night-rhythm in feeding activity. Interestingly, a hepatic sympathectomy or parasympathectomy did abolish the plasma glucose rhythm, demonstrating that a "unilateral" denervation of the liver is more deleterious to maintaining the rhythmic liver metabolism than a complete removal of both branches. This observation supports the notion that an unbalanced ANS in obesity and diabetes accounts for the disturbed glucose balance in these disorders.

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day- and night-time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N-acetyltransferase (AA-NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h-pattern of melatonin secretion in SCN-lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN-lesioned and ganglionectomised rats, AA-NAT levels and pineal melatonin content were low (i.e. 12% of night-time control levels) for both day- and night-time periods. In SCN-lesioned rats, AA-NAT levels were intermediate (i.e. 30% of night-time control levels) and the 48-h secretion of melatonin presented constant levels not exceeding 20% of night-time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day-length (i.e. adaptation to seasons).

BACKGROUND: Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine. METHODS: Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages. RESULTS: Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers. CONCLUSION: In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages. ispartof: Plos One vol:9 issue:1 ispartof: location:United States status: Published online

Together with the paraventricular nucleus (PVN), the dorsomedial nucleus of the hypothalamus (DMH) acts as one of the hypothalamic centers that integrate autonomic and central information. The DMH in the rat brain has extensive intrahypothalamic connections and is implicated in a wide variety of functions. Up until now, no knowledge has been available to indicate that the human DMH might have functions similar to those of the rat DMH. In the present study, intrahypothalamic efferent projections of the human DMH were revealed by a recently developed in vitro postmortem tracing method. It was found that the most densely innervated areas are the PVN, the ventromedial nucleus of the hypothalamus, and the area below the PVN. Other significant terminal fields include the periventricular nucleus, the lateral hypothalamic area, and the medial part of the anteroventral hypothalamic area. Scarce fibers project to the suprachiasmatic nucleus, infundibular nucleus, posterior hypothalamic nucleus, and posterior part of the bed nucleus of the stria terminals. The projections of the ventral and dorsal part of the DMH show some differences. The dorsal part of the DMH has denser projections to the dorsal part of the PVN than to the ventral part of the PVN. In contrast, the ventral part of the DMH has denser projections to the ventral part of the PVN. Labeled fibers in the PVN from ventral and dorsal DMH appear to run near many vasopressin and oxytocin neurons of different sizes, and also near some corticotropin- releasing hormone neurons, suggesting that the DMH neurons may directly affect the functioning of these PVN neurons. In many aspects, the observed projections of the human DMH resemble those of the rat, indicating that the organization of DMH intrahypothalamic projections of human is similar to that of rat. The functional significance of DMH intrahypothalamic connections is discussed.

The suprachiasmatic nucleus (SCN) is a small structure considered to be the site of the major circadian pacemaker of the mammalian brain. Disturbances in human biological clock function may occur in several diseases, such as Alzheimer's disease, sleep problems, and seasonal depression. Since basic knowledge of the anatomical connections of the human SCN is limited due to the lack of suitable neuroanatomical tracing methods, the understanding of physiological mechanisms of human SCN function has obviously been hampered. In the present study, the hypothalamic connections of the human SCN were revealed for the first time with a newly developed in vitro postmortem anterograde tracing method. The human SCN was found to be connected with nuclei in the hypothalamus that are involved in hormone secretion, cardiovascular regulation, and behavior activity. These human SCN projections appear to follow the same general patterns as those in the rodent brain. This homology may indicate an evolutionary conservation of the SCN projections from rodent to human. Through these connections, the human SCN may transmit its circadian information to regulate hormone secretion, body temperature, and behavioral functions as it does in animal species. In addition, the postmortem tracing technique may be a valuable tool that will contribute to our understanding of anatomical connections in the human brain, and may have other applications in the research on the physiology and pathology of the human brain.

SPM 8 was applied to test anatomical regions of interest in the pINS, aINS and the aMCC while maintaining a family-wise error rate at p

BACKGROUND: We previously showed that the mast cell stabilizer ketotifen reduces IBS symptoms and increases the threshold of discomfort in hypersensitive IBS-patients. However, as ketotifen also has Histamine 1-receptor antagonistic properties and is known to penetrate the blood-brain barrier, its beneficial effect observed in this trial could also result from H1 receptor blockade, either at the peripheral or central level. AIM: To establish whether fexofenadine, a peripherally restricted 2nd generation Histamine 1-receptor antagonist, can mimic the effect of ketotifen and A) reverse stress-induced, mast cell dependent visceral hypersensitivity in a rat model of maternal separation (MS), and B) modulate gastrointestinal transit in MS and non-handled (NH) rats. METHODS: Adult MS and NH rats (n=9 in all groups) were subjected to acute stress (1 hr water avoidance,WA). The visceromotor response (VMR) to colorectal distension (1, 11⁄2, 2ml) was established preand 24 hours post-WA and expressed as area-under-curve (AUC, volume-vs-response, significant difference when P

SummarySmall cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, and no effective treatment is available to date. Mouse models of SCLC based on the inactivation of Rb1 and Trp53 show frequent amplifications of the Nfib and Mycl genes. Here, we report that, although overexpression of either transcription factor accelerates tumor growth, NFIB specifically promotes metastatic spread. High NFIB levels are associated with expansive growth of a poorly differentiated and almost exclusively E-cadherin (CDH1)-negative invasive tumor cell population. Consistent with the mouse data, we find that NFIB is overexpressed in almost all tested human metastatic high-grade neuroendocrine lung tumors, warranting further assessment of NFIB as a tumor progression marker in a clinical setting.