implantation-cranial-window-for-repeated-vivo-imaging-awake

Implantation of a Cranial Window for Repeated <em>In Vivo</em> Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

University of Nebraska Medical Center

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS results from a mutation that causes silencing of the FMR1 gene, which encodes the fragile X mental retardation protein. Patients with FXS exhibit a range of neurological deficits, including motor skill deficits. Here, we have investigated motor skill learning and its synaptic correlates in the fmr1 knock-out (KO) mouse. We find that fmr1 KO mice have impaired motor skill learning of a forelimb-reaching task, compared with their wild-type (WT) littermate controls. Electrophysiological recordings from the forelimb region of the primary motor cortex demonstrated reduced, training-induced synaptic strengthening in the trained hemisphere. Moreover, long-term potentiation (LTP) is impaired in the fmr1 KO mouse, and motor skill training does not occlude LTP as it does in the WT mice. Whereas motor skill training induces an increase of synaptic AMPA-type glutamate receptor subunit 1 (GluA1), there is a delay in GluA1 increase in the trained hemisphere of the fmr1 KO mice. Using transcranial in vivo multiphoton microscopy, we find that fmr1 KO mice have similar spine density but increased dendritic spine turnover compared with WT mice. Finally, we report that motor skill training-induced formation of dendritic spines is impaired in fmr1 KO mice. We conclude that FMRP plays a role in motor skill learning and that reduced functional and structural synaptic plasticity might underlie the behavioral deficit in the fmr1 KO mouse.

Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome.

Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma-axon recordings combined with axonal Na(+) and Ca(2+) imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na(+) channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale. 

Neuronal adaptation involves rapid expansion of the action potential initiation site.

Both genetic and environmental factors are thought to contribute to neurodevelopmental and neuropsychiatric disorders with maternal immune activation (MIA) being a risk factor for both autism spectrum disorders and schizophrenia. Although MIA mouse offspring exhibit behavioral impairments, the synaptic alterations in vivo that mediate these behaviors are not known. Here we employed in vivo multiphoton imaging to determine that in the cortex of young MIA offspring there is a reduction in number and turnover rates of dendritic spines, sites of majority of excitatory synaptic inputs. Significantly, spine impairments persisted into adulthood and correlated with increased repetitive behavior, an ASD relevant behavioral phenotype. Structural analysis of synaptic inputs revealed a reorganization of presynaptic inputs with a larger proportion of spines being contacted by both excitatory and inhibitory presynaptic terminals. These structural impairments were accompanied by altered excitatory and inhibitory synaptic transmission. Finally, we report that a postnatal treatment of MIA offspring with the anti-inflammatory drug ibudilast, prevented both synaptic and behavioral impairments. Our results suggest that a possible altered inflammatory state associated with maternal immune activation results in impaired synaptic development that persists into adulthood but which can be prevented with early anti-inflammatory treatment. 

Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders.

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 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. 

Motor-Skill Learning Is Dependent on Astrocytic Activity.

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.

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.

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

Motor skill training induces functional and structural changes in the primary motor cortex. New dendritic spines are formed with training and the horizontal connections in the layer II/III area of the primary motor cortex are strengthened. Here we investigated the functional synaptic properties of pyramidal neurons following motor skill training. We trained mice on a single forelimb-reaching task for five days and performed whole cell recordings from layer II/III pyramidal neurons in the forelimb representation area of the primary motor cortex in the ipsilateral (untrained) and contralateral (trained) hemispheres in acute brain slices. Success rate in the forelimb-reaching task rapidly improved over the first 3 days and stabilized on subsequent days. After five days of training, a time at which learning has peaked and synaptic strengthening with field potential recordings show enhancement, we observed an increase in mEPSC frequency while increases in mEPSC amplitudes was only observed in 20% of the cells. Increase in excitatory synaptic properties were correlated with improved motor skill. Measurement of miniature IPSC (mIPSC) after five days of training showed no difference in either frequency or amplitude between the trained and untrained hemispheres. Our present results indicate dynamic changes in excitatory but not inhibitory synapses in M1 layer II/III pyramidal neurons at the late stages of motor skill learning.

Modulation of excitatory but not inhibitory synaptic inputs in the mouse primary motor cortex in the late phase of motor learning.

Astrocytes, a highly heterogeneous population of glial cells, serve as essential regulators of brain development and homeostasis. The heterogeneity of astrocyte populations underlies the diversity in their functions. In addition to the typical mammalian astrocyte architecture, the cerebral cortex of humans exhibits a radial distribution of interlaminar astrocytes in the supragranular layers. These primate-specific interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. However, due to the lack of accessible experimental models, their functional properties and their role in regulating neuronal circuits remain unclear. Here we modeled human interlaminar astrocytes in humanized glial chimeric mice by engrafting astrocytes differentiated from human-induced pluripotent stem cells into the mouse cortex. This model provides a novel platform for understanding neuron-glial interaction and its alterations in neurological diseases.

Article	Year
Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24336735	2013
Neuronal adaptation involves rapid expansion of the action potential initiation site. Nature communications\| PubMed ID: 24851940	2014
Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders. Brain, behavior, and immunity\| PubMed ID: 26218293	2015
Motor-Skill Learning Is Dependent on Astrocytic Activity. Neural plasticity\| PubMed ID: 26346977	2015
Maternal Immune Activation Causes Behavioral Impairments and Altered Cerebellar Cytokine and Synaptic Protein Expression. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology\| PubMed ID: 28102228	2017
Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins. Stem cell reports\| PubMed ID: 31091434	2019
Modulation of excitatory but not inhibitory synaptic inputs in the mouse primary motor cortex in the late phase of motor learning. Neuroscience letters\| PubMed ID: 31283966	2019
Modeling human-specific interlaminar astrocytes in the mouse cerebral cortex. The Journal of comparative neurology\| PubMed ID: 32639590	2021

Ragunathan Padmashri

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