{"cards":[{"_id":"7598757828800fd976000082","treeId":"7598756228800fd976000080","seq":9443976,"position":1,"parentId":null,"content":"GLP1/PPG Neurons are activated by metabolically relevant neurotransmitters and peptides"},{"_id":"7598796a28800fd9760000bb","treeId":"7598756228800fd976000080","seq":9443986,"position":1,"parentId":"7598757828800fd976000082","content":"Intro"},{"_id":"76badf912b8b04e496000054","treeId":"7598756228800fd976000080","seq":9612606,"position":4,"parentId":"7598796a28800fd9760000bb","content":"Stock Info\n"},{"_id":"76bad9412b8b04e496000050","treeId":"7598756228800fd976000080","seq":9612431,"position":0.125,"parentId":"76badf912b8b04e496000054","content":"## Relevant background material\n"},{"_id":"76bad5da2b8b04e49600004c","treeId":"7598756228800fd976000080","seq":9612429,"position":0.25,"parentId":"76badf912b8b04e496000054","content":"- GLP1\n - Role peripherally\n - effects\n - central sites of PPG, and GLP1R\n - use of agonists in diabetes and obesity\n - GPCR, biased agonism (http://molpharm.aspetjournals.org/content/89/3/335), different effects to glucagon, GLP1, Ex4, Liraglutide. However explanation only to justify importance of GLP1 research as experiments all concern PPG; One step down the line."},{"_id":"75bf99b597fd4e706500003b","treeId":"7598756228800fd976000080","seq":9612376,"position":1,"parentId":"76bad5da2b8b04e49600004c","content":"## PPG Neurons Themselves and GLP1\n\n- `Could they maybe overlap with CCK neurons? probably not but worth asking to get clear understanding\n\n- Role in satiety, energy expenditure\n- Central versus peripheral controls\n Lead into the Anatomy section\n- Discuss use of exendin 4 and liraglutide in diabetes; give grounding in humans for importance of understanding GLP1\n - Maybe describe problems with GLP1 to indicate need for alternative solutions.\n\n- GLP1\n - Role peripherally\n - Vagal afferents from L cells, to NTS etc;\n - \"suggest that PPG neurons are Second order neurons with direct contact from vagal afferents\" Hisadome, Leptin (find real reference)\n - effects\n - central sites of PPG, and GLP1R\n - use of agonists in diabetes and obesity\n- PPG neurons more fleshed out\n - The NTS and its connections to the AP (circumventricular)\n - Projections of PPG Neurons\n - different regions and effect of GLP1R activation"},{"_id":"75c2643097fd4e7065000048","treeId":"7598756228800fd976000080","seq":9475699,"position":1,"parentId":"75bf99b597fd4e706500003b","content":"## GLP-1R Activation effects\n- GLP1 concentrated in hypothalamus, thalamus and lower brain stem (NTS and IRt)"},{"_id":"766c979bf4bf5128bb00004b","treeId":"7598756228800fd976000080","seq":9576411,"position":2,"parentId":"75bf99b597fd4e706500003b","content":"# PPG INFO\n\nGlucagon is a 29-amino acid pancreatic hormone which counteracts the blood glucose-lowering action of insulin by stimulating hepatic glycogenolysis and gluconeogenesis1. The structure of the hamster pancreatic glucagon precursor has recently been determined from the sequence of a cloned cDNA2. Hamster preproglucagon is a 180-amino acid protein which contains five functional regions; a signal or pre-peptide, an NH2-terminal peptide (also called glicentin-related pancreatic peptide, GRPP), glucagon, and two carboxy-terminal glucagon-like peptides (GLP-1 and GLP-2). The sequences of two non-allelic anglerfish pancreatic glucagon precursors3–5 have also been determined and their organization is similar but not identical to the hamster protein; they lack the polypeptide segment corresponding to hamster GLP-2. The presence of three regions possessing internal homology, that is, glucagon, GLP-1 and GLP-2, within proglucagon, and the absence of GLP-2 in the anglerfish precursors suggests that the structure of the preproglucagon gene might provide insight into the evolution of this polyprotein. We have isolated and sequenced the human preproglucagon gene and report here that the organization of the human precursor deduced from this sequence is identical to the hamster protein. The gene contains at least three intervening sequences which divide the protein-coding portion of the gene into four regions corresponding to the signal peptide and part of the NH2-terminal peptide, the remainder of the NH2-terminal peptide and glucagon, GLP-1, and GLP-2. The data suggest that triplication and subsequent sequence divergence of an exon encoding glucagon or a glucagon-like peptide produced this polyprotein precursor.\n\nhttp://www.nature.com/nature/journal/v304/n5924/abs/304368a0.html"},{"_id":"76bad7a92b8b04e49600004d","treeId":"7598756228800fd976000080","seq":9612430,"position":0.375,"parentId":"76badf912b8b04e496000054","content":"- PPG neurons more fleshed out\n - *The NTS* and maybe mention the IRt?\n - and its connections to the AP (circumventricular)\n - Projections of PPG Neurons\n - different regions and effect of GLP1R activation"},{"_id":"759bbb8128800fd9760000cd","treeId":"7598756228800fd976000080","seq":9612380,"position":1,"parentId":"76bad7a92b8b04e49600004d","content":"## Anatomy of relevant regions\n- NTS etc, discuss connection to area postrema (circumventricular organ), Arc and projections of PPG neurons, also? amount on GLP1R location? Maybe small amount on projections of those neurons?\n\n\nFROM TRAPP AND CORK\n"},{"_id":"759bd60828800fd9760000ce","treeId":"7598756228800fd976000080","seq":9474348,"position":1,"parentId":"759bbb8128800fd9760000cd","content":"## Solitary Tract\n\nThe solitary tract conveys afferent information from stretch receptors and chemoreceptors in the walls of the cardiovascular, respiratory, and intestinal tracts. Afferent fibers from cranial nerves 7, 9 and 10 convey taste (SVA) in its rostral portion, and general visceral sense (GVA) in its caudal part. \n\nContact with Area Postrema (circumventricular organs) ??weakened BBB?? (not right phrasing, but almost campling of contents of blood?\n\"PPG neurons; some dendrites project into the area postrema\" proves connectivity."},{"_id":"759be2ea28800fd9760000d0","treeId":"7598756228800fd976000080","seq":9447781,"position":2,"parentId":"759bbb8128800fd9760000cd","content":"##Nucleus of Solitary Tract in Brain Stem\n\n\"Information goes from the NTS to a large number of other regions of the brain including the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala, as well as to other nuclei in the brainstem (such as the parabrachial area, the Locus coeruleus, the Dorsal raphe nucleus , and other visceral motor or respiratory networks).\" Wiki"},{"_id":"759beb9428800fd9760000d1","treeId":"7598756228800fd976000080","seq":9447812,"position":3,"parentId":"759bbb8128800fd9760000cd","content":"## Central Canal\n\n\"the cerebrospinal fluid-filled space that runs longitudinally through the length of the entire spinal cord. The central canal is continuous with the ventricular system of the brain. The fourth ventricle narrows at a region called the obex to become the central canal of the spinal cord. The central canal helps to transport nutrients to the spinal cord as well as protect it by cushioning the impact of a force when the spine is affected\""},{"_id":"75bf847697fd4e7065000039","treeId":"7598756228800fd976000080","seq":9474208,"position":4,"parentId":"759bbb8128800fd9760000cd","content":"- if PPG neurons in NTS are stimulated by these NT/Ps, then these NTs/Ps might modulate activation of GLP1Rs in known areas of PPG projection and modulate the role and effect of GLP1R activation in those areas."},{"_id":"76bad82d2b8b04e49600004e","treeId":"7598756228800fd976000080","seq":9612410,"position":0.5,"parentId":"76badf912b8b04e496000054","content":"- Metabolically relevant compounds\n - Describe previous experiments by Hisadome et al. and the Swedes et al. investigating leptin and CCK on PPG neurons in the same mouse. \n - Describe why the following are relevant based on known actions/effects, receptor location etc.\n - CART\n - Histamine \n - Anandamide\n - Not really worth carbachol description?"},{"_id":"759bb3f428800fd9760000cc","treeId":"7598756228800fd976000080","seq":9612381,"position":1,"parentId":"76bad82d2b8b04e49600004e","content":"## Messengers"},{"_id":"759baccc28800fd9760000ca","treeId":"7598756228800fd976000080","seq":9447554,"position":1,"parentId":"759bb3f428800fd9760000cc","content":"Histamine\n"},{"_id":"759bacf528800fd9760000cb","treeId":"7598756228800fd976000080","seq":9475168,"position":1,"parentId":"759baccc28800fd9760000ca","content":"\"Mutual interactions with other transmitter systems form a network that links basic homeostatic and higher brain functions, including sleep-wake regulation, circadian and feeding rhythms, immunity, learning, and memory in health and disease.\"\n\nhttps://www.ncbi.nlm.nih.gov/pubmed/18626069"},{"_id":"75c168d397fd4e7065000042","treeId":"7598756228800fd976000080","seq":9475215,"position":2,"parentId":"759baccc28800fd9760000ca","content":"\"The source of histamine in the CNS is the tuberomammillary nucleus (TMN) of the hypothalamus, which provides widespread innervation to the forebrain, brainstem, and spinal cord. Histamine, acting via H1, H2, and H3 receptors, controls neuronal excitability, synaptic transmission, and synaptic plasticity, both directly and via interactions with other neurotransmitter systems. Histaminergic TMN neurons are active during wakefulness and exert multiple functions. Studies on knockout mice indicate that histamine has a major role in maintenance of arousal and contributes to modulation of circadian rhythms, homeostasis, motor behavior, and cognition. The histaminergic system is involved in narcolepsy and may also contribute to the manifestations of Alzheimer disease (AD), Parkinson disease (PD), Tourette syndrome, and other neurologic disorders. Drugs that affect histamine receptors have therapeutic potential for sleep disorders, pain, epilepsy, and cognitive disorders.\"\n\nhttp://ovidsp.tx.ovid.com.libproxy.ucl.ac.uk/sp-3.23.1b/ovidweb.cgi?QS2=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"},{"_id":"75c17bd497fd4e7065000043","treeId":"7598756228800fd976000080","seq":9475247,"position":3,"parentId":"759baccc28800fd9760000ca","content":"\"Orexigenic actions of orexins/hypocretins (310) and\nanorexigenic effects of leptin (453, 758) and glucagon-like peptide-1 (GLP-1), which depend on CRH released by\nPVN neurons (214), are all blunted or absent by pharma-\ncological or genetic loss of H1R function.\"\n\nHISTAMINE CHAPTER, p1212\n"},{"_id":"75989bfa28800fd9760000c6","treeId":"7598756228800fd976000080","seq":9447555,"position":2,"parentId":"759bb3f428800fd9760000cc","content":"### Anandamide\n\nUsing methyl-anandamide (more stable? Paper showing that no difference or even yes difference)"},{"_id":"75bf4f5597fd4e7065000036","treeId":"7598756228800fd976000080","seq":9474261,"position":1,"parentId":"75989bfa28800fd9760000c6","content":"https://www.ncbi.nlm.nih.gov/pubmed/19083471\n\n\"recent studies implicate CB1 as the primary receptor that functions in appetite regulation, insulin sensitivity, lipid metabolism, and futile cycling.\"\n\n\"Fourth, when the vagal neurons that mediate cholecystokinin were eliminated with capsaicin (a neurotoxin), AEA administration failed to promote hyperphagia [62]. \" CCK and PPG neurons? Shown by hisadome? are these the same neurons?\nNo as not activated by melanotan and cck neurons are?\n\n\n\" Fasted rats had increased levels of both AEA and 2-AG in the limbic forebrain and increased levels of 2-AG in the hypothalamus\" opposed to satiety effect of GLP1 so potentially aligns with inhibitory effect we expected to see? Limbic region associated with hedonic aspect; GLP1 in hedonic control?"},{"_id":"759898c228800fd9760000c5","treeId":"7598756228800fd976000080","seq":9447570,"position":3,"parentId":"759bb3f428800fd9760000cc","content":"CART\n\n- Precursor protein, 2 active fragments\n- No known receptors (?)\n- Anorectic\n- Feeding relation identified due to localization in feeding regions (VMH, DMN, LH, Arc, PVN and NucAcc)\n- Association with POMC?\n\n\n\nhttp://www.cell.com/trends/endocrinology-metabolism/fulltext/S1043-2760(04)00220-6\n"},{"_id":"7598b94a28800fd9760000c7","treeId":"7598756228800fd976000080","seq":9444328,"position":1,"parentId":"759898c228800fd9760000c5","content":"\n\"The human propeptide is 89 amino acids in length and is processed into two fragments of known biological activity, residues 42–89 and 49–89, which correspond to residues 55–102 and 62–102, respectively, in the long form expressed in the rat. The amino acid homology between the rat and human forms of the peptide is 95% (Figure 1). Interestingly, the two active forms of the peptide seem to have different effects on several behaviors, including feeding [6, 9, 10]. Moreover, the two peptides have different distribution profiles, with some regions expressing both or, as in the pituitary, only one (42–89 in the anterior and 49–89 in the posterior) [8].\n"},{"_id":"75c2301597fd4e7065000045","treeId":"7598756228800fd976000080","seq":9475548,"position":1.5,"parentId":"759898c228800fd9760000c5","content":"\"CART peptides, in particular, CART (55–102), seem to have an important function in the regulation of energy homeostasis, and interact with several hypothalamic appetite circuits. CART expression is regulated by several peripheral peptide hormones involved in appetite regulation, including leptin,[5] cholecystokinin and ghrelin,[6] with CART and cholecystokinin having synergistic effects on appetite regulation.[7]\" Wiki"},{"_id":"7598b99a28800fd9760000c8","treeId":"7598756228800fd976000080","seq":9444332,"position":2,"parentId":"759898c228800fd9760000c5","content":"\n CART immunoreactivity was localized to brain regions associated with feeding behavior [11, 12], notably the ventromedial nucleus (VMN), dorsomedial nucleus (DMN), lateral hypothalamus (LH), arcuate nucleus (Arc), paraventricular nucleus of the hypothalamus (PVN) and nucleus accumbens, which is a neural substrate for the expression of reward for behaviors such as food intake [13]. It has since been demonstrated that CART can be found in several locations, both centrally and peripherally, that are involved in feeding \""},{"_id":"7598b9f728800fd9760000c9","treeId":"7598756228800fd976000080","seq":9444334,"position":3,"parentId":"759898c228800fd9760000c5","content":"\nIn a way coloacted with POMC? \"CART peptides are colocalized with a range of other neurotransmitters and peptides that are involved in the regulation of feeding, such as melanin-concentrating hormone in the DMN and LH [22], corticotrophin-releasing factor (CRF) in the PVN [23, 24, 25] and α-melanocyte-stimulating hormone from pro-opiomelanocortin in the Arc\""},{"_id":"76bad89a2b8b04e49600004f","treeId":"7598756228800fd976000080","seq":9612407,"position":1,"parentId":"76badf912b8b04e496000054","content":"\n- Experimental background, approach and aims\n - Ex vivo brain slices allow for direct imaging of cell population \n - Why calcium imaging? Brief (Very brief) description of calcium release in response to cell depolarisation (VGCC and Ca release for sarcoplasmic reticulum.)\n - Many other pathways; glutamatergic activation (Ca permeable AMPAR, NMDAR, mGLUR, also Ca permeable nAChRs)\n - Describe the PPG-Cre GCaMP mouse (flxd repressor for GCaMP so that Cre activates, not removes) and how this leads to selective GCaMP expression\n - Validate model using expression maps?\n - reliability of system\n - Briefly described\n - Aims; to screen for responses to relevant compounds in order to identify candidates worthy of further scrutiny"},{"_id":"75bfb8fa97fd4e706500003d","treeId":"7598756228800fd976000080","seq":9612382,"position":1,"parentId":"76bad89a2b8b04e49600004f","content":"Explain Cre mouse and floxd repressor for GCaMP? or is it FLEXed?\nCre-loxp in mouse;\n\nassess expression shown in mouse of choice?"},{"_id":"75a72dec3f0f6b8206000035","treeId":"7598756228800fd976000080","seq":9612384,"position":2,"parentId":"76bad89a2b8b04e49600004f","content":"GCaMP as a GECI\nSo measuring fluorescence due to calcium binding to EF hands in GCaMP; this is a measure of the intracellular calcium concentration and in turn"},{"_id":"75cd3d59e0e1026419000053","treeId":"7598756228800fd976000080","seq":9482565,"position":1,"parentId":"75a72dec3f0f6b8206000035","content":"FRET Based\nSlow kinetics compared to chemical calcium indicator? So time lapse not particularly clear; more advantageous to do patch clamps vs further calcium recordings tho."},{"_id":"76bae2a62b8b04e496000055","treeId":"7598756228800fd976000080","seq":9612432,"position":5,"parentId":"7598796a28800fd9760000bb","content":"New"},{"_id":"76bae2c12b8b04e496000056","treeId":"7598756228800fd976000080","seq":9612437,"position":1,"parentId":"76bae2a62b8b04e496000055","content":"## GLP-1 Matters"},{"_id":"76bb52a92b8b04e496000060","treeId":"7598756228800fd976000080","seq":9612756,"position":1,"parentId":"76bae2c12b8b04e496000056","content":"## 1\n\nIt was first shown in 1996 that glucagon-like peptide 1 (GLP-1) reduces food intake through actions within the central nervous system (CNS) (Turton et al. 1996). Originally marginalised as a gut hormone with an important role in gastric emptying, glucose homeostasis and appetite control, current research is focused on illuminating how and where GLP-1 acts in the CNS, as well as where it originates from. GLP-1 is released peripherally by specialised endocrine cells in the intestinal epithelium and centrally by neurons in the lower brain stem, which are innervated by vagal afferents (Holst 2007). "},{"_id":"76bb70b12b8b04e496000062","treeId":"7598756228800fd976000080","seq":9612742,"position":1,"parentId":"76bb52a92b8b04e496000060","content":"Glucagon-like Peptide 1 (GLP1) is a neuropeptide which has been a focus of research for several decades due to its role as an incretin and satiety signal (Turton et al. 1996). GLP-1 is released by intestinal L-cells as well as by neurons in the central nervous system (CNS)."},{"_id":"76bb54b12b8b04e496000061","treeId":"7598756228800fd976000080","seq":9612740,"position":2,"parentId":"76bb52a92b8b04e496000060","content":"increased satiety due to action on hypothalamic sites, as well as through triggering of visceral malaise and conditioned taste aversion via activity in the amygdala and brain stem"},{"_id":"76bb86372b8b04e496000064","treeId":"7598756228800fd976000080","seq":9614012,"position":2,"parentId":"76bae2c12b8b04e496000056","content":"Glucagon-like Peptide 1 (GLP1) is a neuropeptide which has been a focus of research for several decades due to its role as an incretin and satiety signal (Turton et al. 1996). GLP-1 is released by intestinal L-cells as well as by neurons in the central nervous system (CNS). Its physiological effects include the modulation of gastric emptying, glucose homeostasis, and appetite control, with both central and peripheral mechanisms likely contributing to its role in food intake control (Holst 2007; Sandoval 2008; Trapp & Richards 2013).\n\nProglucagon is a precursor peptide encoded for by the *GCG* gene and processed by prohormone convertases to produce various metabolically important hormones. GLP1 is produced by posttranslational processing of proglucagon, which is derived from preproglucagon (PPG) and encoded for by the GCG gene. Proglucagon is processed by either prohormone convertase 2 (PC2), which is expressed in pancreatic alpha cells and converts proglucagon into glucagon (reference), or by prohormone convertase 1/3 (PC1/3) which is expressed in specialised intestinal endothelial cells (L-cells) as well as by neurones in the lower brain stem (Merchenthaler et al. 1999; Orskov et al. 1987; Trapp & Cork 2015). In these neurones, processing by PC1/3 produces GLP-1, Glucagon-like Peptide 2 and Oxyntomodulin (Holst 2007)(reference). \n\nGLP1 is produced in the brain stem by a population of glutamatergic cells , which in mice are identified based on the activity of the GCG gene and are therefore referred to as PPG neurones.\n"},{"_id":"76bae34a2b8b04e496000057","treeId":"7598756228800fd976000080","seq":9612451,"position":2,"parentId":"76bae2a62b8b04e496000055","content":"## GLP-1 Does This At These Sites"},{"_id":"76bae4762b8b04e496000058","treeId":"7598756228800fd976000080","seq":9612640,"position":3,"parentId":"76bae2a62b8b04e496000055","content":"## Central GLP-1 Supply"},{"_id":"76bb500b2b8b04e49600005d","treeId":"7598756228800fd976000080","seq":9612642,"position":2,"parentId":"76bae4762b8b04e496000058","content":"There is controversy as to whether the centrally-mediated effects are produced by GLP-1 released in the periphery by L-cells reaching GLP-1Rs in the CNS, or if it is due to the activity of a small population of neurons in the lower brain stem. (Trapp & Cork 2015)"},{"_id":"76bb4efa2b8b04e49600005c","treeId":"7598756228800fd976000080","seq":9612643,"position":1,"parentId":"76bb500b2b8b04e49600005d","content":"GLP-1 receptors have been localised in the CNS using in situ hybridisation techniques (Merchenthaler et al., 1999; Shughrue et al., 1996). They are found in many parts of the brain, with exception of the cortex and the cerebellum. Particularly high levels are detected in hypothalamic nuclei and in the lower brainstem (Merchenthaler et al., 1999; Shughrue et al., 1996). Most of these receptors are located behind the blood-brain barrier and it is under debate whether GLP-1 can or cannot cross this barrier (Kastin et al., 2002; Orskov et al., 1996). In a very convincing study Orskov et al (1996) addressed this issue by assessing receptor binding of radiolabeled GLP-1 with autoradiography and concluded that only GLP-1 receptors in the subfornical organ and the area postrema (AP) are accessible to circulating GLP-1. Both of these regions, however, have a leaky blood- brain barrier. Considering this together with the short half-life of GLP- 1 in the circulation (Holst and Deacon, 2005), it might be questionable whether GLP-1 released from enteroendocrine epithelial cells can reach receptors inside the blood-brain barrier. It appears more feasible that a source of GLP-1 within the brain is serving GLP-1 receptors in the CNS. Cells expressing GLP-1 have been located in the lower brainstem, particularly within the nucleus of the solitary tract (NTS), and in the olfactory bulb, using in situ hybridisation or immunohistochemistry techniques (Jin et al., 1988; Larsen et al., 1997; Merchenthaler et al., 1999). Recently, these results have been confirmed in a transgenic mouse-model where yellow fluorescent protein (YFP; Venus™) has been expressed under the control of the PPG promoter (Reimann et al., 2008). These mGLU-YFP mice showed YFP fluorescence in pancreatic α-cells, in L-cells of the gut, and an YFP expression pattern in the brain that was largely identical to the distribution of GLP-1 positive cell bodies found with immunocytochemistry in rat (Hisa- dome et al., 2010b; Larsen et al., 1997; Reimann et al., 2008; Trapp et al., 2010). Consequently, these neurons would be the most likely source of GLP-1 acting within the brain, with the possible exception of receptors located in AP or subfornical organ. If this were the case, the question remains whether these GLP-1 neurons release GLP-1 in a postprandial fashion like enteroendocrine L-cells. And if so, what is the exact signal that triggers their GLP-1 release? Could it be peripheral"},{"_id":"76bae4fa2b8b04e496000059","treeId":"7598756228800fd976000080","seq":9612458,"position":4,"parentId":"76bae2a62b8b04e496000055","content":"## PPG Neurons in Central GLP-1R Activation"},{"_id":"76bb51502b8b04e49600005f","treeId":"7598756228800fd976000080","seq":9612646,"position":1,"parentId":"76bae4fa2b8b04e496000059","content":"Centrally mediated satiety signalling has been studied using icv application of GLP-1 analogs and antagonists and shows a clear effect which is not blocked by peripheral (intraperitoneal) application of GLP-1 antagonists. Activation of central GLP-1 receptors seems likely to require release of GLP-1 from PPG neurons. In support of this hypothesis, a number of findings have suggested the involvement of the brain stem PPG neurons in appetite control. The immediate early gene cFOS, for example, is activated in PPG cells by peripheral satiety signals, such as gastric distension or systemically administered leptin."},{"_id":"76bae5972b8b04e49600005a","treeId":"7598756228800fd976000080","seq":9612467,"position":5,"parentId":"76bae2a62b8b04e496000055","content":"## PPG Neurons, The Periphery and What Causes Activation"},{"_id":"76bae6ce2b8b04e49600005b","treeId":"7598756228800fd976000080","seq":9612652,"position":6,"parentId":"76bae2a62b8b04e496000055","content":"## How To Find Out? Experimental Information"},{"_id":"7598798928800fd9760000bc","treeId":"7598756228800fd976000080","seq":9443987,"position":2,"parentId":"7598757828800fd976000082","content":"Methods"},{"_id":"759879f428800fd9760000bf","treeId":"7598756228800fd976000080","seq":9443991,"position":1,"parentId":"7598798928800fd9760000bc","content":"Slicing"},{"_id":"75bfe25797fd4e706500003e","treeId":"7598756228800fd976000080","seq":9474328,"position":1,"parentId":"759879f428800fd9760000bf","content":"Prep etc,\nKey is locating NTS?"},{"_id":"75987a1a28800fd9760000c0","treeId":"7598756228800fd976000080","seq":9443992,"position":2,"parentId":"7598798928800fd9760000bc","content":"Imaging"},{"_id":"75987a3328800fd9760000c1","treeId":"7598756228800fd976000080","seq":9483513,"position":3,"parentId":"7598798928800fd9760000bc","content":"Processing/Analysis"},{"_id":"759879a128800fd9760000bd","treeId":"7598756228800fd976000080","seq":9457244,"position":3,"parentId":"7598757828800fd976000082","content":"Results"},{"_id":"75a721d13f0f6b8206000034","treeId":"7598756228800fd976000080","seq":9614101,"position":1,"parentId":"759879a128800fd9760000bd","content":"\n\n- Histamine Concentration experiments?\n"},{"_id":"75c1016597fd4e7065000041","treeId":"7598756228800fd976000080","seq":9614104,"position":2,"parentId":"759879a128800fd9760000bd","content":"Analysis Plans\n- Comment on n numbers with Carbachol; reliability of response; and consistency\n - Comment on signs of death within the slice?\n - Any issues which might be caused by stackreg? none come to mind......\n - Comparative bar chart for difference in dose response with carbachol\n- Select Prime graphs to exemplify responses for each\n - Select choice cell? average and show trace with n number? Can you combine across slices? (should be fine even if brightness etc adjusted.)\n- Spiking analysis for CART? \n\n"},{"_id":"76bd49862495e23fd8000100","treeId":"7598756228800fd976000080","seq":9619355,"position":3,"parentId":"759879a128800fd9760000bd","content":"## Carbachol and Glutamate\nInitial set, validate experimental data?\nWhat literature can you refer to?"},{"_id":"759879c128800fd9760000be","treeId":"7598756228800fd976000080","seq":9483500,"position":4,"parentId":"7598757828800fd976000082","content":"Discussion\n- Is there an effect on [Ca] or not\n - all that should be stated\n- Extrapolate on this to connectivity between these circuits (circadian with histamine etc.)"},{"_id":"75a71f253f0f6b8206000031","treeId":"7598756228800fd976000080","seq":9483499,"position":1,"parentId":"759879c128800fd9760000be","content":"Discussion: This section is where you discuss and interpret your results, setting them in the\ncontext of what is already known. You have the opportunity here to be more speculative and\nyou should include suggestions for further studies that might develop from your work.\n\nAssess Carbachol, reliability of response and dose responsiveness; KCl, any statistical test that might be possible? Compare height of fluorescence increase\nExplain why carbachol is a valid control; presence of cholinergic neurons via ChAT staining, previously shown effects of ACh on GLP1R/PPG neurons? Keep looking\n\nGENERAL COMMENT ON FLUORESCENCE RESULT\nDue to the wide range of mechanisms which lead to calcium influx the effect of these neurotransmitters can not be linked to anything mechanistic\nActivation of any cell with a histamine receptor (for example) can lead to glutamatergic signalling and produce calcium response; alternatively PPG neurons might have histamine receptors.\n\nOnly comment is really that histaminergic signalling is implicated in the activity of PPG neurons. "},{"_id":"75ce37ade0e1026419000056","treeId":"7598756228800fd976000080","seq":9482902,"position":1.25,"parentId":"759879c128800fd9760000be","content":"Glutamate response;\nBiphasic due to activation of slow mGluRs and PLC/IP3 pathway to trigger Ca release."},{"_id":"75a71f3e3f0f6b8206000032","treeId":"7598756228800fd976000080","seq":9457207,"position":2,"parentId":"759879c128800fd9760000be","content":"Histamine Positive Result\nDiscuss central circadian link to GLP1?\n"},{"_id":"75c0348397fd4e7065000040","treeId":"7598756228800fd976000080","seq":9474844,"position":2.5,"parentId":"759879c128800fd9760000be","content":"CART\n- No response\n- One odd response; clear, consistent however did not wash off?\n- Significance of not washing off??\n\nDid I leave sufficient time maybe? 30 minutes tho....\n\nNot washing off suggests long lasting metabotropic effect maybe?"},{"_id":"75a720b53f0f6b8206000033","treeId":"7598756228800fd976000080","seq":9457209,"position":3,"parentId":"759879c128800fd9760000be","content":"Anandamide (requires more data)\nSlight inhibition; "},{"_id":"75c030f097fd4e706500003f","treeId":"7598756228800fd976000080","seq":9474496,"position":4,"parentId":"759879c128800fd9760000be","content":"Compare size of response to KCl response however overly variable due to various factors?\n - maybe positioning in the slice\n - exact duration of exposure (though this should not have been an issue.....)\n - Variability in concentration (again should not have been)"},{"_id":"75c2587c97fd4e7065000046","treeId":"7598756228800fd976000080","seq":9475655,"position":6,"parentId":"759879c128800fd9760000be","content":"## Critique"},{"_id":"75c1842297fd4e7065000044","treeId":"7598756228800fd976000080","seq":9475653,"position":1,"parentId":"75c2587c97fd4e7065000046","content":"Experimental comments\nhttp://www.nature.com/articles/srep05309\nAND \nSome Slice It Hot?"},{"_id":"75c2592197fd4e7065000047","treeId":"7598756228800fd976000080","seq":9475693,"position":2,"parentId":"75c2587c97fd4e7065000046","content":"Barrera et al KD PPG in NTS using lentiviral mediated RNA interference; did not effect energy expenditure or glucose tolerance independent of body weight. There was an increase in night time feeding however (histamine connection?). However casts doubt on potential connection between NT/P examined and central GLP1RA effects. "},{"_id":"773364941d53c0c3f8000065","treeId":"7598756228800fd976000080","seq":9707010,"position":7,"parentId":"759879c128800fd9760000be","content":""},{"_id":"75bf6bef97fd4e7065000037","treeId":"7598756228800fd976000080","seq":9515884,"position":2,"parentId":null,"content":"Questions\n- Why no pyruvate?\n- Details of GFP Filter STEFAN\n- Mouse info; \n - should the be intro or methods?\n- Rig info? CoolLED? Blue light wavelength?\n - Numerical Aperture and Magnification (should be on Lense)\n- Sourcing of NTs; Methyl-Anandamide (sigma) But carb? Glu? His? CART (also what CART am i using?? full precurser?) ASK STEFAN\n- Double check use of bregma/lambda +\n- Carbachol validates through positive effect; would it not also be good to show difference via no observed effect from null compound, then KCl spike?\n- n Numbers; is this n off slices or n of cells?\n- Does PPG neuron stimulation but with someway of blocking GLP1 have any effect? That would indicate an external role in the GLP1 that may connect\n- Validity of PPG as GLP1 marker? GLP1 versus GLP2? Also Oxyntomodulin? Could be determined by presence of cleaving enzymes in the neurons.\n\nNeeds checking\n- Reservoir temperature"},{"_id":"75ce3d35e0e1026419000057","treeId":"7598756228800fd976000080","seq":9482907,"position":3,"parentId":null,"content":"Comments\n- Source for physiological range of concentrations of NT/P"},{"_id":"76031e51704eda3a3f0000e4","treeId":"7598756228800fd976000080","seq":9534745,"position":4,"parentId":null,"content":"Frank Reimann made ppG Cre RFP, then crossed Zariwala (Cre dependent GCaMP3 reporter mouse)"}],"tree":{"_id":"7598756228800fd976000080","name":"Dissertation","publicUrl":"7598756228800fd976000080"}}