RESUMO
Astroglial interlaminar processes are unique features of the cerebral cortex of adult primates, including man. The functional role of these processes in the primate cerebral cortex is largely unknown. The development and standardization of procedures that could maximize the utilization of primate brain samples is required for the experimental analysis of the individual and collective dynamic properties of interlaminar glial processes. With this aim and in order to assess the relative stability of these glial processes in ex vivo conditions, "tissue printing" procedures were applied. "Tissue printing" allows for the acute transfer of cellular elements from fresh tissue onto an artificial substrate. Human, monkey (Cebus apella), and rat brain samples were subjected to "tissue printing" procedures followed by cell culture and immunohistochemistry. For the purpose of comparing the efficiency of this procedure on the transfer of other long glial processes, "tissue prints" of radial glial processes from neonatal rats and of Bergmann glia from cerebellar samples of adult rats were included. Nitrocellulose (with and without added fibronectin or laminin) produced the best attachment results. Interlaminar processes were not modified following 24-h incubation in a cell culture medium, with the addition of agents known to modify astroglial morphotypes in vitro (cyclic adenosine monophosphate, 40 mM K(+), or fetal calf serum). It is concluded that glia with interlaminar processes can be detached from fresh tissue using "tissue printing" procedures, can be maintained for at least 24 h in standard culture conditions, and showed a stable morphological phenotype.
Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Membranas Artificiais , Animais , Cebus , Adesão Celular , Diferenciação Celular , Criança , Colódio , Técnicas Citológicas , Fibronectinas , Humanos , Laminina , RatosRESUMO
Subcultured astroglial cells from striatum, cerebral cortex and ventral mesencephalon obtained from primary cultures of fetal (E14, E17 and E21) or postnatal (days 5-6) rats showed different regional, age-dependent morphological response (stellation) to cyclic AMP. While most of the cerebral cortex and ventral mesencephalic astroglial cell population was responsive at all ages tested, striatal cells at E14 and E17 were not. At age E21 striatal astroglia showed a significant shift toward a mature-like type of response to cyclic AMP. Postnatal striatal astroglia responded to cyclic AMP as the cortical and ventral mesencephalic astroglia did, with generalized stellation. Prenatal striatal astroglia was characterized immunocytochemically as A2B5+, fibronectin+, vimentin+, S-100+ and GFAP-. Failure of early prenatal (E14, E17) striatal astroglia to differentiate in response to cyclic AMP, was overcome by previous (5-7 days) co-culture with primary cell dissociates from postnatal-, but not from prenatal donors, from all brain regions tested including a non-target region for striatal cells, such as septum. This effect was duplicated when striatal astroglia was co-cultured with cell populations enriched in neurons through Percoll gradients. Only cell-to-cell contact co-cultures were able to induce a change in the studied response. Dead neuron-enriched populations obtained following various types of physical treatments were also able to change significantly striatal cell response toward cyclic AMP. Enriched astroglial populations from postnatal donors did not change striatal astroglial response toward cyclic AMP, except for ventral mesencephalic astroglia which induced a comparatively reduced but significant increase in striatal cell responsiveness. It is concluded that astroglial maturation and potential for phenotype expression during brain development proceeds with regional heterochrony. Also, that maturation of prenatal striatal astroglia responsiveness toward cyclic AMP is inducible by non-diffusible factors, probably of neuronal origin, expressed in live or dead primary cultures from various, homotopic and heterotopic, postnatal brain regions. It is further suggested that striatal afferents and/or mature local striatal neurons express membrane associated molecules that regulate responsiveness for phenotype expression of striatal glial cells, thus reinforcing the concept of a highly interactive, continuous neuron-glial developmental process that takes place during brain organization.
Assuntos
Diferenciação Celular/fisiologia , Corpo Estriado/citologia , Neuroglia/citologia , Neurônios/citologia , Animais , Antígenos de Diferenciação/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Técnicas de Cocultura , Corpo Estriado/embriologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Mesencéfalo/citologia , Mesencéfalo/embriologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Septo do Cérebro/citologia , Septo do Cérebro/embriologiaRESUMO
El conjunto de proyectos desarrollados en PRUNA se basan en la implementación de diferentes enfoques con el objetivo global de estudiar y analizar procesos de organización y reorganización en el sistema nervioso central, así como la participación de componentes no neuronales en los mismos y su posible implementación terapéutica en los procesos de reorganización luego del daño cerebral. Se ilustran tales aspectos comparando las distintas líneas experimentales desarrolladas en PRUNA: la difusión de factores tróficos que actúan modificando el fenotipo de diferentes procesos celulares y la provisión de señales para su organización; la modulación de información neural a través de procesos autocrínicos y paracrínicos; la expresión de moléculas específicas de distintas regiones corticales en función de variaciones amientales; la modulación de la respuesta astroglial ante procesos de lesión y su modulación por parte de factores tróficos; la funcionalidad de la astroglUa como prótesis biológica a transplantar; y el hallaazgo, la expresión, caracterrización citoarquitectónica y funcionamiento postlesional de componentes astrogliales específicos de la corteza cerebral de los primates. En síntesis, la naturaleza hterogénea de los componentes y funciones astrogliales implica tenerlos en consideración obligada para cualquier estudio del SNC que contemple su desarrollo en condiciones normales y patológicas
Assuntos
Animais , Humanos , Sistema Nervoso Central/fisiopatologia , Fenômenos PsicológicosRESUMO
El conjunto de proyectos desarrollados en PRUNA se basan en la implementación de diferentes enfoques con el objetivo global de estudiar y analizar procesos de organización y reorganización en el sistema nervioso central, así como la participación de componentes no neuronales en los mismos y su posible implementación terapéutica en los procesos de reorganización luego del daño cerebral. Se ilustran tales aspectos comparando las distintas líneas experimentales desarrolladas en PRUNA: la difusión de factores tróficos que actúan modificando el fenotipo de diferentes procesos celulares y la provisión de señales para su organización; la modulación de información neural a través de procesos autocrínicos y paracrínicos; la expresión de moléculas específicas de distintas regiones corticales en función de variaciones amientales; la modulación de la respuesta astroglial ante procesos de lesión y su modulación por parte de factores tróficos; la funcionalidad de la astroglUa como prótesis biológica a transplantar; y el hallaazgo, la expresión, caracterrización citoarquitectónica y funcionamiento postlesional de componentes astrogliales específicos de la corteza cerebral de los primates. En síntesis, la naturaleza hterogénea de los componentes y funciones astrogliales implica tenerlos en consideración obligada para cualquier estudio del SNC que contemple su desarrollo en condiciones normales y patológicas
Assuntos
Animais , Humanos , Sistema Nervoso Central/fisiopatologia , Fenômenos PsicológicosRESUMO
Cerebrospinal fluid from L-dopa-treated Parkinson's disease patients and subjects without neurodegenerative diseases (controls) was explored in its trophic properties as culture medium on a variety of cells from neural origin. Primary cultures of regional brain dissociates from rat and Cebus apella monkey fetuses, immature rat adrenal chromaffin cells, phaeochromocytoma (PC12), and neuroblastoma (NB69) cell lines as well as subcultured fetal rat astroglia were used as target cells for 24- to 48-h culture periods. Most cerebrospinal fluid samples from L-dopa-treated patients had a general dystrophic effect. This phenomenon was more apparent on striatum and ventral mesencephalon than on cerebral cortex cell dissociates. The deleterious effect of these samples was abolished by previous exposure to fetal astroglial cells. Neuroblastoma cells showed no differential response when exposed to samples from control and L-dopa-treated patients. Phaeochromocytoma cells did not grow processes under any of the samples assayed in the time interval explored, but neither showed evidence of dystrophy. The relevance of these findings to the transplantation of different cell types as one of the possible therapies for Parkinson's disease is discussed. The suggestion is made that CSF testing prior to transplantation may aid in anticipating its possible outcome. Cotransplantation of neuronal cells with subcultured astroglia may foster survival and growth of the former cells.
Assuntos
Antiparkinsonianos/administração & dosagem , Astrócitos/efeitos dos fármacos , Proteínas do Líquido Cefalorraquidiano/farmacologia , Levodopa/administração & dosagem , Neurônios/efeitos dos fármacos , Doença de Parkinson/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Cebus , Córtex Cerebral/citologia , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Humanos , Mesencéfalo/citologia , Pessoa de Meia-Idade , Neostriado/citologia , Neurônios/citologia , Células PC12 , Doença de Parkinson/tratamento farmacológico , Ratos , Ratos Sprague-DawleyRESUMO
We studied the influence of maternal deprivation on the RNA biosynthesis in the brain cortex of 10 day-old rats. Mother-deprived pups, placed at 25 degrees C showed a reduction in body temperature of 6 +/- 1 degree C. After mother retrieval, RNA biosynthesis decreased 27% and 34% in total brain cortex and in isolated neurons, respectively. This fall is proportional to the body temperature reduction and can be avoided placing the pups at 37 degrees C immediately after the separation. Rethermostatization of offsprings, after one hour at 25 degrees C, showed an overshoot of RNA biosynthesis (145%) with further stabilization of synthesis rates to normal levels after 100 min. This classical physiological mechanism was further studied in vitro. Comparing in vivo and in vitro experiments, it is concluded that overshooting can not be observed in vitro if temperature reduction was not previously performed in vivo. Thus, this phenomenon seems to respond to humoral factors in order to be triggered. Afterwards, in vitro overshooting following cold stress in vivo, demonstrates that the depressed tissue by itself has the capability to turn back to normal RNA levels in the same way as observed in vivo.
Assuntos
Animais Recém-Nascidos/metabolismo , Córtex Cerebral/metabolismo , Temperatura Baixa/efeitos adversos , Privação Materna , RNA/biossíntese , Estresse Fisiológico/metabolismo , Animais , Animais Recém-Nascidos/genética , Regulação da Temperatura Corporal , Cinética , Ratos , Ratos WistarRESUMO
Ex vivo induction of radial-like glia has been previously reported to occur following exposure of cerebral cortex subcultures from fetal origin to cerebral cortex astroglial-conditioned medium. The present report further confirms similarities between in vivo and ex vivo radial glia, using additional criteria: adhesion of primary cell dissociates to glial processes, with presumptive cell migration along them, punctuate labelling for laminin, and immunolabelling with Rat-401 antisera.
Assuntos
Astrócitos/química , Astrócitos/citologia , Animais , Especificidade de Anticorpos , Astrócitos/metabolismo , Biomarcadores , Adesão Celular/fisiologia , Células Cultivadas/química , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/imunologia , Imuno-Histoquímica , Laminina/análise , Laminina/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Vimentina/análise , Vimentina/imunologiaRESUMO
A population of subcultured astroglia from rat fetal cortex was transformed into radial-like cells after exposure to cerebral cortex astroglial conditioned medium in vitro. Such changes were also induced by basal medium modified by fetal leptomeningeal subcultures, but not by postnatal leptomeninges nor by fetal skin fibroblasts. The radializing effects of astroglial conditioned medium were inhibited by previous heat treatment. The addition of protease inhibitors to the basal medium did not cause spontaneous radialization of subcultured cortical astroglia, but increased the length of cell processes and incidence of radial-like forms when added to cortical astroglial conditioned medium. It is concluded that cortical astroglia and leptomeningeal cells share the capability of synthesizing and releasing diffusible molecules into the culture medium which act as morphogenetic inducers in vitro. Based on the present results, it is suggested that such effects would depend on the presence of instructive factor(s) in the conditioned medium which are able to induce rearrangement of the cytoskeleton, rather than on secreted molecules able to modify cell adhesion to the substrate.
Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Meninges/citologia , Inibidores de Proteases/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Feminino , Feto/citologia , Feto/efeitos dos fármacos , Microscopia de Contraste de Fase , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Primary cell cultures from cerebral cortex, striatum and ventral mesencephalon obtained from rat fetal (embryonic day 17, E17) or postnatal (day 2, PN2) donors were grown either in media conditioned by subcultured astroglia from the same regions, an artificial trophic medium, normal human amniotic fluid, or in normal human cerebrospinal fluid. To estimate the presence of neuronal-like and non-neuronal cells, cell morphology and immunocytochemistry against microtubule-associated proteins and beta-tubulin were taken into consideration. The percentage of emitting neural cells and length of cell processes were determined after 24 hr in culture. Growth of cell processes in neuronal and non-neuronal cells from prenatal striatum was minimal compared with that in cerebral cortex and ventral mesencephalon, regardless of the culture condition. Nerve growth factor, basic fibroblast growth factor or epidermal growth factor did not significantly modify cell growth in E17 cultures, except for epidermal growth factor, which reduced the number of emitting cells in striatal cultures and increased it in cerebral cortex ones. Cultures derived from postnatal striatum showed a significant increase in neurite length when grown in an astroglial conditioned medium as compared to cultures derived from prenatal (E17) striatum. Results suggest significant regional differences in the brain regarding growth of cell processes at age E17, and reversal of striatal ability to grow cell processes by postnatal day 2. Reduced growth of cell processes showed by E17 striatum cultures was rather independent of the culture media. This fact could suggest that such early regional differences would depend on characteristics of sublineages present at this developmental stage, which would modulate the organization of regional neuropils. The restricted growth of cell processes in cultures from E17 striatum, no longer present in postnatal striatum, suggests that inputs to the striatum may modify expression of cell lineages at later stages of development.
Assuntos
Neuritos/fisiologia , Neurônios/citologia , Líquido Amniótico/química , Animais , Astrócitos/metabolismo , Contagem de Células , Divisão Celular/fisiologia , Tamanho Celular , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Proteínas do Líquido Cefalorraquidiano/farmacologia , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Mesencéfalo/citologia , Mesencéfalo/embriologia , Neostriado/citologia , Neostriado/embriologia , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , RatosRESUMO
Cerebral cortex and striatal cell dissociates obtained from rat fetuses (E 17) were subcultured and enriched in astroglial cells before being grown in regional (cerebral cortex, striatum) astroglial conditioned media (CM) or defined basal medium. Incidence of radial-like astroglia (vimentin+ or glial fibrillary acid protein, GFAP+) and length of processes in cortical cell subcultures showed a greater increase when exposed to cerebral cortex CM than to striatal CM or basal medium. Stellate (GFAP+) forms prevailed in subcultures grown in basal medium while striatal cells exposed to CM of either origin remained undifferentiated. Additionally, cultures were treated with various concentrations of cAMP (0.25 and 0.5 mM) and calcitonin gene related peptide (CGRP) (0.1, 0.5, and 1.0 microM). Under these conditions CM-exposed cultures (with predominant "radial-like" forms) did not increase stellate glial numbers, while fetal calf serum (FCS)-exposed cultures (morphologically undifferentiated) underwent significant degrees of stellate transformation. When CM-exposed cultures were shifted to FCS supplemented basal medium for 24-48 hr and then to basal medium alone prior to treatment, cAMP and CGRP were effective in transforming flat astroglia into stellate morphology. Results are indicative of the existence of astroglial diffusible factors affecting the in vitro expression of astroglial morphotypes from the cerebral cortex. Previous exposure to CM interferes with cytoskeletal astrocytic changes induced by cAMP and CGRP. It is speculated that astroglial factors could act in vivo to maintain the expression of radial-like cells during early developmental stages of the cerebral cortex, but it would not be effective in E 17 striatum.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Astrócitos/citologia , Diferenciação Celular/fisiologia , Animais , Astrócitos/ultraestrutura , Calcitonina/farmacologia , Divisão Celular/fisiologia , Córtex Cerebral/citologia , AMP Cíclico/farmacologia , Citoesqueleto , RatosRESUMO
Leptomeningeal and skin fetal (E16-17) fibroblasts were subcultured in vitro either in DMEM/F12 basal medium (with or without 10% FCS) or in astroglial conditioned medium (ACM). Both populations were characteristically composed of flat, undifferentiated, fibronectin(+), GFAP(-)cells where cultured in fetal serum supplemented basal media. When exposed to ACM leptomeningeal cells developed a population of thin, elongated, fibronectin(+) cells with radial type long processes while skin fibroblasts did not show significant changes in their characteristic morphotype. Exposure to db cAMP in basal medium resulted within 3 hr in their transformation to an astrocytic-like morphotype characterized by a condensed soma and multiple, short processes. Twenty-four hours later skin fibroblasts had returned to their flat appearance while leptomeningeal ones showed elongated, radial-like forms. Results indicate the possible existence of different receptors (to ACM factors) and/or cytoskeletal properties, and suggest that ACM-reactive fibroblasts of leptomeningeal origin represent a different cell type from those of skin origin. The hypothetical role of leptomeningeal cells during brain development is considered.
Assuntos
Aracnoide-Máter/citologia , Pia-Máter/citologia , Pele/citologia , Animais , Astrócitos/metabolismo , Bucladesina/farmacologia , Bovinos/sangue , Meios de Cultura , Meios de Cultivo Condicionados , Sangue Fetal , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-HistoquímicaRESUMO
Exposure of "in vitro" grown immature and adult adrenal chromaffin cells to concentrations of 10(-3) or 10(-5) M but not 10(-7) M GM1 ganglioside, resulted in significant increase in cell diameter, coupled with reduction of adhesion to substrate within 48 hrs of exposure. None of the GM1 concentrations, with or without serum supplementation, did significantly increase neuritogenesis in chromaffin cells. Immature chromaffin cells underwent neuritogenesis when grown in co-cultures with actively growing astroglia from striatum or cerebral cortex, an effect that was potentiated by NGF administration and blocked by anti-NGF. In neither of the former conditions did 10(-6) M GM1 prove to increase the number of neurite emitting cells nor their mean neuritic length further. It is speculated that GM1 does not perform the neuritogenic role described for central neurons in chromaffin cells, nor does it potentiate NGF effect on neuritogenesis observed in other peripheral neurons.
Assuntos
Grânulos Cromafim/efeitos dos fármacos , Gangliosídeo G(M1)/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Masculino , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Adrenal chromaffin cells from immature or adult rats were grown in one of the following 'in vitro' conditions: (1) on coverslips placed on top of confluent, fetal, regional glia cultures; (2) in conditioned media from similar confluent cultures; (3) after direct seeding on top of such confluent cultures. Astroglia was obtained from cerebral cortex, septum, striatum and ventral mesen-cephalon from El6-17 pregnant-dated rats. All regions succesfully generated conditions for the early (less than 24 h) expression of neuritogenesis in about 15% of cells, which was more apparent in immature adrenal cell dissociates than in adult ones. The former grew long neurites compared with their adult counterparts. In addition to the known effects of glioma conditioned medium and isolated trophic factors described by other authors, it is concluded that adrenal chromaffin cells are responsive to the neuritogenic activity of (central) astroglial diffusible factor(s) in non-supplemented, defined culture media conditioned by astrocytes from various brain regions. Additionally, evidence is offered that adult chromaffin cells show a reduced responsiveness towards such astroglial factor(s). Possible implications for cell trasplantation chimeras are discussed.
RESUMO
Normal human (week 17-20) and rat (E16-17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures. Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype. Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.
Assuntos
Líquido Amniótico/fisiologia , Encéfalo/embriologia , Líquido Amniótico/química , Animais , Encéfalo/citologia , Células Cultivadas , Meios de Cultura , Fator de Crescimento Epidérmico/imunologia , Feminino , Humanos , Fatores de Crescimento Neural/imunologia , Neuroglia/fisiologia , Neurônios/fisiologia , Gravidez , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Esteroides/análise , Esteroides/metabolismoRESUMO
Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.
Assuntos
Acetilcolinesterase/análise , Colinesterases/análise , Fígado/enzimologia , Acetilcolinesterase/isolamento & purificação , Animais , Membrana Celular/enzimologia , Centrifugação com Gradiente de Concentração , Colinesterases/isolamento & purificação , Membranas Intracelulares/enzimologia , Ratos , Ratos Endogâmicos , Solubilidade , Frações Subcelulares/enzimologiaRESUMO
The allozymic variation at at the esterase-6 locus examined in fourteen samples of natural populations of Drosophila simulans collected in two different localities. The samples were collected from different species of fruits in the two localities and from banana baits located some meters apart from the fruits. The analysis was done by sequential electrophoresis, using varied gel concentrations and buffers. By this technique, we detected 27 alleles at the esterase-6 locus, where only five alleles were detected by the usual method. The results obtained suggest that habitat choice alone may not be a selective factor, but may be selective in combination with other environmental factors.