As alterações nutricionais no período neonatal são capazes de comprometer o controle hipotalâmico da ingestão alimentar e o metabolismo do indivíduo em fases posteriores do desenvolvimento. Avaliamos as alterações decorrentes do modelo de programação nutricional neonatal em células gliais hipotalâmicas, devido ao seu importante papel na homeostase energética. Os astrócitos possuem função metabólica ativa, e por sua vez, fornecem substrato energético aos neurônios por meio das conexinas 30 (CX30) e 43 (CX43). A CX30, por sua vez, exerce função, também, na manutenção morfológica astrocitária, contribuindo na inserção astrocitária na fenda sináptica, portanto interferindo na neurotransmissão. A TCPTP (T-cell protein tyrosine phosphatase) proteína contra-reguladora da sinalização celular da leptina e a insulina participam nos mecanismos de resistência a esses hormônios e está presente em células da glia e possui ação moduladora na atividade de CX43. Sendo assim, a hipótese do presente trabalho é de que alterações em células da glia no hipotálamo participam nos efeitos da programação nutricional neonatal na modulação do balanço energético na vida juvenil e adulta. Para investigarmos essa hipótese, utilizamos o modelo de programação nutricional neonatal de alteração do tamanho da ninhada, sendo a quantidade de filhotes por lactante formada da seguinte maneira: 3 filhotes, ninhada pequena (SL), 10 filhotes, ninhada normal (NL) e de 16 filhotes, ninhada grande (LL). O peso corporal da ninhada foi verificado semanalmente até o desmame, realizado no 21º dia de vida (PN21). Após o desmame, o peso corporal foi verificado a cada cinco dias até o 60º dia de vida (PN60). A ingestão alimentar individual foi determinada entre o PN50 e PN60. Os animais SL apresentaram maior peso corporal (72,3 ± 2,08g) ao desmame, quando comparados aos grupos NL (57,2 ± 3,5g) e LL (36,3 ± 1,8g) e essa diferença entre os grupos foi mantida até o PN60. Observou-se, porém, que a ingestão alimentar dos animais adultos SL, não foi diferente do grupo NL. Todavia, os animais LL apresentaram um ganho de peso reduzido ao desmame, porém, esses animais alcançaram o ganho de peso corporal dos animais NL (NL: 165 ± 3,97g; LL: 145,4 ± 4,5g), a partir do PN35, fenômeno esse associado ao comportamento hiperfágico. No PN21, observou-se no grupo SL um aumento nas concentrações plasmáticas de leptina (6,4 ? 0,9ng/ml) e insulina (1,9 ? 0,15 ng/ml), quando comparado aos grupos NL (leptina: 3,8 ? 0,3ng/ml; insulina: 1,3 ? 0,2 ng/ml) e LL (leptina: 1,2 ? 0,1ng/ml; 9 insulina: 1,0 ? 0,1ng/ml). No PN60, ambos os grupos SL (leptina: 5,2 ? 1,15ng/ml; insulina: 2,5 ? 0,4ng/ml) e LL (leptina: 4,3 ? 0,5ng/ml; insulina: 3,4 ? 0,5ng/ml) apresentaram aumento nas concentrações plasmáticas de leptina e insulina, comparados ao grupo NL (leptina: 1,8 ? 0,4ng/ml; insulina: 1,2 ? 0,1ng/ml). Quando avaliada a expressão do RNAm de Ptpn2, gene que codifica TCPTP, e a expressão dessa proteína no núcleo arqueado (ARC), observamos um aumento no PN21 no grupo SL e em ambos os grupos no PN60, quando comparados ao grupo NL. O grupo SL apresentou aumento na imunorreatividade para GFAP no PN21 e ambos os grupos apresentaram essa mesma resposta no PN60. O mesmo resultado foi observado na imunorreatividade para a molécula adaptadora ligante de cálcio inonizado-1 (IBA-1) no PN21 e PN60 nos grupos SL e LL. Houve colocalização da TCPTP com GFAP, porém não com IBA-1. A TCPTP possui ação demonstrada na modulação de CX43, ao investigá-la observou-se no PN21, um aumento na expressão do RNAm de Gja1, gene que codifica CX43, assim como na imunorreatividade para CX43 apenas no grupo SL. No PN21 e PN60 observou-se redução da expressão do RNAm de Gja6, gene que codifica CX30, em ambos os grupos SL e LL. Observou-se redução na imunorreatividade de CX30 em ambos os grupos, SL e LL no PN60. No PN21, a expressão do RNAm de Il1b aumentou no ARC em ambos os grupos SL e LL. No entanto, no PN60, apenas o grupo LL apresentou um aumento da expressão do RNAm de Il1b. Adicionalmente, no PN60 ambos os grupos SL e LL apresentaram um aumento na expressão do RNAm de Tnfa no ARC. Na análise morfológica das células da glia, no PN21, observou-se no grupo SL um aumento na imunorreatividade do soma da microglia e do astrócito, assim como, nos processos de extensão de ambas as células. No PN60 ambos os grupos apresentaram um aumento na imunorreatividade do soma e dos processos de extensão astrocitários, no entanto, apenas o grupo SL apresentou um aumento na imunorreatividade do soma microglial. Para analisarmos o efeito da leptina na morfologia dos astrócitos e a participação da TCPTP nesse processo, realizamos a cultura primária de astrócitos hipotalâmicos de ratos neonatos que foram estimulados com leptina [1000ng/ml], [5000ng/ml] e LPS [500ng/ml]. O LPS foi utilizado como controle positivo do protocolo. Observamos que os estímulos com leptina e LPS, aumentaram a expressão do RNAm de Ptpn2, a imunorreatividade para TCPTP e a área astrocitária. O tratamento com LPS foi capaz de promover um aumento na expressão do RNAm de Gja1 e o inverso foi observado na expressão de Gja6. Todavia, tanto o tratamento com leptina e LPS promoveu aumento na imunorreatividade para CX43 e o inverso observou-se na imunorreatividade para CX30. Para avaliarmos a participação da TCPTP nos efeitos da leptina na morfologia dos astrócitos, realizamos o silenciamento de seu gene, utilizando o siRNA Ptpn2. O silenciamento de Ptpn2 foi capaz de reverter os efeitos da leptina tanto na expressão gênica, na imunorreatividade assim como na morfologia astrocitária. O silenciamento de Ptpn2 reverteu também as respostas de redução de CX30 e o aumento de CX43 promovidas pelo LPS pela leptina. De maneira inédita esses dados sugerem a importância da TCPTP na modulação das conexinas nos efeitos da leptina e LPS na morfologia astrocitária hipotalâmica. Observamos que apenas o tratamento com LPS foi capaz de promover um aumento na expressão do RNAm de Ptpn1, e o silenciamento de Ptpn2 intensificou esse aumento da expressão de Ptpn1, demonstrando de forma inédita 10 que a TCPTP exerce ação contra regulatória sobre a PTP1B. Como esperado o estímulo dos astrócitos com LPS aumentou a expressão do RNAm de Il6, Il1b e Tnfa. Interessantemente, o silenciamento de Ptpn2 intensificou esse aumento da expressão do RNAm de Il6, Il1b e Tnfa, demonstrando desse modo que a TCPTP possui ação contra regulatória na secreção dessas citocinas. O conjunto de dados demonstra que a alteração nutricional neonatal é capaz de promover alterações no balanço energético na vida juvenil e adulta. Estas alterações estão associadas a modificações morfológicas das células da glia e ao aumento de citocinas inflamatórias, caracterizando um estado reativo glial. Adicionalmente, demonstramos em cultura primária de astrócitos hipotalâmicos que a leptina altera a morfologia destas células e pela primeira vez demonstramos, também, que a TCPTP modula esses efeitos da leptina, por meio de suas ações na conexina CX30. A CX30 participa na modulação da morfologia dos astrócitos e sua redução está associada ao aumento na área e nos processos de extensão destas células. Em conclusão, o presente estudo demonstra que alterações na disponibilidade nutricional na vida neonatal acarretam alterações no comportamento alimentar e no peso corporal na vida juvenil e adulta em ratos. Demonstramos, também, que tais alterações nutricionais neonatais estão associadas a alterações em células da glia. A leptina induz alterações morfológicas em astrócitos, sendo este efeito mediado pela TCPTP e sua regulação sobre a expressão da proteína CX30. O conjunto dos dados indica a importância das células não neuronais no controle central da homeostase energética em modelo de programação nutricional neonatal. / Nutritional changes in the neonatal period can affect the hypothalamic control of food intake and metabolism in later life. We evaluated the influence of the neonatal nutritional programming on hypothalamic glial cells, known to play an important role in the energy homeostasis. Astrocytes have active metabolic function and provide energy substrate for the neurons through connexin 43 (CX43). CX30 is important in the maintenance of astrocyte morphology, contributing to the insertion of its process into the synaptic cleft. The TCPTP (T-cell protein tyrosine phosphatase) is a counterregulator of cellular signaling of leptin and insulin, contributing to the molecular mechanisms of resistance to these hormones and it is expressed in glial and modulates CX43 activity. We hypothesized that alterations in the hypothalamic glial cells participate in the long-lasting effects on energy balance induced by neonatal nutritional programming. For this purpose, we used the model of neonatal nutritional programming induced by changing the litter size, according to the number of offspring per dam: 3 offsprings, small litter (SL), 10 offsprings, normal litter (NL) and 16 offsprings, large litter (LL). The body weight of the litter was determined weekly until weaning on the 21st day of life (PN21). After weaning, body weight was determined every five days until the 60th day of life (PN60). Individual dietary intake was determined between PN50 and PN60. The SL animals presented higher body weight (72.3 ± 2.08g) at weaning, when compared with the NL (57.19 ± 3.49g) and LL (36.27 ± 1.79g) groups and the difference between these groups were maintained until the PN60. However, the food intake of adult SL animals was not different from the NL group. On the other hand, LL animals presented a reduced weight gain at weaning but they had a catch up of reaching the vody weight of NL animals (NL: 165 ± 3.97g; LL: 145.42 ± 4.55g) from PN35 on, and this response was associated with higher food inatke. At PN21, there was an increase in plasma leptin (6.41 ± 0.90 ng/ml) and insulin (1.97 ± 0.11ng/ml) concentrations in the SL group, when compared with the NL group (leptin: 3.79 ± 0,35ng/ml; insulin: 1.32 ± 0.21ng/ml) and LL (leptin: 1.23 ± 0.10ng/ml; insulin: 0.99 ± 0.10 ng/ml). At PN60, both SL (leptin: 5,26 ± 1.15ng/ml, insulin: 2,53 ± 0,36ng/ml) and LL (leptin: 4.30 ± 0.51ng/ml, insulin: 3.39 ± 0.47ng/ml) groups presented increased plasma leptin and insulin concentrations compared with the group NL (leptin: 1.79 ± 0.41ng/ml; insulin: 1.19 ± 0.09ng/ml). The mRNA expression of Ptpn2 mRNA, gene encoding TCPTP, and its protein in the arcuate nucleus (ARC) was increase at PN21 in the SL group and in both groups at PN60, compared with the NL group. The SL group showed an increased immunoreactivity for GFAP at PN21 and 12 both groups showed this increased response at PN60. Similar response was observed for ionized calcium binding adaptor molecule 1 (IBA-1) immunoreactivity at PN21 and PN60. There was an overlap of TCPTP with GFAP immunoreactivity, but not with IBA-1. At PN21 there was an increase in the mRNA expression of Gja1, gene coding for CX43, as well as in the immunoreactivity of CX43 in the SL group only. At PN21 and PN60, mRNA expression of the Gja6, gene encoding for CX30, was reduced in both SL and LL groups. However, at PN60 it was reduction of CX30 immunoreactivity in both groups, SL and LL. At PN21, Il1b mRNA expression was increased in the ARC in both SL and LL groups. However, at PN60, only the LL group showed an increased Il1b mRNA expression. Additionally, at PN60 both SL and LL groups showed an increase in the Tnfa mRNA expression in the ARC. In the morphological analysis of glia cells, at PN21, there was an increase in the immunoreactivity of the microglia and astrocyte in the SL group, as well as in the extension processes of both cells. At PN60, both groups showed an increase in the soma immunoreactivity and astrocytic processe extension, however, only the SL group showed an increase in the immunoreactivity of the microglial soma. To analyze the effect of leptin on astrocyte morphology and the participation of TCPTP in this process, we performed the primary culture of hypothalamic astrocytes from neonatal rats that were stimulated with leptin [1000ng/ml], [5000ng/ml] and LPS [500ng /ml]. The LPS was used as a positive control of the protocol. We observed that the leptin and LPS stimuli increased the Ptpn2 mRNA expression, the TCPTP immunoreactivity and the astrocyte area. The LPS treatment increased the Gja1 mRNA expression and the opposite was observed in the Gja6 expression. On the other hand, both treatment with leptin and LPS increased the immunoreactivity for CX43 and the opposite was observed for the CX30 immunoreactivity. In order to evaluate the participation of TCPTP in the effects of leptin on the astrocyte morphology, we performed the silencing of its gene using the siRNA Ptpn2. The silencing of Ptpn2 was able to reverse the effects of leptin and LPS on gene expression, immunoreactivity as well as astrocyte morphology. The silencing of Ptpn2 was able to revert the reduction of CX30 and the increase of CX43 immunoreactivity and the its gene expression promoted by LPS leptin. These data are the first to show the importance of TCPTP in the modulation of connexins on the leptin and LPS effects on the morphology of hypothalamic astrocytes. Additionally, only LPS treatment was able to promote an increase in the Ptpn1 mRNA expression and Ptpn2 silencing enhanced this increase in Ptpn1 mRNA expression.These data demonstrate an unprecedented way that Ptpn2 exerts regulatory action against Ptpn1. As expected, the stimulation with LPS increased the mRNA expression of the Il6, Il1b and Tnfa. The silencing of Ptpn2 amplified this effect of LPS on cytokine gene expression, demonstrating that TCPTP has a counterregulatory action on the secretion of IL6, IL1? and Tnf?. Taken together these data demonstrate that the neonatal nutritional changes are able to promote alterations in the energy balance in the juvenile and adult life. These effects are associated with morphological changes in glial cells and increase of inflammatory cytokines, characterizing a glial reactive state. Additionally, using primary cell culture, we demonstrated that leptin alters the morphology of hypothalamic astrocytes. We also demonstrate for the first time that TCPTP modulates these effects of 13 leptin, through its actions regulating the expression of CX30. The data shown indicate the importance of non-neuronal cells in the central control of energy homeostasis in a model of neonatal nutritional programming.

As alterações nutricionais no período neonatal são capazes de comprometer o controle hipotalâmico da ingestão alimentar e o metabolismo do indivíduo em fases posteriores do desenvolvimento. Avaliamos as alterações decorrentes do modelo de programação nutricional neonatal em células gliais hipotalâmicas, devido ao seu importante papel na homeostase energética. Os astrócitos possuem função metabólica ativa, e por sua vez, fornecem substrato energético aos neurônios por meio das conexinas 30 (CX30) e 43 (CX43). A CX30, por sua vez, exerce função, também, na manutenção morfológica astrocitária, contribuindo na inserção astrocitária na fenda sináptica, portanto interferindo na neurotransmissão. A TCPTP (T-cell protein tyrosine phosphatase) proteína contra-reguladora da sinalização celular da leptina e a insulina participam nos mecanismos de resistência a esses hormônios e está presente em células da glia e possui ação moduladora na atividade de CX43. Sendo assim, a hipótese do presente trabalho é de que alterações em células da glia no hipotálamo participam nos efeitos da programação nutricional neonatal na modulação do balanço energético na vida juvenil e adulta. Para investigarmos essa hipótese, utilizamos o modelo de programação nutricional neonatal de alteração do tamanho da ninhada, sendo a quantidade de filhotes por lactante formada da seguinte maneira: 3 filhotes, ninhada pequena (SL), 10 filhotes, ninhada normal (NL) e de 16 filhotes, ninhada grande (LL). O peso corporal da ninhada foi verificado semanalmente até o desmame, realizado no 21º dia de vida (PN21). Após o desmame, o peso corporal foi verificado a cada cinco dias até o 60º dia de vida (PN60). A ingestão alimentar individual foi determinada entre o PN50 e PN60. Os animais SL apresentaram maior peso corporal (72,3 ± 2,08g) ao desmame, quando comparados aos grupos NL (57,2 ± 3,5g) e LL (36,3 ± 1,8g) e essa diferença entre os grupos foi mantida até o PN60. Observou-se, porém, que a ingestão alimentar dos animais adultos SL, não foi diferente do grupo NL. Todavia, os animais LL apresentaram um ganho de peso reduzido ao desmame, porém, esses animais alcançaram o ganho de peso corporal dos animais NL (NL: 165 ± 3,97g; LL: 145,4 ± 4,5g), a partir do PN35, fenômeno esse associado ao comportamento hiperfágico. No PN21, observou-se no grupo SL um aumento nas concentrações plasmáticas de leptina (6,4 ? 0,9ng/ml) e insulina (1,9 ? 0,15 ng/ml), quando comparado aos grupos NL (leptina: 3,8 ? 0,3ng/ml; insulina: 1,3 ? 0,2 ng/ml) e LL (leptina: 1,2 ? 0,1ng/ml; 9 insulina: 1,0 ? 0,1ng/ml). No PN60, ambos os grupos SL (leptina: 5,2 ? 1,15ng/ml; insulina: 2,5 ? 0,4ng/ml) e LL (leptina: 4,3 ? 0,5ng/ml; insulina: 3,4 ? 0,5ng/ml) apresentaram aumento nas concentrações plasmáticas de leptina e insulina, comparados ao grupo NL (leptina: 1,8 ? 0,4ng/ml; insulina: 1,2 ? 0,1ng/ml). Quando avaliada a expressão do RNAm de Ptpn2, gene que codifica TCPTP, e a expressão dessa proteína no núcleo arqueado (ARC), observamos um aumento no PN21 no grupo SL e em ambos os grupos no PN60, quando comparados ao grupo NL. O grupo SL apresentou aumento na imunorreatividade para GFAP no PN21 e ambos os grupos apresentaram essa mesma resposta no PN60. O mesmo resultado foi observado na imunorreatividade para a molécula adaptadora ligante de cálcio inonizado-1 (IBA-1) no PN21 e PN60 nos grupos SL e LL. Houve colocalização da TCPTP com GFAP, porém não com IBA-1. A TCPTP possui ação demonstrada na modulação de CX43, ao investigá-la observou-se no PN21, um aumento na expressão do RNAm de Gja1, gene que codifica CX43, assim como na imunorreatividade para CX43 apenas no grupo SL. No PN21 e PN60 observou-se redução da expressão do RNAm de Gja6, gene que codifica CX30, em ambos os grupos SL e LL. Observou-se redução na imunorreatividade de CX30 em ambos os grupos, SL e LL no PN60. No PN21, a expressão do RNAm de Il1b aumentou no ARC em ambos os grupos SL e LL. No entanto, no PN60, apenas o grupo LL apresentou um aumento da expressão do RNAm de Il1b. Adicionalmente, no PN60 ambos os grupos SL e LL apresentaram um aumento na expressão do RNAm de Tnfa no ARC. Na análise morfológica das células da glia, no PN21, observou-se no grupo SL um aumento na imunorreatividade do soma da microglia e do astrócito, assim como, nos processos de extensão de ambas as células. No PN60 ambos os grupos apresentaram um aumento na imunorreatividade do soma e dos processos de extensão astrocitários, no entanto, apenas o grupo SL apresentou um aumento na imunorreatividade do soma microglial. Para analisarmos o efeito da leptina na morfologia dos astrócitos e a participação da TCPTP nesse processo, realizamos a cultura primária de astrócitos hipotalâmicos de ratos neonatos que foram estimulados com leptina [1000ng/ml], [5000ng/ml] e LPS [500ng/ml]. O LPS foi utilizado como controle positivo do protocolo. Observamos que os estímulos com leptina e LPS, aumentaram a expressão do RNAm de Ptpn2, a imunorreatividade para TCPTP e a área astrocitária. O tratamento com LPS foi capaz de promover um aumento na expressão do RNAm de Gja1 e o inverso foi observado na expressão de Gja6. Todavia, tanto o tratamento com leptina e LPS promoveu aumento na imunorreatividade para CX43 e o inverso observou-se na imunorreatividade para CX30. Para avaliarmos a participação da TCPTP nos efeitos da leptina na morfologia dos astrócitos, realizamos o silenciamento de seu gene, utilizando o siRNA Ptpn2. O silenciamento de Ptpn2 foi capaz de reverter os efeitos da leptina tanto na expressão gênica, na imunorreatividade assim como na morfologia astrocitária. O silenciamento de Ptpn2 reverteu também as respostas de redução de CX30 e o aumento de CX43 promovidas pelo LPS pela leptina. De maneira inédita esses dados sugerem a importância da TCPTP na modulação das conexinas nos efeitos da leptina e LPS na morfologia astrocitária hipotalâmica. Observamos que apenas o tratamento com LPS foi capaz de promover um aumento na expressão do RNAm de Ptpn1, e o silenciamento de Ptpn2 intensificou esse aumento da expressão de Ptpn1, demonstrando de forma inédita 10 que a TCPTP exerce ação contra regulatória sobre a PTP1B. Como esperado o estímulo dos astrócitos com LPS aumentou a expressão do RNAm de Il6, Il1b e Tnfa. Interessantemente, o silenciamento de Ptpn2 intensificou esse aumento da expressão do RNAm de Il6, Il1b e Tnfa, demonstrando desse modo que a TCPTP possui ação contra regulatória na secreção dessas citocinas. O conjunto de dados demonstra que a alteração nutricional neonatal é capaz de promover alterações no balanço energético na vida juvenil e adulta. Estas alterações estão associadas a modificações morfológicas das células da glia e ao aumento de citocinas inflamatórias, caracterizando um estado reativo glial. Adicionalmente, demonstramos em cultura primária de astrócitos hipotalâmicos que a leptina altera a morfologia destas células e pela primeira vez demonstramos, também, que a TCPTP modula esses efeitos da leptina, por meio de suas ações na conexina CX30. A CX30 participa na modulação da morfologia dos astrócitos e sua redução está associada ao aumento na área e nos processos de extensão destas células. Em conclusão, o presente estudo demonstra que alterações na disponibilidade nutricional na vida neonatal acarretam alterações no comportamento alimentar e no peso corporal na vida juvenil e adulta em ratos. Demonstramos, também, que tais alterações nutricionais neonatais estão associadas a alterações em células da glia. A leptina induz alterações morfológicas em astrócitos, sendo este efeito mediado pela TCPTP e sua regulação sobre a expressão da proteína CX30. O conjunto dos dados indica a importância das células não neuronais no controle central da homeostase energética em modelo de programação nutricional neonatal. / Nutritional changes in the neonatal period can affect the hypothalamic control of food intake and metabolism in later life. We evaluated the influence of the neonatal nutritional programming on hypothalamic glial cells, known to play an important role in the energy homeostasis. Astrocytes have active metabolic function and provide energy substrate for the neurons through connexin 43 (CX43). CX30 is important in the maintenance of astrocyte morphology, contributing to the insertion of its process into the synaptic cleft. The TCPTP (T-cell protein tyrosine phosphatase) is a counterregulator of cellular signaling of leptin and insulin, contributing to the molecular mechanisms of resistance to these hormones and it is expressed in glial and modulates CX43 activity. We hypothesized that alterations in the hypothalamic glial cells participate in the long-lasting effects on energy balance induced by neonatal nutritional programming. For this purpose, we used the model of neonatal nutritional programming induced by changing the litter size, according to the number of offspring per dam: 3 offsprings, small litter (SL), 10 offsprings, normal litter (NL) and 16 offsprings, large litter (LL). The body weight of the litter was determined weekly until weaning on the 21st day of life (PN21). After weaning, body weight was determined every five days until the 60th day of life (PN60). Individual dietary intake was determined between PN50 and PN60. The SL animals presented higher body weight (72.3 ± 2.08g) at weaning, when compared with the NL (57.19 ± 3.49g) and LL (36.27 ± 1.79g) groups and the difference between these groups were maintained until the PN60. However, the food intake of adult SL animals was not different from the NL group. On the other hand, LL animals presented a reduced weight gain at weaning but they had a catch up of reaching the vody weight of NL animals (NL: 165 ± 3.97g; LL: 145.42 ± 4.55g) from PN35 on, and this response was associated with higher food inatke. At PN21, there was an increase in plasma leptin (6.41 ± 0.90 ng/ml) and insulin (1.97 ± 0.11ng/ml) concentrations in the SL group, when compared with the NL group (leptin: 3.79 ± 0,35ng/ml; insulin: 1.32 ± 0.21ng/ml) and LL (leptin: 1.23 ± 0.10ng/ml; insulin: 0.99 ± 0.10 ng/ml). At PN60, both SL (leptin: 5,26 ± 1.15ng/ml, insulin: 2,53 ± 0,36ng/ml) and LL (leptin: 4.30 ± 0.51ng/ml, insulin: 3.39 ± 0.47ng/ml) groups presented increased plasma leptin and insulin concentrations compared with the group NL (leptin: 1.79 ± 0.41ng/ml; insulin: 1.19 ± 0.09ng/ml). The mRNA expression of Ptpn2 mRNA, gene encoding TCPTP, and its protein in the arcuate nucleus (ARC) was increase at PN21 in the SL group and in both groups at PN60, compared with the NL group. The SL group showed an increased immunoreactivity for GFAP at PN21 and 12 both groups showed this increased response at PN60. Similar response was observed for ionized calcium binding adaptor molecule 1 (IBA-1) immunoreactivity at PN21 and PN60. There was an overlap of TCPTP with GFAP immunoreactivity, but not with IBA-1. At PN21 there was an increase in the mRNA expression of Gja1, gene coding for CX43, as well as in the immunoreactivity of CX43 in the SL group only. At PN21 and PN60, mRNA expression of the Gja6, gene encoding for CX30, was reduced in both SL and LL groups. However, at PN60 it was reduction of CX30 immunoreactivity in both groups, SL and LL. At PN21, Il1b mRNA expression was increased in the ARC in both SL and LL groups. However, at PN60, only the LL group showed an increased Il1b mRNA expression. Additionally, at PN60 both SL and LL groups showed an increase in the Tnfa mRNA expression in the ARC. In the morphological analysis of glia cells, at PN21, there was an increase in the immunoreactivity of the microglia and astrocyte in the SL group, as well as in the extension processes of both cells. At PN60, both groups showed an increase in the soma immunoreactivity and astrocytic processe extension, however, only the SL group showed an increase in the immunoreactivity of the microglial soma. To analyze the effect of leptin on astrocyte morphology and the participation of TCPTP in this process, we performed the primary culture of hypothalamic astrocytes from neonatal rats that were stimulated with leptin [1000ng/ml], [5000ng/ml] and LPS [500ng /ml]. The LPS was used as a positive control of the protocol. We observed that the leptin and LPS stimuli increased the Ptpn2 mRNA expression, the TCPTP immunoreactivity and the astrocyte area. The LPS treatment increased the Gja1 mRNA expression and the opposite was observed in the Gja6 expression. On the other hand, both treatment with leptin and LPS increased the immunoreactivity for CX43 and the opposite was observed for the CX30 immunoreactivity. In order to evaluate the participation of TCPTP in the effects of leptin on the astrocyte morphology, we performed the silencing of its gene using the siRNA Ptpn2. The silencing of Ptpn2 was able to reverse the effects of leptin and LPS on gene expression, immunoreactivity as well as astrocyte morphology. The silencing of Ptpn2 was able to revert the reduction of CX30 and the increase of CX43 immunoreactivity and the its gene expression promoted by LPS leptin. These data are the first to show the importance of TCPTP in the modulation of connexins on the leptin and LPS effects on the morphology of hypothalamic astrocytes. Additionally, only LPS treatment was able to promote an increase in the Ptpn1 mRNA expression and Ptpn2 silencing enhanced this increase in Ptpn1 mRNA expression.These data demonstrate an unprecedented way that Ptpn2 exerts regulatory action against Ptpn1. As expected, the stimulation with LPS increased the mRNA expression of the Il6, Il1b and Tnfa. The silencing of Ptpn2 amplified this effect of LPS on cytokine gene expression, demonstrating that TCPTP has a counterregulatory action on the secretion of IL6, IL1? and Tnf?. Taken together these data demonstrate that the neonatal nutritional changes are able to promote alterations in the energy balance in the juvenile and adult life. These effects are associated with morphological changes in glial cells and increase of inflammatory cytokines, characterizing a glial reactive state. Additionally, using primary cell culture, we demonstrated that leptin alters the morphology of hypothalamic astrocytes. We also demonstrate for the first time that TCPTP modulates these effects of 13 leptin, through its actions regulating the expression of CX30. The data shown indicate the importance of non-neuronal cells in the central control of energy homeostasis in a model of neonatal nutritional programming.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.