Maternal Immune Activation Causes Behavioral Impairments and Altered Cerebellar Cytokine and Synaptic Protein Expression

Emerging epidemiology studies indicate that maternal immune activation (MIA) resulting from inflammatory stimuli such as viral or bacterial infections during pregnancy serves as a risk factor for multiple neurodevelopmental disorders including autism spectrum disorders and schizophrenia. Although alterations in the cortex and hippocampus of MIA offspring have been described, less evidence exists on the impact on the cerebellum. Here, we report altered expression of cytokines and chemokines in the cerebellum of MIA offspring, including increase in the neuroinflammatory cytokine TNFα and its receptor TNFR1. We also report reduced expression of the synaptic organizing proteins cerebellin-1 and GluRδ2. These synaptic protein alterations are associated with a deficit in the ability of cerebellar neurons to form synapses and an increased number of dendritic spines that are not in contact with a presynaptic terminal. These impairments are likely contributing to the behavioral deficits in the MIA exposed offspring

Maternal Immune Activation Causes Behavioral Impairments and Altered Cerebellar Cytokine and Synaptic Protein Expression

Emerging epidemiology studies indicate that maternal immune activation (MIA) resulting from inflammatory stimuli such as viral or bacterial infections during pregnancy serves as a risk factor for multiple neurodevelopmental disorders including autism spectrum disorders and schizophrenia. Although alterations in the cortex and hippocampus of MIA offspring have been described, less evidence exists on the impact on the cerebellum. Here, we report altered expression of cytokines and chemokines in the cerebellum of MIA offspring, including increase in the neuroinflammatory cytokine TNFα and its receptor TNFR1. We also report reduced expression of the synaptic organizing proteins cerebellin-1 and GluRδ2. These synaptic protein alterations are associated with a deficit in the ability of cerebellar neurons to form synapses and an increased number of dendritic spines that are not in contact with a presynaptic terminal. These impairments are likely contributing to the behavioral deficits in the MIA exposed offspring

Glutamate pretreatment affects Ca2+Ca^{2+} signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and $Ca^{2+}$ imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular $Ca^{2+}([Ca^{2+}]_i)$ in the processes of cultured rat cortical astrocytes existing as pairs. Transient $Ca^{2+}$ changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, $Ca^{2+}$ transients were recorded following pretreatment of cells with glutamate (500 lM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, \alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation f local $Ca^{2+}$ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the $Ca^{2+}$ transients in the heparin-loaded astrocyte but attenuated the $Ca^{2+}([Ca^{2+}]_i)$ responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate $(IP_3)$ may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the $Ca^{2+}([Ca^{2+}]_i)$ responses in both the control and lutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamateinduced alterations in $Ca^{2+}$ signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), $IP_3$, internal $Ca^{2+}$ stores, VGCC and gap junction channels appear to play an important role

Glutamate pretreatment affects Ca<SUP>2+</SUP> signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca<SUP>2+</SUP> imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular C<SUP>a2+</SUP> ([Ca<SUP>2+</SUP>]i) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca<SUP>2+</SUP> changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca<SUP>2+</SUP> transients were recorded following pretreatment of cells with glutamate (500 μm for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, -methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca<SUP>2+</SUP> diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca<SUP>2+</SUP> transients in the heparin-loaded astrocyte but attenuated the [Ca<SUP>2+</SUP>]i responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP<SUB>3</SUB>) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca<SUP>2+</SUP>]i responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca<SUP>2+ </SUP>signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP<SUB>3</SUB>, internal Ca<SUP>2+</SUP> stores, VGCC and gap junction channels appear to play an important role

Motor-Skill Learning Is Dependent on Astrocytic Activity

Motor-skill learning induces changes in synaptic structure and function in the primary motor cortex through the involvement of a long-term potentiation- (LTP-) like mechanism. Although there is evidence that calcium-dependent release of gliotransmitters by astrocytes plays an important role in synaptic transmission and plasticity, the role of astrocytes in motor-skill learning is not known. To test the hypothesis that astrocytic activity is necessary for motor-skill learning, we perturbed astrocytic function using pharmacological and genetic approaches. We find that perturbation of astrocytes either by selectively attenuating IP3R2 mediated astrocyte Ca2+ signaling or using an astrocyte specific metabolic inhibitor fluorocitrate (FC) results in impaired motor-skill learning of a forelimb reaching-task in mice. Moreover, the learning impairment caused by blocking astrocytic activity using FC was rescued by administration of the gliotransmitter D-serine. The learning impairments are likely caused by impaired LTP as FC blocked LTP in slices and prevented motor-skill training-induced increases in synaptic AMPA-type glutamate receptor in vivo. These results support the conclusion that normal astrocytic Ca2+ signaling during a reaching task is necessary for motor-skill learning

Kynurenate treatment of autaptic hippocampal microcultures affect localized voltage-dependent calcium diffusion in the dendrites

It is not clear how different spatial compartments in the neuron are affected during epileptiform activity. In the present study we have examined the spatial and temporal profiles of depolarization induced changes in the intracellular Ca^2^+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally by treatment with kynurenate (2 mM) and Mg^2^+ (11.3 mM) in culture (treated neurons). This was examined with simultaneous somatic patch-pipette recording and Ca^2^+ imaging experiments using the Ca^2^+ indicator Oregon Green 488 BAPTA-1. Neurons stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca^2^+ entry at localized sites in the dendrites. Ca^2^+ transients were observed even in the presence of NMDA and AMPA receptor antagonists suggesting that the opening of voltage gated calcium channels primarily triggered the local Ca^2^+ changes. Peak Ca^2^+ transients in the dendrites of treated neurons were larger compared to the signals recorded from the control neurons. Dendritic Ca^2^+ transients in treated neurons showed a distance dependent scaling. Estimation of dendritic local Ca^2^+ diffusion coefficients indicated higher values in the treated neurons and a higher availability of free Ca^2^+. Simulation studies of Ca^2^+ dynamics in these localized dendritic compartments indicate that local Ca^2^+ buffering and removal mechanisms may be affected in treated neurons. Our studies indicate that small dendritic compartments are rendered more vulnerable to changes in intracellular Ca^2^+ following induction of epileptiform activity. This can have important cellular consequences including local membrane excitability through mechanisms that remain to be elucidated

Motor-Skill Learning Is Dependent on Astrocytic Activity

Motor-skill learning induces changes in synaptic structure and function in the primary motor cortex through the involvement of a long-term potentiation-(LTP-) like mechanism. Although there is evidence that calcium-dependent release of gliotransmitters by astrocytes plays an important role in synaptic transmission and plasticity, the role of astrocytes in motor-skill learning is not known. To test the hypothesis that astrocytic activity is necessary for motor-skill learning, we perturbed astrocytic function using pharmacological and genetic approaches. We find that perturbation of astrocytes either by selectively attenuating IP 3 R2 mediated astrocyte Ca 2+ signaling or using an astrocyte specific metabolic inhibitor fluorocitrate (FC) results in impaired motor-skill learning of a forelimb reaching-task in mice. Moreover, the learning impairment caused by blocking astrocytic activity using FC was rescued by administration of the gliotransmitter D-serine. The learning impairments are likely caused by impaired LTP as FC blocked LTP in slices and prevented motor-skill training-induced increases in synaptic AMPA-type glutamate receptor in vivo. These results support the conclusion that normal astrocytic Ca 2+ signaling during a reaching task is necessary for motor-skill learning

Kynurenate treatment of autaptic hippocampal microcultures affect localized voltage-dependent calcium diffusion in the dendrites

It is not clear how different spatial compartments in the neuron are affected during epileptiform activity. In the present study we have examined the spatial and temporal profiles of depolarization induced changes in the intracellular Ca^2^+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally by treatment with kynurenate (2 mM) and Mg^2^+ (11.3 mM) in culture (treated neurons). This was examined with simultaneous somatic patch-pipette recording and Ca^2^+ imaging experiments using the Ca^2^+ indicator Oregon Green 488 BAPTA-1. Neurons stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca^2^+ entry at localized sites in the dendrites. Ca^2^+ transients were observed even in the presence of NMDA and AMPA receptor antagonists suggesting that the opening of voltage gated calcium channels primarily triggered the local Ca^2^+ changes. Peak Ca^2^+ transients in the dendrites of treated neurons were larger compared to the signals recorded from the control neurons. Dendritic Ca^2^+ transients in treated neurons showed a distance dependent scaling. Estimation of dendritic local Ca^2^+ diffusion coefficients indicated higher values in the treated neurons and a higher availability of free Ca^2^+. Simulation studies of Ca^2^+ dynamics in these localized dendritic compartments indicate that local Ca^2^+ buffering and removal mechanisms may be affected in treated neurons. Our studies indicate that small dendritic compartments are rendered more vulnerable to changes in intracellular Ca^2^+ following induction of epileptiform activity. This can have important cellular consequences including local membrane excitability through mechanisms that remain to be elucidated

Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins

Summary: The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However, it remains unknown whether analogous developmental processes are manifested in the human brain. Here we report oligodendrogenesis in forebrain organoids, generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) versus dorsal forebrain organoids (DFOs). Interestingly, oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore, dorsally derived oligodendroglial cells outcompete ventrally derived oligodendroglia and become dominant in FFOs after long-term culture. Thus, our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally and dorsally derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects. : Using OLIG2-GFP knockin human pluripotent stem cell reporter lines, Jiang and colleagues demonstrate oligodendrogenesis in dorsal and ventral forebrain organoids. Furthermore, fused dorsal and ventral forebrain organoids recapitulate the developmental interactions between dorsally and ventrally derived oligodendroglia. These organoid models will serve to study heterogeneity of human oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects. Keywords: human pluripotent stem cells, oligodendrogenesis, OLIG2, forebrain organoids, fused organoids, oligodendroglial heterogeneit