Effects of lithium on the proliferation and migration of neuroblasts in the rostral migratory stream in adult mice

The discovery of neurogenesis in adult brains opened the possibility of cellular therapy strategies for the treatment of neurodegenerative diseases, such as Alzheimer’s disease. Neurogenesis in the adult brain occurs in two areas: subgranular zone of the hippocampus and subventricular zone (SVZ) of the lateral ventricles. Neurons that originate from the SVZ migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). In Alzheimer’s disease, there is a progressive neuronal dysfunction and degeneration, resulting in brain atrophy and cognitive impairments including olfactory dysfunction. Several studies have demonstrated that pharmacological treatment with lithium exerts positive effects on adult neurogenesis, and one pathway seems to be the modulation of factors that regulate the migration of neuroblasts. The objective of this study was to investigate whether treatment with lithium promotes the increase of migratory neuroblasts using as parameter the RMS. Adult male C57BL/6 mice were divided into control and lithium-treated groups. The animals were treated for 6 weeks and, at four different time points, i.e., 10 days, 7 days, 3 days and 1 day before the end of treatments, they received an injection of BrdU (cell proliferation marker). The animals were sacrificed by perfusion fixation and the brains were immunohistochemically labeled for BrdU for analysis of migrating neuroblasts in the RMS. The results showed that the number of BrdU+ cells in the RMS was not significantly different between the two groups, suggesting that lithium, alone, is not capable of increasing the number of neuroblasts migrating from the SVZ to the OB

Electrophysiological properties of rostral ventrolateral medulla presympathetic neurons modulated by the respiratory network in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cardiovascular responses to glutamate microinjection in the dorsomedial periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Glutamate (L-Glu) is an abundant excitatory amino acid in the central nervous system (CNS) and is present in the rat PAG. Moreover, data in the literature indicate its involvement in central blood pressure control. Here we report on the cardiovascular effects caused by microinjection of L-Glu into the dorsomedial PAG (dmPAG) of rats and the glutamatergic receptors as well as the peripheral mechanism involved in their mediation. The microinjection of L-Glu into the dmPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. The cardiovascular response was significantly reduced by pretreatment of the dmPAG with a glutamatergic M-methyl-D-aspartate (NMDA) receptor antagonist (LY235959) and was not affected by pretreatment with a non-NMDA receptor antagonist (NBQX), suggesting a mediation of that response by the activation of NMDA receptors. Furthermore, the pressor response was blocked by pretreatment with the ganglion blocker pentolinium (5 mg/kg, intravenously), suggesting an involvement of the sympathetic nervous system in this response. Our results indicate that the microinjection of L-Glu into the dmPAG causes sympathetic-mediated pressor responses in unanesthetized rats, which are mediated by glutamatergic NMDA receptors in the dmPAG. (c) 2012 Wiley Periodicals, Inc.

Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats. (C) 2012 Elsevier B.V. All rights reserved.

Opioid mu-receptors in the rostral medullary raphe modulate hypoxia-induced hyperpnea in unanesthetized rats

Aim: It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As opioid mu-receptors have been found in the MR, we studied the putative role of opioid mu-receptors in the rostral MR (rMR) region on ventilation in normal and 7% hypoxic conditions. Methods: We measured pulmonary ventilation ((V) over dotE) and the body temperatures (Tb) of male Wistar rats before and after the selective opioid l-receptor antagonist CTAP ( d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2, cyclic, 0.1 mu g per 0.1 mu L) was microinjected into the rMR during normoxia or after 60 min of hypoxia. Results: The animals treated with intra-rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia ( 430 +/- 86 mL kg) 1 min) 1) compared with the control group ( 790 +/- 82 mL kg) 1 min) 1) ( P < 0.05). No differences in the Tb were observed between groups during hypoxia. Conclusion: These data suggest that opioids acting on l-receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.

Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures

Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P<0.001) and FGF8 (3.8 fold; P<0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells (P<0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells (P<0.05) or control cells (P<0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

Transplantation of fetal dopaminergic (DA) neurons offers an experimental therapy for Parkinson's disease (PD). The low availability and the poor survival and integration of transplanted cells in the host brain are major obstacles in this approach. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with growth- and survival-promoting capabilities for developing DA neurons. In the present study, we examined whether pretreatment of ventral mesencephalic (VM) free-floating roller tube (FFRT) cultures with GDNF would improve graft survival and function. For that purpose organotypic cultures of E14 rat VM were grown for 2, 4 or 8 days in the absence (control) or presence of GDNF [10 ng/ml] and transplanted into the striatum of 6-hydroxydopamine-lesioned rats. While all groups of rats showed a significant reduction in d-amphetamine-induced rotations at 6 weeks posttransplantation a significantly improved graft function was observed only in the days in vitro (DIV) 4 GDNF pretreated group compared to the control group. In addition, no statistical significant differences between groups were found in the number of surviving tyrosine hydroxylase-immunoreactive (TH-ir) neurons assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts. In sum, our findings demonstrate that GDNF pretreatment was not disadvantageous for transplants of embryonic rat VM with the FFRT culture technique but only marginally improved graft survival and function.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.