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Effect of maternal stress on excitability of hippocampal neurons isolated from newborn rats
P.1.b. Basic and clinical neuroscience − Neuroanatomy and neurophysiology and expression of yellow fluorescent protein (YFP) in adult nestin-expressing stem cells and their progeny. To assess which neurogenic stages were affected by elimination of CB1, YFP SGZ cells were assigned to categories based on expression of immunohistochemistry (IHC) markers. Furthermore, we examined the effect of CB1 deletion on adult neurogenesis-related behavior (open field test, light dark test, spatial object recognition test, and forced swim test) and investigated activity-dependent synaptic plasticity on acute hippocampal slices four weeks after TAM injection. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region by stimulating Schaffer collateral axons of area CA3. Statistical significances were ascertained by Student’s t-test. Results: We found that neural stem cell-specific deletion of the CB1 receptor led to a reduced number of total YFP cells in the dentate gyrus four weeks after TAM injection (p = 0.007) and to a decrease in neural stem cell proliferation, as assessed by BrdU staining (p = 0.042). Animals lacking a functional CB1 receptor in newborn neurons displayed a decrease in spatial memory function in the spatial object recognition test (p = 0.038) and an increase in immobility in the forced swim test (p = 0.037). Anxiety-like behavior in the light dark test and spontaneous activity in the open field was not affected. Acute slices of CB1 flox/flox mice displayed a LTP curve that is different to that of littermate controls and averaged potentiation levels of the last 5 minutes of LTP were significantly lower (p = 0.042). Conclusion: The present study shows that the proliferation and survival of newborn neurons critically depends on the activation of the CB1 receptor, reflecting the importance of the functional connectivity and the involvement on the behavioral level. Identification of underlying molecular mechanisms via RNA sequencing of FACS sorted neural stem cells and investigation of the complex dendritic structure of newborn neurons via 3D reconstruction are currently addressed. References [1] Schlicker, E., Kathmann, M., 2001. Modulation of transmitter release via presynaptic cannabinoid receptors. Trends Pharmacol Sci 22:565−72. [2] Aguado, T., K. Monory, J. Palazuelos, N. Stella, B. Cravatt, B. Lutz, G. Marsicano, Z. Kokaia, M. Guzman, I. Galve-Roperh, 2005. The endocannabinoid system drives neural progenitor proliferation. FASEB J 19(12): 1704−6. [3] Aguado, T., J. Palazuelos, K. Monory, N. Stella, B. Cravatt, B. Lutz, G. Marsicano, Z. Kokaia, M. Guzman, I. Galve-Roperh, 2006. The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor cells. J Neurosci 26(5): 1551−61.
P.1.b.004 Effect of maternal stress on excitability of hippocampal neurons isolated from newborn rats L. Lap´ınov´a1 , E. Dremencov1,2 ° , K. Melicherˇc´ıkov´a3 , E. Cs´asz´ar3 , M. Dubovick´y3 , L. Lacinov´a1 1 Institute of Molecular Physiology and Genetics of Slovak Academy of Sciences, Department of Cellular Physiology and Genetics, Bratislava, Slovak Republic; 2 Biomedical Center of Slovak Academy of Sciences, Institute of Experimental Endocrinology, Bratislava, Slovak Republic; 3 Institute of Experimental Pharmacology and Toxicology of Slovak Academy of Sciences, Department of Reproductive Toxicology, Bratislava, Slovak Republic Background: It was previously reported that maternal stress increases the risk of development of certain brain disorders in
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offsprings, such as depression and related mood and anxiety disorders, autism spectrum disorders, and psychotic illnesses, such as schizophrenia [1]. Since hippocampus plays an important role in pathophysiology of these disorders, it is possible that maternal stress attenuates the development and functioning of hippocampal neurons in offsprings. The aim of the current study was to investigate the effect of maternal stress on the excitability of hippocampal neurons in primary cultures isolated from new-born rats. Methods: Adult female Wistar rats (200–250 g) were exposed to an unpredictable chronic stress for a period of two weeks. Control females were kept under the standard laboratory conditions. After this period the females were mated with male rats. Gestation was confirmed by the presence of semen in a vaginal smear. Pups were sacrificed by decapitation within 24 hours after the birth. Their hippocampi were isolated and the primary cultures of hippocampal neurons were prepared, as previously described [2]. Neurons were cultivated for periods from 4 to 16 days and wholecell patch-clamp electrophysiological experiments were performed with borosilicate glass electrodes (impedance 3−4 MW), using HEKA EPC-10 amplifier. Spontaneous and depolarizing currentevoked action potential firing as well as sodium currents were measured. Statistical differences between days of in vitro cultivation and between groups were determined using two-way analysis of variance (ANOVA). The probability of p < 0.05 was considered as statistically significant. Results: Maternal exposure to the unpredictable chronic stress significantly decreased the evoked firing activity of offsprings’ hippocampal neurons measured at days 10, 13, and 16 of in vitro cultivation (p < 0.01, p < 0.01, and p < 0.001, in comparison with controls, respectively). At a day 16 of in vitro cultivation, decreased resting potential (from −55±1 mV to −59±1 mV) of the cellular membrane was observed in the neurons isolated from the offspring of the dams exposed to the unpredictable chronic stress (p < 0.05, in comparison with controls). Higher depolarization was required to induce action potential firing in these neurons (p < 0.001, in comparison with controls). The amplitude of the sodium current was reduced (from −78±15 pA/pF to −39±5 pA/pF) in the neurons isolated from the offsprings of the dams exposed to the unpredictable chronic stress (p < 0.01, in comparison with controls). Conclusions: Our findings suggest that hippocampal neurons in primary cultures isolated from the offspring of the dams exposed to the unpredictable chronic stress are characterized by decreased excitability, in comparison with controls. To the decreased excitability contribute both reduced sodium conductivity and more hyperpolarized resting membrane potential. Further studies should be performed to examine neurochemical alterations responsible for these physiological abnormalities. References [1] Babenko, O., Kovalchuk, I., Metz, G.A., 2015. Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health. Neurosci Biobehav R 48, 70−91. [2] Caro, A., Tarabova, B., Rojo-Ruiz, J., Lacinova, L., 2011. Nimodipine inhibits AP firing in cultured hippocampal neurons predominantly due to block of voltage-dependent potassium channels. Gen Physiol Biophys 30, S44-S53. Disclosure statement: This work was supported by the Slovak Academy of Sciences (SAS) Scholarship, VEGA grant 2/0024/15 and APVV grant 0212−10.
P.1.b.004 Effect of maternal stress on excitability of hippocampal neurons isolated from newborn rats L. Lap´ınov´a1 , E. Dremencov1,2 ° , K. Melicherˇc´ıkov´a3 , E. Cs´asz´ar3 , M. Dubovick´y3 , L. Lacinov´a1 1 Institute of Molecular Physiology and Genetics of Slovak Academy of Sciences, Department of Cellular Physiology and Genetics, Bratislava, Slovak Republic; 2 Biomedical Center of Slovak Academy of Sciences, Institute of Experimental Endocrinology, Bratislava, Slovak Republic; 3 Institute of Experimental Pharmacology and Toxicology of Slovak Academy of Sciences, Department of Reproductive Toxicology, Bratislava, Slovak Republic Background: It was previously reported that maternal stress increases the risk of development of certain brain disorders in
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offsprings, such as depression and related mood and anxiety disorders, autism spectrum disorders, and psychotic illnesses, such as schizophrenia [1]. Since hippocampus plays an important role in pathophysiology of these disorders, it is possible that maternal stress attenuates the development and functioning of hippocampal neurons in offsprings. The aim of the current study was to investigate the effect of maternal stress on the excitability of hippocampal neurons in primary cultures isolated from new-born rats. Methods: Adult female Wistar rats (200–250 g) were exposed to an unpredictable chronic stress for a period of two weeks. Control females were kept under the standard laboratory conditions. After this period the females were mated with male rats. Gestation was confirmed by the presence of semen in a vaginal smear. Pups were sacrificed by decapitation within 24 hours after the birth. Their hippocampi were isolated and the primary cultures of hippocampal neurons were prepared, as previously described [2]. Neurons were cultivated for periods from 4 to 16 days and wholecell patch-clamp electrophysiological experiments were performed with borosilicate glass electrodes (impedance 3−4 MW), using HEKA EPC-10 amplifier. Spontaneous and depolarizing currentevoked action potential firing as well as sodium currents were measured. Statistical differences between days of in vitro cultivation and between groups were determined using two-way analysis of variance (ANOVA). The probability of p < 0.05 was considered as statistically significant. Results: Maternal exposure to the unpredictable chronic stress significantly decreased the evoked firing activity of offsprings’ hippocampal neurons measured at days 10, 13, and 16 of in vitro cultivation (p < 0.01, p < 0.01, and p < 0.001, in comparison with controls, respectively). At a day 16 of in vitro cultivation, decreased resting potential (from −55±1 mV to −59±1 mV) of the cellular membrane was observed in the neurons isolated from the offspring of the dams exposed to the unpredictable chronic stress (p < 0.05, in comparison with controls). Higher depolarization was required to induce action potential firing in these neurons (p < 0.001, in comparison with controls). The amplitude of the sodium current was reduced (from −78±15 pA/pF to −39±5 pA/pF) in the neurons isolated from the offsprings of the dams exposed to the unpredictable chronic stress (p < 0.01, in comparison with controls). Conclusions: Our findings suggest that hippocampal neurons in primary cultures isolated from the offspring of the dams exposed to the unpredictable chronic stress are characterized by decreased excitability, in comparison with controls. To the decreased excitability contribute both reduced sodium conductivity and more hyperpolarized resting membrane potential. Further studies should be performed to examine neurochemical alterations responsible for these physiological abnormalities. References [1] Babenko, O., Kovalchuk, I., Metz, G.A., 2015. Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health. Neurosci Biobehav R 48, 70−91. [2] Caro, A., Tarabova, B., Rojo-Ruiz, J., Lacinova, L., 2011. Nimodipine inhibits AP firing in cultured hippocampal neurons predominantly due to block of voltage-dependent potassium channels. Gen Physiol Biophys 30, S44-S53. Disclosure statement: This work was supported by the Slovak Academy of Sciences (SAS) Scholarship, VEGA grant 2/0024/15 and APVV grant 0212−10.