ABSTRACT
Fetal alcohol spectrum disorders are one of the leading causes of developmental abnormalities worldwide. Maternal consumption of alcohol during pregnancy leads to a diverse range of cognitive and neurobehavioral deficits. Although moderate-to-heavy levels of prenatal alcohol exposure (PAE) have been associated with adverse offspring outcomes, there is limited data on the consequences of chronic low-level PAE. Here, we use a model of maternal voluntary alcohol consumption throughout gestation in a mouse model to investigate the effects of PAE on behavioral phenotypes during late adolescence and early adulthood in male and female offspring. Body composition was measured by dual-energy X-ray absorptiometry. Baseline behaviors, including feeding, drinking, and movement, were examined by performing home cage monitoring studies. The impact of PAE on motor function, motor skill learning, hyperactivity, acoustic reactivity, and sensorimotor gating was investigated by performing a battery of behavioral tests. PAE was found to be associated with altered body composition. No differences in overall movement, food, or water consumption were observed between control and PAE mice. Although PAE offspring of both sexes exhibited deficits in motor skill learning, no differences were observed in basic motor skills such as grip strength and motor coordination. PAE females exhibited a hyperactive phenotype in a novel environment. PAE mice exhibited increased reactivity to acoustic stimuli, and PAE females showed disrupted short-term habituation. Sensorimotor gating was not altered in PAE mice. Collectively, our data show that chronic low-level exposure to alcohol in utero results in behavioral impairments.
Subject(s)
Fetal Alcohol Spectrum Disorders , Prenatal Exposure Delayed Effects , Humans , Mice , Pregnancy , Animals , Female , Male , Learning , Ethanol/toxicity , PhenotypeABSTRACT
To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.
Subject(s)
Skull , Wakefulness , Animals , Cerebral Cortex , Diagnostic Imaging , Mice , Neurons , Skull/diagnostic imagingABSTRACT
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.
Subject(s)
Astrocytes/chemistry , Astrocytes/physiology , Cerebral Cortex/chemistry , Cerebral Cortex/physiology , Induced Pluripotent Stem Cells/chemistry , Induced Pluripotent Stem Cells/physiology , Adolescent , Animals , Cells, Cultured , Cerebral Cortex/cytology , Female , Humans , Male , Mice , Mice, 129 Strain , Mice, TransgenicABSTRACT
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.
Subject(s)
Excitatory Postsynaptic Potentials/physiology , Inhibitory Postsynaptic Potentials/physiology , Learning/physiology , Motor Cortex/physiology , Motor Skills/physiology , Synapses/physiology , Animals , Male , Mice , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Neural Stem Cells/cytology , Oligodendroglia/cytology , Organoids/cytology , Pluripotent Stem Cells/cytology , Cell Differentiation/genetics , Gene Expression Profiling/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Neural Stem Cells/metabolism , Neurogenesis/genetics , Oligodendrocyte Transcription Factor 2/genetics , Oligodendrocyte Transcription Factor 2/metabolism , Oligodendroglia/metabolism , Organoids/metabolism , Pluripotent Stem Cells/metabolismABSTRACT
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.
Subject(s)
Cerebellum/immunology , Cytokines/immunology , Nerve Tissue Proteins/immunology , Prenatal Exposure Delayed Effects/immunology , Protein Precursors/immunology , Receptors, Glutamate/immunology , Synapses/immunology , Animals , Cerebellum/metabolism , Cytokines/biosynthesis , Female , Male , Maternal Exposure/adverse effects , Mice, Transgenic , Nerve Tissue Proteins/biosynthesis , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , Protein Biosynthesis/physiology , Protein Precursors/biosynthesis , Receptors, Glutamate/biosynthesis , Synapses/metabolismABSTRACT
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.
Subject(s)
Astrocytes/physiology , Learning/physiology , Motor Skills/physiology , Animals , Antimetabolites/pharmacology , Astrocytes/drug effects , Citrates/pharmacology , Estrogen Antagonists/pharmacology , Forelimb , In Vitro Techniques , Injections, Intraventricular , Inositol 1,4,5-Trisphosphate Receptors/drug effects , Inositol 1,4,5-Trisphosphate Receptors/genetics , Learning/drug effects , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Mice , Mice, Inbred C57BL , Motor Skills/drug effects , Mutation , Psychomotor Performance/drug effects , Receptors, AMPA/drug effects , Serine/pharmacology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Tamoxifen/pharmacologyABSTRACT
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.
Subject(s)
Dendritic Spines/immunology , Maternal-Fetal Exchange , Neurodevelopmental Disorders/immunology , Synapses/immunology , Animals , Anti-Inflammatory Agents/pharmacology , Dendritic Spines/drug effects , Disease Models, Animal , Excitatory Postsynaptic Potentials , Female , Mice , Mice, Inbred C57BL , Neurodevelopmental Disorders/etiology , Neurons/drug effects , Neurons/immunology , Pregnancy , Pyridines/pharmacology , Somatosensory Cortex/drug effects , Somatosensory Cortex/growth & development , Somatosensory Cortex/immunology , Synapses/drug effectsABSTRACT
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.
Subject(s)
Action Potentials/physiology , Adaptation, Physiological , Neurons/physiology , Animals , Axons/physiology , Biophysical Phenomena , Dendrites/physiology , Dentate Gyrus/cytology , Fluorescence , Ion Channel Gating , Male , Models, Neurological , Rats, Sprague-Dawley , Sodium Channels/metabolism , Synapses/physiologyABSTRACT
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.
Subject(s)
Fragile X Syndrome/physiopathology , Learning/physiology , Motor Skills/physiology , Neuronal Plasticity/physiology , Synapses/physiology , Animals , Disease Models, Animal , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , Long-Term Potentiation/genetics , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Receptors, AMPA/metabolism , Synapses/metabolismABSTRACT
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 microM 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 of 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+)](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+)](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(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.
Subject(s)
Astrocytes/drug effects , Calcium Signaling/drug effects , Calcium/metabolism , Gap Junctions/drug effects , Glutamic Acid/pharmacology , 1-Octanol/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Astrocytes/metabolism , Brain/cytology , Calcium Signaling/physiology , Cells, Cultured , Electric Stimulation/methods , Excitatory Amino Acid Antagonists/pharmacology , Gap Junctions/physiology , Gap Junctions/radiation effects , Glial Fibrillary Acidic Protein/metabolism , Glycine/analogs & derivatives , Glycine/pharmacology , Immunohistochemistry/methods , Inositol 1,4,5-Trisphosphate/pharmacology , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Models, Biological , Patch-Clamp Techniques/methods , Rats , Rats, WistarABSTRACT
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.