Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse

International audienceTracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse Processing of odors in the main olfactory bulb (MOB) is modulated by higher brain afferents depending on the internal state, motivation, memory and emotions. For example satiety or hunger are known to change the perception of food odors. To shed light on this modulation a greater understanding of the underlying circuitry is required. To this aim we conducted tracing experiments in mice. First, stereotaxic injections of monosynaptic retrograde DiI and choleratoxin subunit B (CTb) into the dorsal olfactory bulb (focusing on the granular layer) were performed. In a second approach, we injected the pseudorabies virus 152 (PRV152) (kindly provided by Prof L. Enquist; Princeton University). As a neuronal tracer, this neurotropic virus can spread in synaptically connected neurons, dissecting the entire circuitry. The temporal analysis of the viral distribution allows to determine the number of synapses crossed. Both DiI and CTb confirm all the main centrifugal afferents to the MOB which were previously described in the literature. Thus, except the olfactory tubercle, all regions belonging to the olfactory primary cortex were labeled. Moreover, in the piriform cortex, labeling was mainly located in its dorsal part, confirming the topographical anatomical organization of cortico-bulbar projections. Direct projections arising from orexinergic neurons in the lateral hypothalamus were also observed. Regarding polysynaptic tracing, mice were sacrificed one, two and three days after PRV152 injections. After one day, only some of the direct neuromodulatory afferents were labelled. Thus, the locus coeruleus, which has a very caudal location in the brain close to the forth ventricle, showed already staining. In contrast, other neuromodulatory afferents (arising from raphe nuclei, ventral tegmental area, basal forebrain) were not labeled at this stage. Two days after injection, comparison with DiI/CTb results indicate that all the first-order connections were labeled. At this stage, second-order projections started to appear (for example, the midline thalamic nuclei as reuniens and rhomboid nuclei). Lastly, after three days, an extensive brain labeling occurred although some brain regions still showed a lack of staining. In conclusion, using DiI/CTb tracers that do not cross synapses allow to stain all primary afferents. In contrast, viral tracer migration depends on timing as well as number of connecting synapses. Thus, labeling occurring already one day after injection indicates a strong connection. Accordingly, our results suggest that the stained cells in the locus coeruleus send strong projections to the MOB. Furthermore this method allows to follow up the circuits involved in olfactory modulation. Data analysis of all the other labeled regions, focusing on hypothalamic nuclei and brain areas involved in arousal and food intake, are in progress

Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction

Aims Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. Methods and results The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29+, CD31−, CD34−, CD44+, CD45−, CD81+, CD90−, CD117−, and Flk-1− and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas. Conclusion Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart

Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction

Aims Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. Methods and results The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29+, CD31−, CD34−, CD44+, CD45−, CD81+, CD90−, CD117−, and Flk-1− and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas. Conclusion Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction

Aims: To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction. Methods: Ninety-five Sprague–Dawley rats underwent left coronary artery ligation and after 1month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence. Results: One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3±9.6% versus 27.7±8% pre-transplant) and tissue viability (64.78±7.2% versus 55.89±6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability. Conclusion: These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction

Aims: To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction. Methods: Ninety-five Sprague–Dawley rats underwent left coronary artery ligation and after 1month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence. Results: One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3±9.6% versus 27.7±8% pre-transplant) and tissue viability (64.78±7.2% versus 55.89±6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability. Conclusion: These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC