The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins.

A comunidade cientifica, ao longo dos anos, comprovou aprendizagem vocal em tres ordens de aves (papagaios, beija-flores e passeriformes oscines). Contudo, mais recentemente, estudos feitos em tres especies de Suboscines, Sayornis Phoebe, Procnias tricarunculata e Chiroxiphia linearis revelaram a existencia de um sistema rudimentar do canto e/ou extensivas variacoes vocais quantitativas e qualitativas entre as populacoes reprodutivas, sugerindo que estas especies sao capazes de aprender o seu canto. Esta evidencia incita a investigacao de outras especies de passaros Suboscines, visto que a distincao entre passaros aprendizes e nao-aprendizes vocais nao e tao obvia quanto se pensava. Uma especie de passaro Suboscines amazonica, Myiozetetes cayanensis, desperta o nosso interesse devido a complexidade estrutural do canto em dueto que pode inferir alguma aprendizagem do canto. Com o intuito de identificar as regioes de controle da producao do canto nestes animais nos utilizamos diferentes tecnicas de marcacao celular coloracao de Nissl, hibridizacao in situ radioativa e injecao de tracadores neuronais; ferramentas neuroanatomicas estas que nos forneceram pistas sobre a presenca de areas cerebrais potencialmente envolvidas no sistema de controle do canto. Dos tres nucleos envolvidos na producao do canto, conseguimos identificar apenas o nucleo hipoglosso - porcao traqueosiringeal (NXIIts) e somente atraves da injecao do tracador neuronal CTB. Identificamos o NXIIts tanto em macho quanto em femeas e ao quantificar os dados verificamos que o volume deste nucleo e 2 vezes maior nos machos do que nas femeas, inferindo um possivel dimorfismo sexual. Ao compararmos o volume total de NXIIts entre o nosso animal experimental e uma especie de passaro Oscine, Taeniopigia guttata, verificamos que este nucleo foi quase 2 vezes maior em Taeniopigia guttata machos e femeas. Nao foram identificados nucleos telencefalicos que se assemelhassem ao HVC e ao RA de Oscines por meio de nenhuma das tecnicas utilizadas, o que nos leva a concluir que, em Myiozetetes cayanensis, nao haja sistema de controle do canto e que seu canto seja inato / Along the years vocal learning has been repeatedly demonstrated in three bird orders (parrots, hummingbirds and the oscine songbirds). But recently some studies developed in three spicies of suboscine birds, Sayornis Phoebe, Procnias tricarunculata e Chiroxiphia linearis, demonstrated the existence of a rudmentary song system and/or extensive qualitative and quantitative vocal variations between reproductives populations suggesting that these species are capable of learning their songs. This evidence instigates the investigation of other suboscine songbirds spieces, since the distinction between learning and non-learning vocal birds is not that clear as thought. An amazon uboscine bird, Myiozetetes cayanensis, has attracted our attention due to the structural complexity of its duet song that may imply some vocal learning. Therefore, we tried to identificate the song control regions in this animals by means of different technics: Nissl staining, radioactive in situ hibridization and injections of neural tracers; neuroanatomical tools that provided important clues of cerebral areas implicated in the song system control. We identified only one of the three nuclei involved in the controlling of song production, the NXIIts and only by the injection of the neural tracer CTB. The NXIIts was identified both in males and females. When we quantified the data, we observed that the NXIIts was 2 times larger in males than in females, indicating a possible sexual dimorphism. We also compared the total volume of NXIIts between our experimental animal and a oscine species, Taeniopygia guttata. The volume of NXIIts was almost 2 times larger both in males and females of Taeniopigia guttata. We didint identified any nucleus that guards some resemblence to the oscine HVC or RA through none of the techniques that we tested. Therefore, we can assume that Myiozetetes cayanensis have no song control system and that their song are inate

A comunidade cientifica, ao longo dos anos, comprovou aprendizagem vocal em tres ordens de aves (papagaios, beija-flores e passeriformes oscines). Contudo, mais recentemente, estudos feitos em tres especies de Suboscines, Sayornis Phoebe, Procnias tricarunculata e Chiroxiphia linearis revelaram a existencia de um sistema rudimentar do canto e/ou extensivas variacoes vocais quantitativas e qualitativas entre as populacoes reprodutivas, sugerindo que estas especies sao capazes de aprender o seu canto. Esta evidencia incita a investigacao de outras especies de passaros Suboscines, visto que a distincao entre passaros aprendizes e nao-aprendizes vocais nao e tao obvia quanto se pensava. Uma especie de passaro Suboscines amazonica, Myiozetetes cayanensis, desperta o nosso interesse devido a complexidade estrutural do canto em dueto que pode inferir alguma aprendizagem do canto. Com o intuito de identificar as regioes de controle da producao do canto nestes animais nos utilizamos diferentes tecnicas de marcacao celular coloracao de Nissl, hibridizacao in situ radioativa e injecao de tracadores neuronais; ferramentas neuroanatomicas estas que nos forneceram pistas sobre a presenca de areas cerebrais potencialmente envolvidas no sistema de controle do canto. Dos tres nucleos envolvidos na producao do canto, conseguimos identificar apenas o nucleo hipoglosso - porcao traqueosiringeal (NXIIts) e somente atraves da injecao do tracador neuronal CTB. Identificamos o NXIIts tanto em macho quanto em femeas e ao quantificar os dados verificamos que o volume deste nucleo e 2 vezes maior nos machos do que nas femeas, inferindo um possivel dimorfismo sexual. Ao compararmos o volume total de NXIIts entre o nosso animal experimental e uma especie de passaro Oscine, Taeniopigia guttata, verificamos que este nucleo foi quase 2 vezes maior em Taeniopigia guttata machos e femeas. Nao foram identificados nucleos telencefalicos que se assemelhassem ao HVC e ao RA de Oscines por meio de nenhuma das tecnicas utilizadas, o que nos leva a concluir que, em Myiozetetes cayanensis, nao haja sistema de controle do canto e que seu canto seja inato / Along the years vocal learning has been repeatedly demonstrated in three bird orders (parrots, hummingbirds and the oscine songbirds). But recently some studies developed in three spicies of suboscine birds, Sayornis Phoebe, Procnias tricarunculata e Chiroxiphia linearis, demonstrated the existence of a rudmentary song system and/or extensive qualitative and quantitative vocal variations between reproductives populations suggesting that these species are capable of learning their songs. This evidence instigates the investigation of other suboscine songbirds spieces, since the distinction between learning and non-learning vocal birds is not that clear as thought. An amazon uboscine bird, Myiozetetes cayanensis, has attracted our attention due to the structural complexity of its duet song that may imply some vocal learning. Therefore, we tried to identificate the song control regions in this animals by means of different technics: Nissl staining, radioactive in situ hibridization and injections of neural tracers; neuroanatomical tools that provided important clues of cerebral areas implicated in the song system control. We identified only one of the three nuclei involved in the controlling of song production, the NXIIts and only by the injection of the neural tracer CTB. The NXIIts was identified both in males and females. When we quantified the data, we observed that the NXIIts was 2 times larger in males than in females, indicating a possible sexual dimorphism. We also compared the total volume of NXIIts between our experimental animal and a oscine species, Taeniopygia guttata. The volume of NXIIts was almost 2 times larger both in males and females of Taeniopigia guttata. We didint identified any nucleus that guards some resemblence to the oscine HVC or RA through none of the techniques that we tested. Therefore, we can assume that Myiozetetes cayanensis have no song control system and that their song are inate

Magister Public Health - MPH / Mycobacterium Tuberculosis (MTB) is an endemic disease in Nigeria. The World Health Organization (WHO) estimates the incidence rate for all forms of Tuberculosis at 322 per 100,000 population in Nigeria in 2014 (WHO, 2015). This figure places Nigeria fourth among the 22-high burden countries in the world after India, Indonesia and China. These 22 countries have been prioritized for intensified Tuberculosis (TB) control at the global level, and together they accounted for over 82% of all estimated forms of Tuberculosis the world over in 2014 (WHO, 2014). The United States Agency for International Development (USAID) estimates the Osun state Tuberculosis Case Notification Rate for all forms of TB to be 54 per 100,000 (USAID, 2014). Osun state also has a total of 30 Local Government Areas (LGAs) with 30 TB and Leprosy Supervisors (TBLS) overseeing TB control at local government level. Osun state TB, Leprosy and Buruli Ulcer programme was established in 1993 and currently comprises of 218 health centres implementing the DOTS (Directly Observed Therapy Short-course) strategy and 55 Acid Fast Bacilli (AFB) diagnostic microscopy centres.

Rotavirus is the leading cause of severe infantile diarrhea worldwide. Most related deaths occur in infants from developing countries. Current vaccines are expensive and not readily available throughout the world. Chloroplast transformation technology can be utilized to generate genetically modified plants that produce large quantities of therapeutic proteins and vaccine antigens within their leaves. Plants that are used as bioreactors for vaccine antigens are economically advantageous because they eliminate the need for purification steps and are cheaper to transport. A genetically modified crop could potentially be grown near an endemic area and harvested as needed. There are many influencing factors for transgene expression levels within plant leaves that must be taken into account prior to their harvest. In this work, we seek to determine the optimal expression of CTB-NSP4 in two different cultivars of tobacco plant that have been previously generated by the Daniell lab. The fusion protein, CTBNSP4, is hoped to confer resistance to both rotavirus and cholera. We will determine how the expression of the protein is affected by different variables such as the lighting conditions during harvest and the relative age of leaf at the time of harvest. This knowledge can be used to raise the productivity of the genetically modified plants, further decreasing the cost. Additionally, as unprocessed leaf cannot be used directly for oral delivery due to an unknown concentration of the vaccine antigen, quantification is an important barrier to overcome. Low cost vaccines can be prepared after optimization of dosage and stability. This project seeks to substantiate and quantify genetically modified tobacco plants producing the rotavirus and cholera vaccine antigens.

A colonização da mucosa respiratória é a primeira etapa na patogênese de Streptococcus pneumoniae, bactéria causadora de pneumonia, meningite, e otite média, responsável por mais de um milhão de mortes por ano no mundo. As proteínas de superfície PsaA e PspA têm sido investigadas como candidatos vacinais para estas doenças. CTB é a porção não tóxica da toxina colérica (CT), responsável pela ligação da toxina ao receptor celular GM1 e descrita como adjuvante de mucosas. Neste trabalho, estes genes de S. pneumoniae foram clonados em pAE, um vetor de expressão de E. coli, que utiliza o promotor T7, ou a 3\' de ctxB no plasmídeo pAE-ctxB. As proteínas recombinantes CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 e CTB-PspA3 foram expressas em E. coli BL21 (SI) e purificadas através de coluna carregada com níquel. Ensaios de ligação ao receptor GM1 mostraram que CTB e a porção CTB das proteínas de fusão foram obtidas na forma funcional. Imunização por via intranasal com CTB-PsaA e, por via intranasal e intradérmica com CTB-PspA1 e CTB-PspA3 induziu a produção de IgG no soro. Em compensação, somente a imunização com a fusão CTB-PsaA induziu produção de IgA nas secreções de mucosa. Ensaios de colonização da nasofaringe de camundongos BALB/C e C57BL/6 mostraram que a imunização intranasal com CTB-PsaA resulta em diminuição de colonização por S. pneumoniae. Desafios letais com linhagem virulenta de S. pneumoniae mostraram que a imunização intradérmica com CTB-PspA3, ao contrário da imunização intranasal, é capaz de proteger os animais. Uma vez que estas proteínas de fusão induziram resposta imune protetora, estas deverão ser investigadas como componentes de uma nova vacina para infecções causadas por S. pneumoniae. / The colonization of the respiratory mucosa is the first step in the pathogenesis of Streptococcus pneumoniae, bacterium that causes pneumonia, meningitis and otitis media. It is responsible for more than one million deaths per year worldwide. The surface proteins PsaA and PspA have been investigated as vaccine candidates against these diseases. CTB is the non-toxic portion of cholera toxin (CT), responsible for the toxin binding to the cellular receptor GM1 and described as mucosal adjuvant. In this study, these genes from S. pneumoniae were cloned in pAE, an E. coli expression vector that uses T7 promoter, or downstream to ctxB gene in the pAE-ctxB plasmid. The recombinant proteins CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 and CTB-PspA3 were expressed in E. coli BL21 (SI) and purified through a chelating resin charged with nickel. GM1 binding assays showed that CTB and CTB portion of the fusion proteins were functional. Intranasal immunization with CTB-PsaA and, intranasal and intradermal administration of CTB-PspA1 and CTB-PspA3 induced IgG production in the serum. On the other hand, only CTB-PsaA fusion protein induced IgA in the mucosal secretions. Nasopharyngeal colonization assays in BALB/C and C57BL/6 mice showed that intranasal immunization with CTB-PsaA results in a decrease of colonization by S. pneumoniae. Lethal challenges with S. pneumoniae virulent strains indicated that intradermal immunization with CTB-PspA3, in contrast to the intranasal immunization, is able to protect mice. Since the fusion proteins induced a specific immune response, they should be further investigated as components of a new vaccine against infections caused by S. pneumoniae.

A colonização da mucosa respiratória é a primeira etapa na patogênese de Streptococcus pneumoniae, bactéria causadora de pneumonia, meningite, e otite média, responsável por mais de um milhão de mortes por ano no mundo. As proteínas de superfície PsaA e PspA têm sido investigadas como candidatos vacinais para estas doenças. CTB é a porção não tóxica da toxina colérica (CT), responsável pela ligação da toxina ao receptor celular GM1 e descrita como adjuvante de mucosas. Neste trabalho, estes genes de S. pneumoniae foram clonados em pAE, um vetor de expressão de E. coli, que utiliza o promotor T7, ou a 3\' de ctxB no plasmídeo pAE-ctxB. As proteínas recombinantes CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 e CTB-PspA3 foram expressas em E. coli BL21 (SI) e purificadas através de coluna carregada com níquel. Ensaios de ligação ao receptor GM1 mostraram que CTB e a porção CTB das proteínas de fusão foram obtidas na forma funcional. Imunização por via intranasal com CTB-PsaA e, por via intranasal e intradérmica com CTB-PspA1 e CTB-PspA3 induziu a produção de IgG no soro. Em compensação, somente a imunização com a fusão CTB-PsaA induziu produção de IgA nas secreções de mucosa. Ensaios de colonização da nasofaringe de camundongos BALB/C e C57BL/6 mostraram que a imunização intranasal com CTB-PsaA resulta em diminuição de colonização por S. pneumoniae. Desafios letais com linhagem virulenta de S. pneumoniae mostraram que a imunização intradérmica com CTB-PspA3, ao contrário da imunização intranasal, é capaz de proteger os animais. Uma vez que estas proteínas de fusão induziram resposta imune protetora, estas deverão ser investigadas como componentes de uma nova vacina para infecções causadas por S. pneumoniae. / The colonization of the respiratory mucosa is the first step in the pathogenesis of Streptococcus pneumoniae, bacterium that causes pneumonia, meningitis and otitis media. It is responsible for more than one million deaths per year worldwide. The surface proteins PsaA and PspA have been investigated as vaccine candidates against these diseases. CTB is the non-toxic portion of cholera toxin (CT), responsible for the toxin binding to the cellular receptor GM1 and described as mucosal adjuvant. In this study, these genes from S. pneumoniae were cloned in pAE, an E. coli expression vector that uses T7 promoter, or downstream to ctxB gene in the pAE-ctxB plasmid. The recombinant proteins CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 and CTB-PspA3 were expressed in E. coli BL21 (SI) and purified through a chelating resin charged with nickel. GM1 binding assays showed that CTB and CTB portion of the fusion proteins were functional. Intranasal immunization with CTB-PsaA and, intranasal and intradermal administration of CTB-PspA1 and CTB-PspA3 induced IgG production in the serum. On the other hand, only CTB-PsaA fusion protein induced IgA in the mucosal secretions. Nasopharyngeal colonization assays in BALB/C and C57BL/6 mice showed that intranasal immunization with CTB-PsaA results in a decrease of colonization by S. pneumoniae. Lethal challenges with S. pneumoniae virulent strains indicated that intradermal immunization with CTB-PspA3, in contrast to the intranasal immunization, is able to protect mice. Since the fusion proteins induced a specific immune response, they should be further investigated as components of a new vaccine against infections caused by S. pneumoniae.

Introdução: o desenvolvimento de uma vacina contra a esquistossomose será um importante avanço no controle desta doença crônica e muitas vezes debilitante, afetando milhões de pessoas em todo o mundo. Neste trabalho, descrevemos o uso da subunidade B da toxina colérica (CTB) geneticamente fusionada com Sm14 - uma proteína ligadora de ácidos graxos de Schistosoma mansoni - como uma tentativa de desenvolver uma vacina antiesquistossomose. Métodos: proteínas recombinantes foram expressas em um sistema procariótico, purificadas por diferentes métodos cromatográficos e caracterizadas tanto por métodos imunoquímicos como por métodos espectroscópicos. Experimentos de imunização foram realizados em camundongos fêmeas, da linhagem BALB/c e a eficácia da vacina determinada através da análise da carga parasitária após o desafio com cercárias de S. mansoni e através da análise histopatológica das reações granulomatosas ao redor dos ovos aprisionados no tecido hepático dos camundongos. Resultados: a administração subcutânea de Sm14 reduziu em 27% a carga parasitária nos animais vacinados. Por outro lado, a vacinação intranasal apenas demonstrou uma redução estatisticamente significativa quando CTB esteve presente na formulação. Além disso, a co-administração de CTB e Sm14 reduziu em média 30% a área das lesões granulomatosas hepáticas. Conclusão: o uso de CTB demonstrou ser um importante adjuvante de mucosas; contudo, quando utilizada juntamente com a proteína Sm14, esta molécula não resultou em níveis satisfatórios de redução parasitária em um modelo murino para infecção esquistossomótica. / Introduction: developing a vaccine against schistosomiasis would be an important advance on the control of this chronic and debilitating disease which afflicts millions of people worldwide. Herein we describe the use of the non-toxic B subunit of cholera toxin (CTB) genetically fused to Sm14 - a fatty-acid binding protein from Schistosoma mansoni - as an attempt to the development of an antischistosomiasis vaccine. Methods: recombinant proteins were expressed on a prokaryotic system, purified by different chromatographic methods and both immunochemically and spectroscopically characterized. Immunization experiments were made on females BALB/c mice and vaccines efficacy was assessed by analyzing the worm-burden after challenge infection with S. mansoni cercariae and by analyzing its effect on the hepatic granulomatous reactions around trapped eggs. Results: subcutaneous administration of Sm14 reduced in 27% the worm burden on animals. On the other hand, intranasally vaccination only displayed a statistically significant reduction when CTB was added to the formulation. Furthermore, coadministrating CTB and Sm14 reduces in 30% the area of hepatic granulomas. Conclusion: the use of CTB may be an important tool for mucosal adjuvanticity; however it did not provide, together with Sm14, satisfactory levels of protection on a murine model for Schistosomiasis infection.

O núcleo dorsal da rafe (DR) envia densas projeções para o prosencéfalo e está implicado em várias funções complexas. Muitos estudos indicam que a atividade neuronal do DR é controlada por neurônios do córtex pré-frontal (PFC). Devido à escassez de informações detalhadas sobre as conexões entre o PFC e o DR, objetivamos mapear sistematicamente as projeções do PFC para o DR. O traçador neuronal toxina colérica subunidade b (CTb) foi injetado nas diferentes partes do DR. Com técnica imunoistoquímica contra a CTb, células CTb-ir foram encontradas em maior escala no córtex polar frontal e na parede medial do PFC. No PFC lateral, o córtex da insula agranular dorsal, apresentou maior densidade de neurônios CTb-ir. No PFC orbital, o córtex orbital lateral e dorso-lateral eferentam principalmente a parte central do DR. Injeções situadas na parte caudal do DR resultaram em marcação atenuada. Os resultados indicam que o PFC envia robustas projeções para o DR de forma diferenciada. Através dessas projeções o PFC pode exercer um controle \"top down\" sobre os neurônios do DR. / The dorsal raphe nucleus (DR) sends massive projections to the forebrain and is implicated in a variety of complex functions. Several studies indicate that neuronal activity in the DR is controlled by the prefrontal cortex (PFC). Since there is no detailed information about the projections from the PFC to the DR, the goal of the present study was to map these projections systematically. The neuronal tracer cholera toxin, subunit b (CTb) was injected at different levels of the DR. By immunohistochemical methods, CTb-ir cells were found highly enriched in the frontal polar cortex and in the medial wall of the PFC. In the lateral PFC, the dorsal lateral insular cortex presented the highest density of CTb-ir neurons. In the orbital PFC, the lateral and dorso-lateral orbital cortex project principally to the central segment of the DR. Injections in the caudal segment of the DR resulted in reduced CTb staining. Our results indicate that the PFC sends massive topographically organized projections to the DR, which may exert a \"top down\" control over neurons in the DR.

O núcleo dorsal da rafe (DR) envia densas projeções para o prosencéfalo e está implicado em várias funções complexas. Muitos estudos indicam que a atividade neuronal do DR é controlada por neurônios do córtex pré-frontal (PFC). Devido à escassez de informações detalhadas sobre as conexões entre o PFC e o DR, objetivamos mapear sistematicamente as projeções do PFC para o DR. O traçador neuronal toxina colérica subunidade b (CTb) foi injetado nas diferentes partes do DR. Com técnica imunoistoquímica contra a CTb, células CTb-ir foram encontradas em maior escala no córtex polar frontal e na parede medial do PFC. No PFC lateral, o córtex da insula agranular dorsal, apresentou maior densidade de neurônios CTb-ir. No PFC orbital, o córtex orbital lateral e dorso-lateral eferentam principalmente a parte central do DR. Injeções situadas na parte caudal do DR resultaram em marcação atenuada. Os resultados indicam que o PFC envia robustas projeções para o DR de forma diferenciada. Através dessas projeções o PFC pode exercer um controle \"top down\" sobre os neurônios do DR. / The dorsal raphe nucleus (DR) sends massive projections to the forebrain and is implicated in a variety of complex functions. Several studies indicate that neuronal activity in the DR is controlled by the prefrontal cortex (PFC). Since there is no detailed information about the projections from the PFC to the DR, the goal of the present study was to map these projections systematically. The neuronal tracer cholera toxin, subunit b (CTb) was injected at different levels of the DR. By immunohistochemical methods, CTb-ir cells were found highly enriched in the frontal polar cortex and in the medial wall of the PFC. In the lateral PFC, the dorsal lateral insular cortex presented the highest density of CTb-ir neurons. In the orbital PFC, the lateral and dorso-lateral orbital cortex project principally to the central segment of the DR. Injections in the caudal segment of the DR resulted in reduced CTb staining. Our results indicate that the PFC sends massive topographically organized projections to the DR, which may exert a \"top down\" control over neurons in the DR.