Evidence of Energy Metabolism Alterations in Cultured Neonatal Astrocytes Derived from the Ts65Dn Mouse Model of Down Syndrome

For many decades, neurons have been the central focus of studies on the mechanisms underlying the neurodevelopmental and neurodegenerative aspects of Down syndrome (DS). Astrocytes, which were once thought to have only a passive role, are now recognized as active participants of a variety of essential physiological processes in the brain. Alterations in their physiological function have, thus, been increasingly acknowledged as likely initiators of or contributors to the pathogenesis of many nervous system disorders and diseases. In this study, we carried out a series of real-time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in hippocampal astrocytes derived from neonatal Ts65Dn and euploid control mice using a Seahorse XFp Flux Analyzer. Our results revealed a significant basal OCR increase in neonatal Ts65Dn astrocytes compared with those from control mice, indicating increased oxidative phosphorylation. ECAR did not differ between the groups. Given the importance of astrocytes in brain metabolic function and the linkage between astrocytic and neuronal energy metabolism, these data provide evidence against a pure “neurocentric” vision of DS pathophysiology and support further investigations on the potential contribution of disturbances in astrocytic energy metabolism to cognitive deficits and neurodegeneration associated with DS.

Abnormal Ca2+ Signals in Reactive Astrocytes as a Common Cause of Brain Diseases

In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca2+ signals in pathological brains and found that reactive astrocytes share abnormal Ca2+ signals, even in different types of diseases. In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca2+ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca2+ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca2+ signals. Because the abnormal astrocytic Ca2+ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP3RII), we reanalyzed these pathological events using IP3RII-deficient mice and found that all abnormal Ca2+ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca2+ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca2+ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.

Hippocampus-sensitive and striatum-sensitive learning one month after morphine or cocaine exposure in male rats

These experiments examined whether exposure to drugs of abuse altered the balance between hippocampal and striatal memory systems as measured long after drug treatments. Male rats received injections of morphine (5 mg/kg), cocaine (20 mg/kg), or saline for five consecutive days. One month later, rats were then trained to find food on a hippocampus-sensitive place task or a striatum-sensitive response task. Relative to saline controls, morphine-treated rats exhibited impaired place learning but enhanced response learning; prior cocaine exposure did not significantly alter learning on either task. Another set of rats was trained on a dual-solution T-maze that can be solved with either place or response strategies. While a majority (67%) of control rats used place solutions in this task, morphine treatment one month prior resulted in a shift to response solutions exclusively (100%). Prior cocaine treatment did not significantly alter strategy selection. Molecular markers related to learning and drug abuse were measured in the hippocampus and striatum one month after drug exposure in behaviorally untested rats. Protein levels of glial-fibrillary acidic protein (GFAP), an intermediate filament specific to astrocytes, increased significantly in the hippocampus after morphine, but not after cocaine exposure. Exposure to morphine or cocaine did not significantly change levels of brain-derived neurotrophic factor (BDNF) or a downstream target of BDNF signaling, glycogen synthase kinase 3β (GSK3β), in the hippocampus or striatum. Thus, exposure to morphine results in a long-lasting shift from hippocampal toward striatal dominance during learning. The effects of prior morphine injections on GFAP suggest that long-lasting alterations in hippocampal astrocytes may be associated with these behavioral strategy shifts.

Glial Chloride Homeostasis Under Transient Ischemic Stress

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl–]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl–]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl–]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl–]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl– cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl– cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl–]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl–]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl–]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl–]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl–]int in any of the tested glial cell types. However, [Cl–]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

Optogenetic Activation of Astrocytes—Effects on Neuronal Network Function

Optogenetics approach is used widely in neurobiology as it allows control of cellular activity with high spatial and temporal resolution. In most studies, optogenetics is used to control neuronal activity. In the present study optogenetics was used to stimulate astrocytes with the aim to modulate neuronal activity. To achieve this goal, light stimulation was applied to astrocytes expressing a version of ChR2 (ionotropic opsin) or Opto-α1AR (metabotropic opsin). Optimal optogenetic stimulation parameters were determined using patch-clamp recordings of hippocampal pyramidal neurons’ spontaneous activity in brain slices as a readout. It was determined that the greatest increase in the number of spontaneous synaptic currents was observed when astrocytes expressing ChR2(H134R) were activated by 5 s of continuous light. For the astrocytes expressing Opto-α1AR, the greatest response was observed in the pulse stimulation mode (T = 1 s, t = 100 ms). It was also observed that activation of the astrocytic Opto-a1AR but not ChR2 results in an increase of the fEPSP slope in hippocampal neurons. Based on these results, we concluded that Opto-a1AR expressed in hippocampal astrocytes provides an opportunity to modulate the long-term synaptic plasticity optogenetically, and may potentially be used to normalize the synaptic transmission and plasticity defects in a variety of neuropathological conditions, including models of Alzheimer’s disease and other neurodegenerative disorders.

Prenatal Zinc Supplementation Ameliorates Hippocampal Astrocytes Activation and Inflammatory Cytokines Expression Induced by Lipopolysaccharide in a Rat Model of Maternal Immune Activation

Objective: There is evidence that gestational exposure to lipopolysaccharide (LPS) results in fetal zinc deficiency, and eventually neurodevelopmental abnormalities. In this study, we utilized a rat model of maternal immune activation (MIA) to investigate the possible neuroprotective effect of zinc supplementation throughout pregnancy on hippocampal astrocytes activation as well as inflammatory cytokines expression in adult offspring. Methods: Pregnant rats received intraperitoneal injections of either LPS (0.5 mg/kg) or saline at gestational day (GD) 15 and 16 and orally gavaged with zinc sulfate (30 mg/kg) throughout pregnancy. Astrocyte density and histological assessment were evaluated in the hippocampus of adult offspring at postnatal day (PND) 60-62. Also, the mRNA levels of IL-6, TNF-α, IL-1β, NF-κB, and glial fibrillary acidic protein (GFAP) were measured using qPCR analysis. Results: Prenatal exposure to LPS resulted in up-regulated expression levels of IL-6, TNF-α, NF-κB, and GFAP in the hippocampus of adult pups. Moreover, offspring from LPS group showed an increased astrocyte density in CA1 region with no histological alterations in CA1 and CA3 areas. Conversely, maternal zinc supplementation ameliorated these inflammatory alterations induced by LPS. Discussion: This study provides support for the premise that zinc supplementation during pregnancy might be an early treatment option to inhibit hippocampal inflammation induced by the maternal immune response to infectious agents.

Hippocampal Astrocytes Encode Reward Location

Astrocytic calcium dynamics have been implicated in the encoding of sensory information, and modulating them has been shown to impact behavior. However, real-time calcium activity of astrocytes in the hippocampus of awake mice has never been investigated. We used 2-photon microscopy to chronically image CA1 astrocytes as mice ran in familiar or novel virtual environments and obtained water rewards. We found that astrocytes exhibit persistent ramping activity towards the reward location in a familiar environment, but not in a novel one. Using linear decoders, we could precisely predict the location of the mouse in a familiar environment from astrocyte activity alone. We could not do the same in the novel environment, suggesting astrocyte spatial activity is experience dependent. This is the first indication that astrocytes can encode location in spatial contexts, thereby extending their known computational capabilities, and their role in cognitive functions.

Glial place cells: complementary encoding of spatial information in hippocampal astrocytes

Calcium dynamics into astrocytes influence the activity of nearby neuronal structures. However, because previous reports show that astrocytic calcium signals largely mirror neighboring neuronal activity, current information coding models neglect astrocytes. Using simultaneous two-photon calcium imaging of astrocytes and neurons in the hippocampus of mice navigating a virtual environment, we demonstrate that astrocytic calcium signals actively encode spatial information. Calcium events carrying spatial information occurred in topographically organized astrocytic subregions. Importantly, astrocytes encoded spatial information that was complementary and synergistic to that carried by neurons, improving spatial position decoding when astrocytic signals were considered alongside neuronal ones. These results suggest that the complementary place-dependence of localized astrocytic calcium signals regulates clusters of nearby synapses, enabling dynamic, context-dependent, variations in population coding within brain circuits.