European Journal of Neuroscience, Vol. 39, pp. 893–903, 2014
doi:10.1111/ejn.12465
NEUROSYSTEMS
Extremely low-frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus Maria V. Podda, Lucia Leone, Saviana A. Barbati, Alessia Mastrodonato, Domenica D. Li Puma, Roberto Piacentini and Claudio Grassi
Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy Institute of Human Physiology, Medical School, Universita Keywords: amyloid-b protein, apoptosis, Bax/Bcl-2 ratio, neurogenesis, spatial memory
Abstract In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low-frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5-bromo-2′-deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro-survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro-apoptotic protein Bax, and increased levels of the anti-apoptotic protein Bcl-2, in the hippocampi of ELFEF-exposed mice as well as in ELFEF-exposed NSC cultures, as compared with their sham-exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.
Introduction Adult neurogenesis occurs in the subgranular zone of the dentate gyrus (DG) of the hippocampus of rodents and primates, including humans (Gage, 2000; Alvarez-Buylla & Garcia-Verdugo, 2002; Ming & Song, 2011). Newborn granule neurons are functionally integrated into pre-existing neuronal circuits (Aimone et al., 2011; Shors et al., 2012), and contribute to the formation of memories, especially spatial memory (Dupret et al., 2008; Garthe et al., 2009). Thousands of new cells are added to the adult hippocampus each day, but only 25–30% of them survive and become mature neurons, because the majority are eliminated through programmed cell death (Cameron & McKay, 2001; Kempermann et al., 2003; Sun et al., 2004). The survival rate of new neurons is regulated in an experience-dependent manner: numerous stimuli, including an enriched environment, running, and, above all, learning, can rescue a significant number of newly generated neurons from apoptosis (Kempermann et al., 1997; Gould et al., 1999; Tashiro et al., 2007; Dupret
Correspondence: Claudio Grassi, as above. E-mail: [email protected] Received 4 July 2013, revised 26 November 2013, accepted 28 November 2013
et al., 2008; Shors et al., 2012). On the other hand, fewer neurons are produced in response to stressful experiences, alcohol, and sleep deprivation (Shors, 2009). Convincing evidence has accrued suggesting that there is a link between failure in some or all steps of adult neurogenesis (i.e. cell proliferation, migration, differentiation, and survival) and age-related cognitive decline, as well as neurodegenerative and neuropsychiatric disorders (Winner et al., 2011; Eisch & Petrik, 2012; Encinas & Sierra, 2012). Owing to the functional consequences of adult hippocampal neurogenesis loss/decline, much effort has been put into identifying approaches to enhance and preserve this endogenous process. As a result, several physiological, environmental and pharmacological stimuli have been described that positively affect the complex process of adult hippocampal neurogenesis as a whole or in its specific steps (Van Praag et al., 1999; Brown et al., 2003; Corvino et al., 2012; Kempermann, 2012). In this context, physical stimuli, such as electromagnetic fields (EFs), have been proposed as efficacious tools for enhancing endogenous neurogenesis (Czeh et al., 2002; Arias-Carri on et al., 2004; Piacentini et al., 2008a; Cuccurazzu et al., 2010). In particular, our previous studies (Cuccurazzu et al., 2010) demonstrated that exposure of C57bl/6 mice to extremely low-frequency EFs (ELFEFs)
© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
894 M. V. Podda et al. (1 mT, 50 Hz) stimulates adult hippocampal neurogenesis. Immunofluorescence analysis revealed that ELFEFs affect neural stem cell (NSC) proliferation and neuronal differentiation, as shown by increased numbers of cells labeled with the proliferation marker 5-bromo-2′-deoxyuridine (BrdU), and double-labeled with BrdU and the immature neuron marker doublecortin (DCX). ELFEF-promoted neurogenesis was associated with increases in the transcription of proneuronal genes. Interestingly, 30 days after the end of the ELFEF stimulation protocol, ~50% of the newborn neurons became mature granule cells that were functionally integrated in the DG network, thus improving synaptic plasticity. Here, we extended our previous studies to check whether ELFEFs, besides enhancing NSC proliferation and neuronal differentiation, can increase the survival of hippocampal newborn cells, which is another key event that dramatically affects the functional impact of adult neurogenesis.
Materials and methods
were similar in sham-exposed and control mice that were not subjected to any manipulation (data not shown). For in vitro experiments, cultured NSCs were continuously exposed to ELFEFs (same device as used for in vivo experiments), and the solenoid was placed inside the CO2 incubator. The surfaces of the culture well plates were parallel to the force lines of the alternating magnetic field in the solenoid. Control cells were grown either in the same setting with the generator supplying the solenoid switched off, or in a different CO2 incubator. Air and culture medium temperatures were continuously monitored for the duration of experiments with thermometric probes (Homeometric Control Unit; Harvard Apparatus, Edenbridge, UK; 0.1 °C accuracy). The maximum temperature increase recorded in the cultures exposed to ELFEFs (as compared with shamexposed cultures) was 0.4 0.1 °C. Analyses of data obtained from NSCs cultured in two different CO2 incubators at temperature settings of 37.0 °C and 37.4 °C allowed us to exclude the possible influence of these small changes in temperature on our results. BrdU injection and ex vivo immunofluorescence assays
Animals and exposure to ELFEFs All animal procedures were approved by the Ethics Committee of the Catholic University, and were fully compliant with the Italian Ministry of Health guidelines (Legislative Decree No. 116/1992) and European Union (Directive No. 86/609/EEC) legislation on animal research. Animals were supplied by the Division of Animal Resources of the Catholic University. Fifty-four juvenile male C57bl/6 mice (aged 4–5 weeks) were used in the study, and were divided into two main groups: (i) shamexposed mice (controls); and (ii) ELFEF-exposed mice. ELFEF stimulation (1 mT; 50 Hz; 3.5 h/day for 6 days) was delivered by means of a solenoid connected to an AC power generator, as previously described (Cuccurazzu et al., 2010). In particular, the solenoid was positioned around a Plexiglas cylinder (diameter, 20 cm; length, 42 cm) with open extremities that allowed to position inside the plastic cage containing freely moving animals (three or four mice in each cage). The cage was of the same size as that used for the normal housing of the mice (33 9 15 9 13 cm). Control mice were placed inside the device for the same amount of time as their exposed counterparts, but the generator supplying the solenoid was not turned on (sham exposure). Great care was taken to avoid any stress and animal discomfort. Mice of both groups never showed unusual behaviors during and after exposure. Additionally, control experiments showed that both the magnitude of neurogenesis and the responses to behavioral tests
A set of mice (control, n = 10; ELFEF, n = 10) was used for immunohistochemical detection of neuronal differentiation and survival. These mice received daily single intraperitoneal injections of BrdU (B9285; Sigma, Milan, Italy; 100 mg/kg in sterile 0.9% NaCl solution) for three consecutive days, and were exposed to ELFEFs 9 days after the last BrdU injection (9 days post-injection; Fig. 1). A subset of mice (n = 5 per group) was killed 3 days after completion of the exposure session (18 days post-injection) for immunohistochemical assessment of immature neuron survival, and another set (n = 5 per group) was killed 6 days later (24 days post-injection; Fig. 1) for evaluation of the number of surviving mature neurons. Mice were deeply anesthetised with a cocktail of ketamine (100 mg/ mL) and medetomidine (1 mg/mL) (ratio, 5 : 3), and perfused transcardially with Ringer’s solution followed by 4% (w/v) paraformaldehyde fixative solution. Brains were postfixed overnight at 4 °C, and then transferred to a solution of 30% sucrose in phosphate-buffered saline (PBS) for 2 days. Tissue was sectioned coronally (40 lm) on a vibratome (VT1000S; Leica Microsystems, Wetzlar, Germany), and stored in cryoprotectant at –20 °C (Podda et al., 2008, 2012). After three 10-min rinses in PBS, the sections were incubated in a blocking solution containing 1% bovine serum albumin, 10% normal goat serum, and 0.5% Triton X-100 (TX-100). Immunohistochemistry for BrdU and immunofluorescent doublelabeling for BrdU and the immature neuron marker DCX or the neuronal marker NeuN were performed on a one-in-six series of
Fig. 1. Schematic representation of the experimental design and the protocol timeline. Adult male C57bl/6 mice underwent ELFEF (n = 27) or sham (n = 27) exposure [3.5 h/day for 6 days (6D 9 3.5H)]. Mice used for immunohistochemical detection of newborn cell survival (n = 20) were injected daily with BrdU (100 mg/kg) to label dividing neural precursor cells. IF, immunofluorescence; WB, Western immunoblot assay. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 893–903
ELFEFs promote newborn neuron survival 895 equidistant (240 lm between sections) free-floating sections as previously described (Cuccurazzu et al., 2010). The following antibodies were used: rat monoclonal anti-BrdU antibody (1 : 400; ab6326; Abcam, Cambridge, UK), rabbit polyclonal anti-DCX antibody (1 : 200; #4604; Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-NeuN antibody (1 : 150; MAB377; Millipore, Billerica, MA, USA). The specificity of each antibody has beeen checked in previous studies (Tanaka et al., 2004; Cuccurazzu et al., 2010; Capilla-Gonzalez et al., 2012; Saaltink et al., 2012; Podda et al., 2013). The fluorescent secondary antibodies used were: Alexa Fluor 488conjugated anti-rat secondary antibody (1 : 500; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (1 : 500; Invitrogen), and Alexa Fluor 546conjugated donkey anti-rabbit antibody (1 : 500; Invitrogen). Apoptosis in the DG was evaluated with the APO-BrdU terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kit (Invitrogen), according to the manufacturer’s instructions, as previously described (Fetoni et al., 2009). Briefly, three control and three ELFEF-exposed mice were transcardially perfused 3 days after the end of the ELFEF stimulation protocol (Fig. 1), and their brains were processed as described above. Fixed tissues were embedded in OCT compound for quick freezing in liquid nitrogen. Sixteen-micrometer coronal sections were cut on an SLEE cryostat type MEV (SLEE Medical, Mainz, Germany) at 20 °C, and cryostatic sections were mounted on slides (Superfrost plus). Sections were incubated in ice-cold 70% (v/v) ethanol overnight, and then in freshly prepared DNA-labeling solution for 3 h at 37 °C. Apoptotic cells were identified by immunofluorescence with antiBrdU antibody labeling with Alexa Fluor 488 dye for 1 h at room temperature. Cell nuclei were identified by means of propidium iodide/RNase staining for 20 min at room temperature. Confocal analysis was performed to check that TUNEL signals overlaid nuclear profiles, thus discriminating false positivity. We used the optical dissector method to estimate the number of labeled cells in the DG (West, 1999; Cuccurazzu et al., 2010). Cells were counted under the 940 objective of an Olympus BX51 microscope. The experimenter was blind to the group of origin of the studied section. All labeled cells within each DG layer were counted separately in every sixth section throughout the entire hippocampus. The sum of these cell counts was then multiplied by 6 for estimatation of the total number of labeled cells within a given layer. Two-dimensional images were created as the maximum intensity projection of Z-stack images (512 9 512 pixels) collected on a TCS-SP2 confocal scanning system (Leica Microsystems) equipped with a 9 40 objective (numerical aperture, 1.4; physical voxel size, 0.73 9 0.73 9 0.5 lm) (Santoro et al., 2013). Fluorophores were excited with an Ar/ArKr laser for 488-nm excitation and an HeNe laser for 543-nm excitation. Confocal optical sections (thickness, 10–15 lm) in hippocampal slices were analysed. Co-labeling was verified throughout the z-axis of the focus. Controls for immunofluorescence were obtained by omitting the primary antibody or substituting the primary antibody with the host IgG from which the antibody was generated. In all cases, no artefactual labeling was ever detected. Novel object recognition and Morris water maze tests A set of 20 mice (n = 10 per group) were tested with hippocampusdependent memory tests, namely, the NOR test and the MWM test (Fig. 1). The NOR test was performed as described previously (Fusco
et al., 2012). The preference index, i.e. the ratio of the amount of time spent exploring any one of the two items (training session) or the novel object (retention session) over the total time spent exploring both objects, was used to measure recognition memory. Mice were trained in the MWM (Kempermann & Gage, 2002) to find a platform hidden 1 cm below the surface of a pool (diameter, 127 cm) filled with water made opaque with white non-toxic paint. The acquisition training session started 4 days before the test session (probe test), and consisted of six trials a day for four consecutive days, during which the mice were allowed to reach the platform within 40 s. Starting points were changed daily. A trial lasted either until the mouse had found the platform, or for a maximum of 40 s. Mice rested on the platform for 10 s after each trial. The time taken to reach the platform (latency) and the total distance traveled (the length of the swim path) were recorded with an automated video tracking system (Smart Video Tracking System, Panlab/Harvard Apparatus, Barcelona, Spain). The probe test session was performed 24 h after the last day of the training. In this session, the platform was removed from the pool, and each mouse was allowed to swim for 60 s; the time spent in each quadrant was measured. NSC culture Hippocampal NSC cultures were isolated according to previously published protocols (Podda et al., 2013). Briefly, brains of newborn (aged 0–1 days) C57bl/6 mice were microdissected to obtain the hippocampal region upon sagittal sectioning. Tissues were finely minced and digested with accutase (in Dulbecco’s PBS and 0.5 mM EDTA; Innovative Cell Technologies, San Diego, CA, USA) at 37 °C for 30 min. After centrifugation (800 9 g for 4 min), cells were carefully dissociated by passaging in fire-polished Pasteur pipettes and resuspended in Neurobasal A medium, supplemented with 2% B27 (Gibco, Invitrogen, Carlsbad, CA, USA), Glutamax (0.5 mM; Invitrogen), mouse fibroblast growth factor 2 (10 ng/mL; Invitrogen), epidermal growth factor (10 ng/mL; Invitrogen), and mouse platelet-derived growth factor bb (10 ng/mL; Invitrogen). Cells were seeded into a 25-cm2 T-flask and incubated at 37 °C in a 5% CO2 atmosphere. During the first week, NSCs began to form neurospheres in vitro. At 2-day intervals, the neurospheres were collected and passaged by gentle enzymatic and mechanical dissociation. These processed NSCs retained the potential to grow infinitely, as demonstrated by the percentage of the cells positive for nestin, a marker for immature neural progenitors, which remained stable throughout the course of cell culture. To obtain monolayer cultures, neurospheres of established cultures were passaged by enzymatic and mechanical dissociation, and plated as single cells onto Matrigel Matrix (Becton Dickinson, Franklin Lakes, NJ, USA)-pre-coated Petri dishes. NSCs cultured in ‘proliferation medium’ remained in an undifferentiated state and proliferated. To induce differentiation, NSCs were cultured in a medium defined as ‘differentiation medium’, in which fibroblast growth factor 2, epidermal growth factor and platelet-derived growth factor bb had been replaced with 1% fetal bovine serum. NSC treatments NSCs that had been grown for 1 day in differentiation medium were continuously exposed for 3 days (days 2–4) to ELFEF or sham stimulation in the presence or absence of amyloid-b protein (Ab42) (500 nM). Ab42 solutions were prepared as described in Ripoli et al. (2013). At the end of treatment, NCSs were processed for immunocytochemistry or Western immunoblotting (Fig. 1).
© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 893–903
896 M. V. Podda et al. Immunocytochemistry Hippocampal NSCs were processed for immunofluorescence as previously described (Piacentini et al., 2008a). Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and rinsed twice in PBS. After being permeabilised [15 min of incubation with 0.1% TX-100 (Sigma) in PBS], the cells were incubated for 20 min with 0.3% bovine serum albumin in PBS to block nonspecific binding sites, and then overnight at 4 °C with mouse antimicrotubule-associated protein 2 (MAP2) antibody (1 : 500, clone HM-2; Sigma) (Podda et al., 2013). On the following day, cells were incubated for 90 min at room temperature with the secondary antibody, donkey anti-mouse Alexa Fluor 488 (1 : 1000; Invitrogen). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.5 lg/mL; Invitrogen), and the cells were finally coverslipped with ProLong Gold antifade reagent (Invitrogen). Apoptosis was evaluated in NSC cultures with the APO-BrdU TUNEL assay kit (Invitrogen), according to the manufacturer’s instructions, as previously described (Piacentini et al., 2008b). Briefly, DNA strand breaks in apoptotic cells were labeled with BrdU by the use of terminal deoxynucleotide transferase. Apoptotic cells were identified immunocytochemically by means of anti-BrdU antibody labeling with Alexa Fluor 488 dye, and cell nuclei were identified by means of propidium iodide/RNase staining. Two-dimensional images for immunofluorescence and TUNEL were created as the maximum intensity projection of Z-stack images (512 9 512 pixels) collected on a TCS-SP2 confocal scanning system (Leica) equipped with a 9 40 objective (numerical aperture, 1.4; physical voxel size, 0.73 9 0.73 9 0.5 lm). Fluorophores were excited with an Ar/ArKr laser for 488-nm excitation and an HeNe laser for 543-nm excitation. DAPI staining was imaged during twophoton excitation (740 nm,
doi:10.1111/ejn.12465
NEUROSYSTEMS
Extremely low-frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus Maria V. Podda, Lucia Leone, Saviana A. Barbati, Alessia Mastrodonato, Domenica D. Li Puma, Roberto Piacentini and Claudio Grassi
Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy Institute of Human Physiology, Medical School, Universita Keywords: amyloid-b protein, apoptosis, Bax/Bcl-2 ratio, neurogenesis, spatial memory
Abstract In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low-frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5-bromo-2′-deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro-survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro-apoptotic protein Bax, and increased levels of the anti-apoptotic protein Bcl-2, in the hippocampi of ELFEF-exposed mice as well as in ELFEF-exposed NSC cultures, as compared with their sham-exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.
Introduction Adult neurogenesis occurs in the subgranular zone of the dentate gyrus (DG) of the hippocampus of rodents and primates, including humans (Gage, 2000; Alvarez-Buylla & Garcia-Verdugo, 2002; Ming & Song, 2011). Newborn granule neurons are functionally integrated into pre-existing neuronal circuits (Aimone et al., 2011; Shors et al., 2012), and contribute to the formation of memories, especially spatial memory (Dupret et al., 2008; Garthe et al., 2009). Thousands of new cells are added to the adult hippocampus each day, but only 25–30% of them survive and become mature neurons, because the majority are eliminated through programmed cell death (Cameron & McKay, 2001; Kempermann et al., 2003; Sun et al., 2004). The survival rate of new neurons is regulated in an experience-dependent manner: numerous stimuli, including an enriched environment, running, and, above all, learning, can rescue a significant number of newly generated neurons from apoptosis (Kempermann et al., 1997; Gould et al., 1999; Tashiro et al., 2007; Dupret
Correspondence: Claudio Grassi, as above. E-mail: [email protected] Received 4 July 2013, revised 26 November 2013, accepted 28 November 2013
et al., 2008; Shors et al., 2012). On the other hand, fewer neurons are produced in response to stressful experiences, alcohol, and sleep deprivation (Shors, 2009). Convincing evidence has accrued suggesting that there is a link between failure in some or all steps of adult neurogenesis (i.e. cell proliferation, migration, differentiation, and survival) and age-related cognitive decline, as well as neurodegenerative and neuropsychiatric disorders (Winner et al., 2011; Eisch & Petrik, 2012; Encinas & Sierra, 2012). Owing to the functional consequences of adult hippocampal neurogenesis loss/decline, much effort has been put into identifying approaches to enhance and preserve this endogenous process. As a result, several physiological, environmental and pharmacological stimuli have been described that positively affect the complex process of adult hippocampal neurogenesis as a whole or in its specific steps (Van Praag et al., 1999; Brown et al., 2003; Corvino et al., 2012; Kempermann, 2012). In this context, physical stimuli, such as electromagnetic fields (EFs), have been proposed as efficacious tools for enhancing endogenous neurogenesis (Czeh et al., 2002; Arias-Carri on et al., 2004; Piacentini et al., 2008a; Cuccurazzu et al., 2010). In particular, our previous studies (Cuccurazzu et al., 2010) demonstrated that exposure of C57bl/6 mice to extremely low-frequency EFs (ELFEFs)
© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
894 M. V. Podda et al. (1 mT, 50 Hz) stimulates adult hippocampal neurogenesis. Immunofluorescence analysis revealed that ELFEFs affect neural stem cell (NSC) proliferation and neuronal differentiation, as shown by increased numbers of cells labeled with the proliferation marker 5-bromo-2′-deoxyuridine (BrdU), and double-labeled with BrdU and the immature neuron marker doublecortin (DCX). ELFEF-promoted neurogenesis was associated with increases in the transcription of proneuronal genes. Interestingly, 30 days after the end of the ELFEF stimulation protocol, ~50% of the newborn neurons became mature granule cells that were functionally integrated in the DG network, thus improving synaptic plasticity. Here, we extended our previous studies to check whether ELFEFs, besides enhancing NSC proliferation and neuronal differentiation, can increase the survival of hippocampal newborn cells, which is another key event that dramatically affects the functional impact of adult neurogenesis.
Materials and methods
were similar in sham-exposed and control mice that were not subjected to any manipulation (data not shown). For in vitro experiments, cultured NSCs were continuously exposed to ELFEFs (same device as used for in vivo experiments), and the solenoid was placed inside the CO2 incubator. The surfaces of the culture well plates were parallel to the force lines of the alternating magnetic field in the solenoid. Control cells were grown either in the same setting with the generator supplying the solenoid switched off, or in a different CO2 incubator. Air and culture medium temperatures were continuously monitored for the duration of experiments with thermometric probes (Homeometric Control Unit; Harvard Apparatus, Edenbridge, UK; 0.1 °C accuracy). The maximum temperature increase recorded in the cultures exposed to ELFEFs (as compared with shamexposed cultures) was 0.4 0.1 °C. Analyses of data obtained from NSCs cultured in two different CO2 incubators at temperature settings of 37.0 °C and 37.4 °C allowed us to exclude the possible influence of these small changes in temperature on our results. BrdU injection and ex vivo immunofluorescence assays
Animals and exposure to ELFEFs All animal procedures were approved by the Ethics Committee of the Catholic University, and were fully compliant with the Italian Ministry of Health guidelines (Legislative Decree No. 116/1992) and European Union (Directive No. 86/609/EEC) legislation on animal research. Animals were supplied by the Division of Animal Resources of the Catholic University. Fifty-four juvenile male C57bl/6 mice (aged 4–5 weeks) were used in the study, and were divided into two main groups: (i) shamexposed mice (controls); and (ii) ELFEF-exposed mice. ELFEF stimulation (1 mT; 50 Hz; 3.5 h/day for 6 days) was delivered by means of a solenoid connected to an AC power generator, as previously described (Cuccurazzu et al., 2010). In particular, the solenoid was positioned around a Plexiglas cylinder (diameter, 20 cm; length, 42 cm) with open extremities that allowed to position inside the plastic cage containing freely moving animals (three or four mice in each cage). The cage was of the same size as that used for the normal housing of the mice (33 9 15 9 13 cm). Control mice were placed inside the device for the same amount of time as their exposed counterparts, but the generator supplying the solenoid was not turned on (sham exposure). Great care was taken to avoid any stress and animal discomfort. Mice of both groups never showed unusual behaviors during and after exposure. Additionally, control experiments showed that both the magnitude of neurogenesis and the responses to behavioral tests
A set of mice (control, n = 10; ELFEF, n = 10) was used for immunohistochemical detection of neuronal differentiation and survival. These mice received daily single intraperitoneal injections of BrdU (B9285; Sigma, Milan, Italy; 100 mg/kg in sterile 0.9% NaCl solution) for three consecutive days, and were exposed to ELFEFs 9 days after the last BrdU injection (9 days post-injection; Fig. 1). A subset of mice (n = 5 per group) was killed 3 days after completion of the exposure session (18 days post-injection) for immunohistochemical assessment of immature neuron survival, and another set (n = 5 per group) was killed 6 days later (24 days post-injection; Fig. 1) for evaluation of the number of surviving mature neurons. Mice were deeply anesthetised with a cocktail of ketamine (100 mg/ mL) and medetomidine (1 mg/mL) (ratio, 5 : 3), and perfused transcardially with Ringer’s solution followed by 4% (w/v) paraformaldehyde fixative solution. Brains were postfixed overnight at 4 °C, and then transferred to a solution of 30% sucrose in phosphate-buffered saline (PBS) for 2 days. Tissue was sectioned coronally (40 lm) on a vibratome (VT1000S; Leica Microsystems, Wetzlar, Germany), and stored in cryoprotectant at –20 °C (Podda et al., 2008, 2012). After three 10-min rinses in PBS, the sections were incubated in a blocking solution containing 1% bovine serum albumin, 10% normal goat serum, and 0.5% Triton X-100 (TX-100). Immunohistochemistry for BrdU and immunofluorescent doublelabeling for BrdU and the immature neuron marker DCX or the neuronal marker NeuN were performed on a one-in-six series of
Fig. 1. Schematic representation of the experimental design and the protocol timeline. Adult male C57bl/6 mice underwent ELFEF (n = 27) or sham (n = 27) exposure [3.5 h/day for 6 days (6D 9 3.5H)]. Mice used for immunohistochemical detection of newborn cell survival (n = 20) were injected daily with BrdU (100 mg/kg) to label dividing neural precursor cells. IF, immunofluorescence; WB, Western immunoblot assay. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 893–903
ELFEFs promote newborn neuron survival 895 equidistant (240 lm between sections) free-floating sections as previously described (Cuccurazzu et al., 2010). The following antibodies were used: rat monoclonal anti-BrdU antibody (1 : 400; ab6326; Abcam, Cambridge, UK), rabbit polyclonal anti-DCX antibody (1 : 200; #4604; Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-NeuN antibody (1 : 150; MAB377; Millipore, Billerica, MA, USA). The specificity of each antibody has beeen checked in previous studies (Tanaka et al., 2004; Cuccurazzu et al., 2010; Capilla-Gonzalez et al., 2012; Saaltink et al., 2012; Podda et al., 2013). The fluorescent secondary antibodies used were: Alexa Fluor 488conjugated anti-rat secondary antibody (1 : 500; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (1 : 500; Invitrogen), and Alexa Fluor 546conjugated donkey anti-rabbit antibody (1 : 500; Invitrogen). Apoptosis in the DG was evaluated with the APO-BrdU terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kit (Invitrogen), according to the manufacturer’s instructions, as previously described (Fetoni et al., 2009). Briefly, three control and three ELFEF-exposed mice were transcardially perfused 3 days after the end of the ELFEF stimulation protocol (Fig. 1), and their brains were processed as described above. Fixed tissues were embedded in OCT compound for quick freezing in liquid nitrogen. Sixteen-micrometer coronal sections were cut on an SLEE cryostat type MEV (SLEE Medical, Mainz, Germany) at 20 °C, and cryostatic sections were mounted on slides (Superfrost plus). Sections were incubated in ice-cold 70% (v/v) ethanol overnight, and then in freshly prepared DNA-labeling solution for 3 h at 37 °C. Apoptotic cells were identified by immunofluorescence with antiBrdU antibody labeling with Alexa Fluor 488 dye for 1 h at room temperature. Cell nuclei were identified by means of propidium iodide/RNase staining for 20 min at room temperature. Confocal analysis was performed to check that TUNEL signals overlaid nuclear profiles, thus discriminating false positivity. We used the optical dissector method to estimate the number of labeled cells in the DG (West, 1999; Cuccurazzu et al., 2010). Cells were counted under the 940 objective of an Olympus BX51 microscope. The experimenter was blind to the group of origin of the studied section. All labeled cells within each DG layer were counted separately in every sixth section throughout the entire hippocampus. The sum of these cell counts was then multiplied by 6 for estimatation of the total number of labeled cells within a given layer. Two-dimensional images were created as the maximum intensity projection of Z-stack images (512 9 512 pixels) collected on a TCS-SP2 confocal scanning system (Leica Microsystems) equipped with a 9 40 objective (numerical aperture, 1.4; physical voxel size, 0.73 9 0.73 9 0.5 lm) (Santoro et al., 2013). Fluorophores were excited with an Ar/ArKr laser for 488-nm excitation and an HeNe laser for 543-nm excitation. Confocal optical sections (thickness, 10–15 lm) in hippocampal slices were analysed. Co-labeling was verified throughout the z-axis of the focus. Controls for immunofluorescence were obtained by omitting the primary antibody or substituting the primary antibody with the host IgG from which the antibody was generated. In all cases, no artefactual labeling was ever detected. Novel object recognition and Morris water maze tests A set of 20 mice (n = 10 per group) were tested with hippocampusdependent memory tests, namely, the NOR test and the MWM test (Fig. 1). The NOR test was performed as described previously (Fusco
et al., 2012). The preference index, i.e. the ratio of the amount of time spent exploring any one of the two items (training session) or the novel object (retention session) over the total time spent exploring both objects, was used to measure recognition memory. Mice were trained in the MWM (Kempermann & Gage, 2002) to find a platform hidden 1 cm below the surface of a pool (diameter, 127 cm) filled with water made opaque with white non-toxic paint. The acquisition training session started 4 days before the test session (probe test), and consisted of six trials a day for four consecutive days, during which the mice were allowed to reach the platform within 40 s. Starting points were changed daily. A trial lasted either until the mouse had found the platform, or for a maximum of 40 s. Mice rested on the platform for 10 s after each trial. The time taken to reach the platform (latency) and the total distance traveled (the length of the swim path) were recorded with an automated video tracking system (Smart Video Tracking System, Panlab/Harvard Apparatus, Barcelona, Spain). The probe test session was performed 24 h after the last day of the training. In this session, the platform was removed from the pool, and each mouse was allowed to swim for 60 s; the time spent in each quadrant was measured. NSC culture Hippocampal NSC cultures were isolated according to previously published protocols (Podda et al., 2013). Briefly, brains of newborn (aged 0–1 days) C57bl/6 mice were microdissected to obtain the hippocampal region upon sagittal sectioning. Tissues were finely minced and digested with accutase (in Dulbecco’s PBS and 0.5 mM EDTA; Innovative Cell Technologies, San Diego, CA, USA) at 37 °C for 30 min. After centrifugation (800 9 g for 4 min), cells were carefully dissociated by passaging in fire-polished Pasteur pipettes and resuspended in Neurobasal A medium, supplemented with 2% B27 (Gibco, Invitrogen, Carlsbad, CA, USA), Glutamax (0.5 mM; Invitrogen), mouse fibroblast growth factor 2 (10 ng/mL; Invitrogen), epidermal growth factor (10 ng/mL; Invitrogen), and mouse platelet-derived growth factor bb (10 ng/mL; Invitrogen). Cells were seeded into a 25-cm2 T-flask and incubated at 37 °C in a 5% CO2 atmosphere. During the first week, NSCs began to form neurospheres in vitro. At 2-day intervals, the neurospheres were collected and passaged by gentle enzymatic and mechanical dissociation. These processed NSCs retained the potential to grow infinitely, as demonstrated by the percentage of the cells positive for nestin, a marker for immature neural progenitors, which remained stable throughout the course of cell culture. To obtain monolayer cultures, neurospheres of established cultures were passaged by enzymatic and mechanical dissociation, and plated as single cells onto Matrigel Matrix (Becton Dickinson, Franklin Lakes, NJ, USA)-pre-coated Petri dishes. NSCs cultured in ‘proliferation medium’ remained in an undifferentiated state and proliferated. To induce differentiation, NSCs were cultured in a medium defined as ‘differentiation medium’, in which fibroblast growth factor 2, epidermal growth factor and platelet-derived growth factor bb had been replaced with 1% fetal bovine serum. NSC treatments NSCs that had been grown for 1 day in differentiation medium were continuously exposed for 3 days (days 2–4) to ELFEF or sham stimulation in the presence or absence of amyloid-b protein (Ab42) (500 nM). Ab42 solutions were prepared as described in Ripoli et al. (2013). At the end of treatment, NCSs were processed for immunocytochemistry or Western immunoblotting (Fig. 1).
© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 893–903
896 M. V. Podda et al. Immunocytochemistry Hippocampal NSCs were processed for immunofluorescence as previously described (Piacentini et al., 2008a). Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and rinsed twice in PBS. After being permeabilised [15 min of incubation with 0.1% TX-100 (Sigma) in PBS], the cells were incubated for 20 min with 0.3% bovine serum albumin in PBS to block nonspecific binding sites, and then overnight at 4 °C with mouse antimicrotubule-associated protein 2 (MAP2) antibody (1 : 500, clone HM-2; Sigma) (Podda et al., 2013). On the following day, cells were incubated for 90 min at room temperature with the secondary antibody, donkey anti-mouse Alexa Fluor 488 (1 : 1000; Invitrogen). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.5 lg/mL; Invitrogen), and the cells were finally coverslipped with ProLong Gold antifade reagent (Invitrogen). Apoptosis was evaluated in NSC cultures with the APO-BrdU TUNEL assay kit (Invitrogen), according to the manufacturer’s instructions, as previously described (Piacentini et al., 2008b). Briefly, DNA strand breaks in apoptotic cells were labeled with BrdU by the use of terminal deoxynucleotide transferase. Apoptotic cells were identified immunocytochemically by means of anti-BrdU antibody labeling with Alexa Fluor 488 dye, and cell nuclei were identified by means of propidium iodide/RNase staining. Two-dimensional images for immunofluorescence and TUNEL were created as the maximum intensity projection of Z-stack images (512 9 512 pixels) collected on a TCS-SP2 confocal scanning system (Leica) equipped with a 9 40 objective (numerical aperture, 1.4; physical voxel size, 0.73 9 0.73 9 0.5 lm). Fluorophores were excited with an Ar/ArKr laser for 488-nm excitation and an HeNe laser for 543-nm excitation. DAPI staining was imaged during twophoton excitation (740 nm,
