Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice methods
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Human astrocytes accentuate excitatory synaptic transmission in the murine hippocampus
A principal function of astrocytes is to monitor local synaptic activity by their expression of metabotropic neurotransmitter receptors for both glutamate and GABA (; ). These receptors activate intracellular signaling pathways, mediated primarily by increases in cytosolic Ca 2+, which are linked to synaptic plasticity ( ). To assess the selective impact of human astrocytes on neural transmission within the host murine neural network, we compared synaptic activity in hippocampal slices prepared from human glial chimeric mice, to that of both their unengrafted and allografted littermate controls. We focused on the hippocampal dentate granule layer, because of the many electrophysiological and behavioral tests by which hippocampal function, learning and LTP could be assessed ( ). In addition, human cells typically densely engrafted this area; these included an admixture of GFAP + /hNu...
Quantitative immunohistochemistry
Chimeric mice and littermate controls (ranging from 2 weeks - 20 months, depending upon experimental endpoint) were perfusion-fixed, processed histologically and analyzed as described in.
Human astrocytes enhance LTP in the adult murine hippocampus
We next asked if human astrocytes might affect synaptic plasticity, by assessing the effect of human glia on long-term potentiation (LTP). Two trains of high frequency stimulation (HFS) potentiated the fEPSP slope to 151.2 ± 8.1% of baseline in chimeric mice, compared with 138.6 ± 7.6% in control littermates (n = 7 mice in both groups; 13.8 ± 1.1 vs. 12.6 ± 0.4 months-old, respectively; ages provided as mean ± SEM) ( ). The enhancement of fEPSP slope persisted at 60 min in humanized chimeric mice (113.6 ± 3.8 %, p < 0.05), whereas fEPSP slope in unengrafted controls fell to 103.2 ± 3.9 % (not significantly different from the fEPSP slope prior to HFS; p =0.169). Mouse allografted controls exhibited an initial increase to 138.5 ± 2.3%, which fell to 103.8 ± 1.3 % at 60 min (not significantly different from the fEPSP slope prior to HFS; n = 7,...
Neither adenosine nor D-serine accounted for the enhancement of LTP by human glia
Several mechanisms exist by which astrocytes can modulate excitatory transmission. Astrocytes release ATP, which after degradation to adenosine by extracellular ectonucleotidases, can suppress both basal synaptic transmission, and activity-dependent increases in synaptic strength (; ). However, it seems unlikely that adenosine contributed to the enhanced synaptic strength observed in the xenografted mice. The A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (; ) did not decrease the threshold for induction of LTP in control mice; in slices exposed to 100 nM DPCPX, the fEPSP slope returned to 101.9 ± 3.6% 60 min after HFS, similar to untreated slices ( ). Thus, it is unlikely that the reduced threshold for LTP in chimeric mice was a consequence of altered adenosine concentrations.
Human glial TNFα potentiates synaptic transmission via an increase in GluR1 receptors
Previous studies have analyzed the effect of TNFα on excitatory transmission in vitro, by adding soluble TNFR1 receptors to scavenge free TNFα (; ). Since soluble TNFR1 would not be expected to be an efficient inhibitor in vivo, we instead administered thalidomide, a potent, BBB permeable inhibitor of TNFα production ( ). Human glial chimeric mice treated with thalidomide exhibited a significant suppression of fEPSP slopes compared to those receiving vehicle (0.5% carboxy-methylcellulose) (1.41 ± 0.15 mV/ms vs. 1.05 ± 0.24 mV/ms at 0.1mA; means ± SEM; p<0.05, n = 12). In contrast, excitatory transmission in unengrafted littermates was unaffected by thalidomide (1.02 ± 0.12 mV/ms vs. 0.97 ± 0.20 mV/ms at 0.1mA; p =0.32, n =12). These observations suggested that thalidomide selectively targeted the potentiation of excitatory transmission mediated...
Enhanced learning in humanized chimeric mice
To specifically assess hippocampus-dependent learning, we next prepared chimeric mice using rag1 immunodeficient mice (maintained on a C57/Bl6 background), which differ from their rag2-null counterparts (on a C3H background) by having normal vision. We first compared the net engraftment of human GPCs, as well as their relative differentiation into hNG2 + GPCs or GFAP+ astroglia, in human glial chimeras established in rag1-null and rag2-null mice. We focused on hippocampal learning, as this region was used for our analysis of LTP (; ). We found that both the engraftment and differentiation of human GPCs in rag1- and rag2-immunodeficient mice were indistinguishable from one another ( ). On that basis, we next assessed the effect of chimerization of rag1-null immunodeficient mice on contextual fear conditioning (CFC), a hippocampal-dependent task in which mice learn to fear a context in...
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A principal function of astrocytes is to monitor local synaptic activity by their expression of metabotropic neurotransmitter receptors for both glutamate and GABA (; ). These receptors activate intracellular signaling pathways, mediated primarily by increases in cytosolic Ca 2+, which are linked to synaptic plasticity ( ). To assess the selective impact of human astrocytes on neural transmission within the host murine neural network, we compared synaptic activity in hippocampal slices prepared from human glial chimeric mice, to that of both their unengrafted and allografted littermate controls. We focused on the hippocampal dentate granule layer, because of the many electrophysiological and behavioral tests by which hippocampal function, learning and LTP could be assessed ( ). In addition, human cells typically densely engrafted this area; these included an admixture of GFAP + /hNuclei + and NG2 + /hNuclei + cells (, ). Stimulation of the medial perforant path ( ) consistently evoked a significantly steeper slope of field excitatory postsynaptic potentials (fEPSP) in the humanized chimeric mice than in either their uninjected littermates or mouse GPC allografted controls (n=...
Chimeric mice and littermate controls (ranging from 2 weeks - 20 months, depending upon experimental endpoint) were perfusion-fixed, processed histologically and analyzed as described in.
We next asked if human astrocytes might affect synaptic plasticity, by assessing the effect of human glia on long-term potentiation (LTP). Two trains of high frequency stimulation (HFS) potentiated the fEPSP slope to 151.2 ± 8.1% of baseline in chimeric mice, compared with 138.6 ± 7.6% in control littermates (n = 7 mice in both groups; 13.8 ± 1.1 vs. 12.6 ± 0.4 months-old, respectively; ages provided as mean ± SEM) ( ). The enhancement of fEPSP slope persisted at 60 min in humanized chimeric mice (113.6 ± 3.8 %, p < 0.05), whereas fEPSP slope in unengrafted controls fell to 103.2 ± 3.9 % (not significantly different from the fEPSP slope prior to HFS; p =0.169). Mouse allografted controls exhibited an initial increase to 138.5 ± 2.3%, which fell to 103.8 ± 1.3 % at 60 min (not significantly different from the fEPSP slope prior to HFS; n = 7, 14.0 ± 0.1 months-old; p = 0.29, t test) ( ). Thus, the observed enhancement of LTP was a specific feature of human glial chimerization, and were not attributable to cell engraftment per se.
Several mechanisms exist by which astrocytes can modulate excitatory transmission. Astrocytes release ATP, which after degradation to adenosine by extracellular ectonucleotidases, can suppress both basal synaptic transmission, and activity-dependent increases in synaptic strength (; ). However, it seems unlikely that adenosine contributed to the enhanced synaptic strength observed in the xenografted mice. The A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (; ) did not decrease the threshold for induction of LTP in control mice; in slices exposed to 100 nM DPCPX, the fEPSP slope returned to 101.9 ± 3.6% 60 min after HFS, similar to untreated slices ( ). Thus, it is unlikely that the reduced threshold for LTP in chimeric mice was a consequence of altered adenosine concentrations.
Previous studies have analyzed the effect of TNFα on excitatory transmission in vitro, by adding soluble TNFR1 receptors to scavenge free TNFα (; ). Since soluble TNFR1 would not be expected to be an efficient inhibitor in vivo, we instead administered thalidomide, a potent, BBB permeable inhibitor of TNFα production ( ). Human glial chimeric mice treated with thalidomide exhibited a significant suppression of fEPSP slopes compared to those receiving vehicle (0.5% carboxy-methylcellulose) (1.41 ± 0.15 mV/ms vs. 1.05 ± 0.24 mV/ms at 0.1mA; means ± SEM; p<0.05, n = 12). In contrast, excitatory transmission in unengrafted littermates was unaffected by thalidomide (1.02 ± 0.12 mV/ms vs. 0.97 ± 0.20 mV/ms at 0.1mA; p =0.32, n =12). These observations suggested that thalidomide selectively targeted the potentiation of excitatory transmission mediated by human glial TNFα. Accordingly, thalidomide also reduced the expression of both TNFα and GluR1 in the human glial chimeras, but not that of NR1 ( ). Importantly, thalidomide also prevented the facilitation of LTP in the human glial chimeras: Two trains of HFS failed to trigger LTP in sl...
To specifically assess hippocampus-dependent learning, we next prepared chimeric mice using rag1 immunodeficient mice (maintained on a C57/Bl6 background), which differ from their rag2-null counterparts (on a C3H background) by having normal vision. We first compared the net engraftment of human GPCs, as well as their relative differentiation into hNG2 + GPCs or GFAP+ astroglia, in human glial chimeras established in rag1-null and rag2-null mice. We focused on hippocampal learning, as this region was used for our analysis of LTP (; ). We found that both the engraftment and differentiation of human GPCs in rag1- and rag2-immunodeficient mice were indistinguishable from one another ( ). On that basis, we next assessed the effect of chimerization of rag1-null immunodeficient mice on contextual fear conditioning (CFC), a hippocampal-dependent task in which mice learn to fear a context in which they receive a foot shock ( ). The human glial-chimeric mice exhibited enhanced performance in CFC throughout all 4 days of training ( ). By just the second day, the human glial-chimeric mice exhibited substantially more rapid and robust CFC than their non-chimeric littermate controls (n=6; 6...
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