Long-lasting anxiolytic effect of neural precursor cells freshly prepared but not neurosphere-derived cell transplantation in newborn rats

Romariz et al. BMC Neuroscience 2014, 15:94 http://www.biomedcentral.com/1471-2202/15/94 RESEARCH ARTICLE Open Access Long-lasting anxiolytic effect of neural precursor cells freshly prepared but not neurosphere-derived cell transplantation in newborn rats Simone Amaro Alves Romariz1, Daisyléa de Souza Paiva1, Maria Fernanda Valente1, Gabriela Filoso Barnabé1, Roberto Frussa-Filho2ˆ, Regina Cláudia Barbosa-Silva3, Maria Elisa Calcagnotto4 and Beatriz Monteiro Longo1* Abstract Background: The GABAergic system plays an important role in modulating levels of anxiety. When transplanted into the brain, precursor cells from the medial ganglionic eminence (MGE) have the ability to differentiate into GABAergic interneurons and modify the inhibitory tone in the host brain. Currently, two methods have been reported for obtaining MGE precursor cells for transplantation: fresh and neurosphere dissociated cells. Here, we investigated the effects generated by transplantation of the two types of cell preparations on anxiety behavior in rats. Results: We transplanted freshly dissociated or neurosphere dissociated cells into the neonate brain of male rats on postnatal (PN) day 2–3. At early adulthood (PN 62–63), transplanted animals were tested in the Elevated Plus Maze (EPM). To verify the differentiation and migration pattern of the transplanted cells in vitro and in vivo, we performed immunohistochemistry for GFP and several interneuron-specific markers: neuropeptide Y (NPY), parvalbumin (PV) and calretinin (CR). Cells from both types of preparations expressed these interneuronal markers. However, an anxiolytic effect on behavior in the EPM was observed in animals that received the MGE-derived freshly dissociated cells but not in those that received the neurosphere dissociated cells. Conclusion: Our results suggest a long-lasting anxiolytic effect of transplanted freshly dissociated cells that reinforces the inhibitory function of the GABAergic neuronal circuitry in the hippocampus related to anxiety-like behavior in rats. Keywords: Anxiety, Medial ganglionic eminence, Neuronal precursor cells, Transplantation Background Neural stem cell-based therapies have recently been proposed as a possible therapeutic strategy targeted to modulate hyperexcitability by increasing inhibitory neuronal activity in the central nervous system [1,2]. Neural precursor cells from the medial ganglionic eminence (MGE) of the telencephalic region in the developing brain are responsible for producing most of the inhibitory interneurons in both the cortex and the hippocampus of the mature brain [3,4]. When transplanted into the newborn cortex, these cells are able to migrate and * Correspondence: ˆDeceased 1 Departamento de Fisiologia, UNIFESP, Rua Botucatu, 862, 5° andar, 04023-062 São Paulo, SP, Brazil Full list of author information is available at the end of the article integrate into the circuitry of the host brain, forming functional synapses that modify the inhibitory input [3-5]. In the central nervous system, GABA-mediated inhibition is critical for controlling synaptic circuits involved in the modulation of anxiety [6]. Studies evaluating the anxiety behavior of animals transplanted with cells derived from the MGE [5] and LGE (lateral ganglionic eminence) [7] have shown that these cells can produce anxiolytic-like effects. Two methods have been reported for obtaining MGE precursor cells before transplantation: freshly dissociated cells [3,8] and cultured as neurosphere dissociated cells [9]. Neurospheres consist of cell aggregates that are cultivated in suspension in the presence of growth factors, and they are able to differentiate into three cell types of © 2014 Romariz et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Romariz et al. BMC Neuroscience 2014, 15:94 http://www.biomedcentral.com/1471-2202/15/94 the central nervous system: neurons, astrocytes and oligodendrocytes [10]. In vitro studies using MGE cells cultured as neurospheres have shown that these cells are able to maintain their regional identity, giving rise to inhibitory interneurons besides presenting other neural lineages such as astrocytes and oligodentrocytes [11-14]. The fresh dissociation method consists of transplanting the fetal brain cells immediately after dissecting them from the tissue. In this method, the cells migrate widely, maintaining their inhibitory phenotype and functionality [3,5,8,15]. When transplanted into the cerebral cortex, these cells are able to migrate and restore hippocampal function [16,17]. Because of the increasing number of emerging studies using MGE cells prepared by different methods and showing discrepant results, the aim of the present study was to compare the two preparations of MGE cells and investigate the long-lasting effects of the transplantation of either freshly dissociated or neurosphere dissociated MGE-derived cells into the neonate brain of male rats on postnatal (PN) day 2–3 on the anxiety behavior of these animals when tested at early adulthood (PN 62–63) using the Elevated Plus Maze (EPM). The EPM is one of the most widely used tests in contemporary preclinical research on anxiety, and is based on an innate fear that rodents have for open and elevated spaces [18]. When in the EPM, rats tend to avoid the open arms and clearly prefer the enclosed arms. The avoidance of the open arms occurs primarily because the open arms prevent the rat from engaging in thigmotaxic behavior [19]. The ratio of open-arm to total arm entries has been used as an index of anxiety [20]. Often, the percentage of time spent in the open arms is also reported. Anxiolytic drugs increase the number of entries into and the total time spent in the open arms, whereas anxiogenic agents do the opposite [21,22]. Our data suggest a long-term anxiolytic effect following transplantation of freshly dissociated MGE cells, but not of cells expanded as neurospheres. We propose that the fresh cells were able to reinforce the inhibitory function of the GABAergic neuronal circuitry related to anxiety-like behavior in rats. Page 2 of 8 Fresh dissociation preparation For tissue extraction and cell dissociation, ventricular and subventricular layers of the MGE were dissected from E14.5 rat embryos expressing enhanced green fluorescent protein (GFP). Briefly, the tissue was removed and mechanically dissociated by pipetting and centrifugation; the cells were washed with DMEM/ F-12 (Dubelco’s Modified Eagle Medium, Gibco) containing DNase I (10 mg/mL), centrifuged, and ressuspended (...truncated)