On the use of transgenic mice and optogenetics to characterize genetically defined subpopulations of neurons

Neuroscientists have a variety of perspectives with which to classify different parts of the brain. With the rise of genetic-based techniques such as optogenetics, it is increasingly important to identify whether a group of cells, defined by morphology, function or anatomical location possesses a distinct pattern of expression of one or more genetic promoters. This would allow for better ways to study of these genetically defined subpopulations of neurons. In this work, I present a theoretical discussion and threeexperimental studies in which this was the main question being addressed. Paper I discusses the issues involved in selecting a promoter to study structures and subpopulations in the Ventral Tegmental Area. Paper II characterizes a subpopulation of cells in the Ventral Tegmental Area that shares the expression of a promoter and is anatomically very restricted, and induces aversion when stimulated. Paper III utilizes a similar strategy to investigate a subpopulation in the subthalamic nucleus that expresses PITX2 and VGLUT2 which, when inactivated, causes hyperlocomotion. Paper IV exploits the fact that a previously identified group of cells in the ventral hippocampus expresses CHRNA2, and indicates that this population may be necessary and sufficient for the establishment of the theta rhythm (2-8 Hz) in the Local Field Potential of anesthetized mice. All of these studies were guided by the same strategy of characterizing and studying the role of a genetically defined subpopulation of cells, and they demonstrate the different ways in which this approach can generate new discoveries.

Influence of laparoscopy and laparotomy on gasometry, leukocytes and cytokines in a rat abdominal sepsis model

Laparoscopic surgery is associated with reduced surgical trauma, and less acute phase response, as compared with open surgery. Cytokines are important regulators of the biological response to surgical and anesthetic stress. The aim of this study was to determine if CO2 pneumoperitoneum would change cytokine expression, gas parameters and leukocyte count in septic rats. Methods: Wistar rats were randomly assigned to five groups: control (anesthesia only), laparotomy, CO2 pneumoperitoneum, cecum ligation and puncture by laparotomy, and laparoscopic cecum ligation and puncture. After 30 min of the procedures, arterial blood samples were obtained to determine leukocytes subpopulations by hemocytometer. TNFα, IL-1β, IL-6 were determined in intraperitoneal fluid (by ELISA). Gas parameters were measured on arterial blood, intraperitoneal and subperitoneal exsudates. Results: Peritoneal TNFα, IL-1β and IL-6 concentrations were lower in pneumoperitoneum rats than in all other groups (p<0.05). TNFα, IL-1β and IL-6 expression was lower in the laparoscopic than in laparotomic sepsis (p<0.05). Rats from laparoscopic cecum ligation and puncture group developed significant hypercarbic acidosis in blood and subperitoneal fluid when compared to open procedure group. Total white blood cells and lymphocytes were significantly lower in laparoscopic cecum ligation and puncture rats than in the laparotomic (p<0.01). Nevertheless, the laparotomic cecum ligation rats had a significant increase in blood neutrophils and eosinophils when compared with controls (p<0.05). Conclusions: This study demonstrates that the CO2 pneumoperitoneum reduced the inflammatory response in an animal model of peritonitis with respect to intraperitoneal cytokines, white blood cell count and clinical correlates of sepsis. The pneumoperitoneum produced hypercarbic acidosis in septic animals

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Influência da laparoscopia e da laparotomia na gasometria, leucometria diferencial e citocinas em modelo de sepse abdominal em ratos

Laparoscopic surgery is associated with reduced surgical trauma, and less acute phase response, as compared with open surgery. Cytokines are important regulators of the biological response to surgical and anesthetic stress. The aim of this study was to determine if CO2 pneumoperitoneum would change cytokine expression, gas parameters and leukocyte count in septic rats. Methods: Wistar rats were randomly assigned to five groups: control (anesthesia only), laparotomy, CO2 pneumoperitoneum, cecum ligation and puncture by laparotomy, and laparoscopic cecum ligation and puncture. After 30 min of the procedures, arterial blood samples were obtained to determine leukocytes subpopulations by hemocytometer. TNFα, IL-1β, IL-6 were determined in intraperitoneal fluid (by ELISA). Gas parameters were measured on arterial blood, intraperitoneal and subperitoneal exsudates. Results: Peritoneal TNFα, IL-1β and IL-6 concentrations were lower in pneumoperitoneum rats than in all other groups (p<0.05). TNFα, IL-1β and IL-6 expression was lower in the laparoscopic than in laparotomic sepsis (p<0.05). Rats from laparoscopic cecum ligation and puncture group developed significant hypercarbic acidosis in blood and subperitoneal fluid when compared to open procedure group. Total white blood cells and lymphocytes were significantly lower in laparoscopic cecum ligation and puncture rats than in the laparotomic (p<0.01). Nevertheless, the laparotomic cecum ligation rats had a significant increase in blood neutrophils and eosinophils when compared with controls (p<0.05). Conclusions: This study demonstrates that the CO2 pneumoperitoneum reduced the inflammatory and immune response in an animal model of peritonitis with respect to intraperitoneal cytokines, white blood cell count and clinical correlates of sepsis. The pneumoperitoneum produced hypercarbic acidosis in septic animals

Estrutura genética de populações de Amphobotrys ricini, agente causal do mofo cinzento da mamoneira

The gray mold, causal organism Amphobotrys ricini, is one of the major diseases of castor bean. Difficulties in managing plant disease arises form the limited understanding of the genetic structure of A. ricini, their complexity and variability make it difficult to control. Genetic structure can be used to infer the relative impact of different forces that influence the evolution of pathogen populations, that allow to predict the potencial for pathogen populations to envolve in agricultural ecosystems. Growers protect their crop by applying fungicides, but there aren t fungicides to provide significant control of gray mold of castor bean. The objectives of this work were use RAPD to determine the genetic structure of A. ricini subpopulations in Paraíba and assay the sensitivity of A. ricini isolates to azoxystrobin and carbendazim. To determine the genetic structure of A. ricini subpopulations in Paraíba, 23 isolates were colleted from two different geographic location (subpopulation). These isolates were analysed by RAPD using 22 random decamer primers, purchased from OPERON, produced a total of 80 markers polimorphics. The resulting matrixes were analysed using PopGene version 1.32. Sensitivity to azoxystrobin and carbendazim of 30 isolates, colleted form Paraíba and Alagoas, was estimated based on spore germination and colony growth inhibition. The stock solutions were added toV8 medium after sterilization to produce final concentrations of 0, 0.01, 0.1, 1, 10, and 100 µg/ml of carbendazim and 0, 0.001, 0.01, 0.1, 1, and 10 µg/ml of azoxystrobin. All statistical analyses were performed using SAS to estimate the dose that inhibited fungal growth by 50% (ED50 values). The genetic diversity within subpopulations (Hs=0,271) accounted for 92% of the total genetic diversity (Ht=0,293), while genetic diversity between subpopulations (Gst = 0,075) represented only 7,5%. The estimated number of migrants per generation (NM ) was 6,15. Nei s average gene identity across 80 RAPD loci was 0,9468. Individual ED50 values, for the 30 isolates screened for their sensitivity to azoxystrobin, ranged From a maximum of 0,168 µg/ml to a minimum of 0,0036 µg/ml. The ED50 values for carbendazim varied within the range of 0,026 to 0,316 µg/ml

Caracterização da resistência do algodoeiro a Ramularia areola e variabilidade molecular do patógeno

This research was conducted with the aim to study the genetic and pathogenic structure of Ramularia areola isolates collected in Brazil and to characterize the resistance response in cotton plants to ramularia spot. The genetic variability of 28 isolates of R. areola was studied using RAPD markers. The pathogenicity evaluation was realized by the inoculation of 6 isolates on cotton varieties Guazuncho-2 (Gossypium hirsutum) and VH8-4602 (Gossypium barbadense). The inheritance of disease resistance was studied using an artificially inoculated population of F2 individuals derived from the intercross of Guazuncho-2 (susceptible variety) end VH8-4602 (resistant variety), and also the parents and F1 individuals. Molecular polymorphism between the G. hisutum varieties DeltaOpal (suscetible) and CNPA CO-11612 (resistant) was estimated by 118 SSR and 24 AFLP markers. The parental genotypes Guazuncho-2 and VH8-4602 were selected for mapping, and then Recombinant Inbred Lines (RIL´s) derived from this crossing were evaluated with SSR 12 markers. The analysis of population structure of R. areola revealed that the three subpopulations were genetically simillar (Gst=0.18), and the isolates from Goiás and Minas Gerais were more similar to each other (0,92). This probability can be related to the relatively high gene flow among the three subpopulations (Nm=2.20). The isolates R. areola 9.1, from Minas Gerais State and 8.1 and 8.3 from Goiás State were the most aggressive ones to the susceptible variety Guazuncho-2. The variety VH8-4602 presented high level of resistance to ramularia spot. No differential interaction was observed between the pathogens and the analyzed varieties, and the resistance was classified as horizontal. The quantification of disease by number of necrotic lesions and number of spores in individual plants of F1 and F2 generations from the crossing between the varieties Guazuncho-2 and VH8-4602 presented continuous distribution, suggesting polygenic resistance. The resistance is probabilly recessive, since necrotic lesions and sporulation were observed on F1 plants. The molecular polymorphism between DeltaOpal e CNPA CO-11612 lineages was low (6%), then would be difficult to accomplish molecular mapping of disease resistance using this intercross. With the genotyping of the RIL s it was verified that 25% of the markers segregated in the proportions proposed by Mendel s Law and 75% of the studied markers presented segregation distortion in favor to the parental G. hirsutum. Both the low genetic variability of the pathogen and the number of resistance genes suggest that durable genetic resitance may be achieved

Receptores de citocinas proinflamatórias na pré-eclâmpsia

Preeclampsia is a disease specific of human pregnancy that affects 3-8% of pregnant women, and it is one of the three leading causes of maternal mortality and morbidity. The disease is characterized by hypertension and proteinuria after the 20th week of gestation. The risk factors for this disease are not completely understood but appear to include dysregulation of the immune response arising from defects in placentation, environmental and genetic factors. This study aimed to determine whether the variation in the amount of proinflammatory cytokine receptors IL-1R2, IL-6R and TNF-αR1 would be involved in preeclampsia. They were recruited women with preeclampsia (n=24) and women who evolved during pregnancy without changes in blood pressure (n=12) were recruited. Clinical and laboratory data were collected. The cytokine receptors (IL-1R2, TNF-αR1 and IL-6R) were assessed in mononuclear cells isolated from peripheral blood using flow cytometry (Control = 8; PE = 24). C-reactive protein (CRP) was determined by CRP ultrasensitive method (Control = 7; PE = 18) was performed using sera pregnant women. Women with preeclampsia had higher weight at the beginning of the pregnancy (p=0.0171) and lower gestational age at delivery (0.0008). Classical monocytes were decreased in preeclampsia but not intermediate or non-classical monocytes. The frequency of IL-1R2 pro inflammatory cytokine receptors is decreased in women with PE only in the subpopulation of non-classical monocytes (p = 0.0011). TNF-αR1 receptor and IL-6R, had a decreased frequency in the three subpopulations of monocyte (classic, intermediate and non-classical) when compared to women with normal pregnancy. An increase in IL-1R2 receptor in TCD4+ lymphocytes, but a decrease in TNF-receptor and IL-6R in women with preeclampsia were found. No differences in the frequency of those receptors in CD3+/CD8+ in preeclampsia. There was no difference in C-reactive protein in preeclampsia. The reduction in the amount of IL-1R2, TNF- αR1 and IL-6R monocytes and lymphocytes can be involved in the regulation of inflammation observed in preeclampsia, contributing to disease.

Influence of laparoscopy and laparotomy on gasometry, leukocytes and cytokines in a rat abdominal sepsis model

Laparoscopic surgery is associated with reduced surgical trauma, and less acute phase response, as compared with open surgery. Cytokines are important regulators of the biological response to surgical and anesthetic stress. The aim of this study was to determine if CO2 pneumoperitoneum would change cytokine expression, gas parameters and leukocyte count in septic rats. Methods: Wistar rats were randomly assigned to five groups: control (anesthesia only), laparotomy, CO2 pneumoperitoneum, cecum ligation and puncture by laparotomy, and laparoscopic cecum ligation and puncture. After 30 min of the procedures, arterial blood samples were obtained to determine leukocytes subpopulations by hemocytometer. TNFα, IL-1β, IL-6 were determined in intraperitoneal fluid (by ELISA). Gas parameters were measured on arterial blood, intraperitoneal and subperitoneal exsudates. Results: Peritoneal TNFα, IL-1β and IL-6 concentrations were lower in pneumoperitoneum rats than in all other groups (p<0.05). TNFα, IL-1β and IL-6 expression was lower in the laparoscopic than in laparotomic sepsis (p<0.05). Rats from laparoscopic cecum ligation and puncture group developed significant hypercarbic acidosis in blood and subperitoneal fluid when compared to open procedure group. Total white blood cells and lymphocytes were significantly lower in laparoscopic cecum ligation and puncture rats than in the laparotomic (p<0.01). Nevertheless, the laparotomic cecum ligation rats had a significant increase in blood neutrophils and eosinophils when compared with controls (p<0.05). Conclusions: This study demonstrates that the CO2 pneumoperitoneum reduced the inflammatory response in an animal model of peritonitis with respect to intraperitoneal cytokines, white blood cell count and clinical correlates of sepsis. The pneumoperitoneum produced hypercarbic acidosis in septic animals

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area