Carola G. Schipke

GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue.

We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP‐positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green‐yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP‐positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage‐gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP‐positive cells enwrapping synapses by their fine membrane processes. EGFP‐positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology. GLIA 33:72–86, 2001.

Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices

Pathologic impacts in the brain lead to a widespread activation of microglial cells far beyond the site of injury. Here, we demonstrate that glial Ca2+ waves can trigger responses in microglial cells. We elicited Ca2+ waves in corpus callosum glial cells by electrical stimulation or local adenosine triphosphate (ATP) ejection in acute brain slices. Macroglial cells, but not microglia, were bulk‐loaded with Ca2+‐sensitive dyes. Using a transgenic animal in which astrocytes were labeled by the enhanced green fluorescence protein (EGFP) allowed us to identify the reacting cell populations: the wave activated a Ca2+ response in both astrocytes and non‐astrocytic glial cells and spread over hundreds of micrometers even into the adjacent cortical and ventricular cell layers. Regenerative ATP release and subsequent activation of metabotropic purinergic receptors caused the propagation of the glial Ca2+ wave: the wave was blocked by the purinergic receptor antagonist Reactive Blue 2 and was not affected by the gap junction blocker octanol, but enhanced in Ca2+ free saline. To test whether microglial cells respond to the wave, microglial cells were labeled with a dye‐coupled lectin and membrane currents were recorded with the patch‐clamp technique. When the wave passed by, a current with the characteristics of a purinergic response was activated. Thus, Ca2+ waves in situ are not restricted to astrocytic cells, but broadly activate different glial cell types.

Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline

The pathology of Alzheimers disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-β (Aβ). Using the APPPS1 Alzheimers disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aβ species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimers disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimers disease pathology and cognitive deficits.

Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors.

In the brain, N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptors are important elements for the manifestation of memory as well as mediators of neurotoxicity, and they are thought to be exclusive to neurons. To test for the expression of functional NMDA receptors on astrocytes, we generated transgenic mice in which glial fibrillary acidic protein (GFAP)‐positive astrocytes are labeled by a green fluorescent protein and tested their responses to NMDA in acute cortical slices by patch‐clamp recording and Ca2+ imaging. The NMDA‐evoked currents reversed at 0 mV; could be blocked by MK‐801; persisted in the absence of synaptic transmission; were sensitive to Mg2+; and were accompanied by focal Ca2+ elevation, indicating the presence of functional NMDA receptors. Furthermore, we detected mRNAs for NMDA receptor subunits in freshly isolated astrocytes purified by fluorescence‐activated cell sorting. We conclude that processes of cortical astrocytes enwrapping synaptic regions express high densities of NMDA receptors that could be involved in neurone‐glia signaling.

Astrocyte responses to neuronal activity

During the past few years, it has been established that astrocytes sense neuronal activity and are involved in signal transmission. Neuronal stimulation triggered electrophysiological and/or Ca2+ responses in astrocyte cultures and in acute brain slices. Present even within one given brain region, different pathways of neuron‐to‐astrocyte communication involving different receptor systems have been described. These mechanisms include glutamatergic and NO‐mediated signaling. Neuron‐to‐astrocyte signaling can be confined to subcellular compartments, the microdomains, or it can activate the entire cell. It can even trigger a multicellular response in astrocytes, a Ca2+ wave. This form of astrocyte long‐range signal propagation can occur independently, in pure astrocyte cultures, but it can also be triggered by neuronal activity. Astrocytes also exhibit spontaneous Ca2+ activity. Neuronal activity in acute brain slices can organize this activity into complex synchronous networks. One of the functional consequences of neuron‐to‐astrocyte signaling might be the neuronal control of microcirculation using astrocytes as a mediator.

Astrocytes Discriminate and Selectively Respond to the Activity of a Subpopulation of Neurons within the Barrel Cortex

Sensory information from single whiskers in rodents projects to defined morphological units in the cortex, the barrels. We found that astrocytes selectively respond with an increase in the cytosolic Ca(2+) concentration to activation of layer 4 neurons, the input cells of the barrel columns. The neuronal Ca(2+) signal also spread across barrel column borders mainly in layer 2/3, but the glutamate-mediated astrocyte response stayed restricted to the barrel column. In contrast, when interfering with inhibitory pathways by blocking either purinergic, adenosine or gamma-aminobutyric acid(A) receptors, the stimulation activated a Ca(2+) response in a much larger astrocyte population no longer restricted to the borders of the barrel column. We also observed spontaneous and evoked Ca(2+) activity in the synaptic target cells of layer 4 neurons, the layer 2/3 pyramidal cells, but again, we never recorded Ca(2+) responses in astrocytes following activity in this neuronal population. Our data show that astrocytes can discriminate and selectively respond to the activity of a subpopulation of excitatory neurons within a given brain region. This selectivity in the astrocyte response describes a new level of complexity and integration in the reaction of astrocytes to neuronal activity.

Astrocyte function is modified by Alzheimer's disease-like pathology in aged mice

Alzheimers disease (AD) may affect all cell types in the central nervous system. Astrocytes have rarely been investigated in the aged brain and the role of astrocytes in AD is poorly understood. In this study, we used acute brain slices from an amyloid-beta overexpressing double transgenic mouse line where astrocytes express the enhanced green fluorescent protein under the control of the glial fibrillary acidic protein promoter. Using the patch-clamp technique, we analyzed cell coupling and glutamate reactivity, two main features of astrocytes, in the living tissue of aged mice in an AD mouse model. We found large astrocytic networks in the aged (20 to 27 months) transgenic animals in the neocortex, but not in the hippocampus. In contrast, coupling was low in all brain regions of aged control mice. We furthermore noticed significant changes in the responses of astrocytes to glutamate. The expression of functional glutamate transporters and AMPA/kainate-type glutamate receptors increases in the amyloid-beta protein precursor overexpressing mice. Thus, exposure to amyloid-beta leads to altered astrocyte properties and this change might be beneficial to maintain synaptic function.

Impact of beta-amyloid-specific florbetaben PET imaging on confidence in early diagnosis of Alzheimer's disease.

Background: Early diagnosis of Alzheimer’s disease (AD) may be corroborated by imaging of beta-amyloid plaques using positron emission tomography (PET). Here, we performed an add-on questionnaire study to evaluate the relevance of florbetaben imaging (BAY 949172) in diagnosis and consecutive management of probable AD patients. Methods: AD patients with a clinical diagnosis in accordance with the NINCDS-ADRDA criteria or controls were imaged using florbetaben. Referring physicians were asked on a voluntary basis about their confidence in initial diagnosis, significance of PET imaging results, and their anticipated consequences for future patient care. Results: 121 questionnaires for probable AD patients and 80 questionnaires for controls were evaluated. In 18% of patients who had initially received the diagnosis of probable AD, PET scans were rated negative, whereas in controls 18% of scans were positive. An increase in confidence in the initial diagnosis was frequently reported (80%). Imaging results had a significant impact on the intended patient care, as judged by the referring physicians; this was most prominent in those patients with a contradicting scan and/or a low confidence in the initial diagnosis. Conclusion: Florbetaben amyloid imaging increases the overall confidence in diagnosis of AD and may frequently influence clinical decisions and patient management.

Antidepressants act on glial cells: SSRIs and serotonin elicit astrocyte calcium signaling in the mouse prefrontal cortex

One important target in the treatment of major depressive disorder (MDD) is the serotonin (5-hydroxytryptamine, 5-HT) system. Selective serotonin reuptake inhibitors (SSRI) are used to treat MDD. Yet, the mode of action of these drugs is not completely understood. There is evolving evidence for a role of glutamate in mood disorder and its signaling. Astrocytes are involved in glutamate metabolism and play an active role in memory processing but their role in mood disorders is still largely unknown. A modulation of astrocytic signaling by SSRIs or 5-HT has not been investigated up to now. We investigated astrocytic calcium signaling with the calcium indicator dye Fluo-4. Using a confocal microscope, we imaged astrocytes in the medial prefrontal cortex of acute mouse brain slices after the application of the SSRIs citalopram and fluoxetine. In the same way, we studied the effects of serotonin and the modulation of this signaling by glutamate in astrocytes. We found that astrocyte calcium signaling can be elicited by 5-HT. Also, the SSRIs citalopram and fluoxetine induce calcium signals in about 1/3 of all astrocytes, even when neuronal signal propagation is inhibited. Astrocytic responses to 5-HT have a unique pattern and they could mostly not be evoked twice. We determined that glutamate is a substance that can interfere with 5-HT-induced calcium signals in astrocytes since after stimulation by glutamate, astrocytes did not show a response to 5-HT. Astrocytic calcium signaling is elicited by SSRIs and 5-HT. They may serve as integrators, linking the serotonergic and glutamatergic signaling pathways.

GABAA receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents

An important function of astrocytes is the clearance of excess extracellular glutamate via specific carriers whose expression has become an astrocytic marker. In the present study, we found that a large population of astrocytes in the supraoptic nucleus (SON) of the rat hypothalamus lacks glutamate uptake currents and receptor responses but expresses GABAA receptors. Patch clamp recordings in acute hypothalamic slices that included the SON showed typical astrocytic membrane currents and demonstrated that GABA, via GABAA receptor activation, triggered a conductance increase with the reversal potential close to the Cl− equilibrium potential and a decrease in resting K+ conductance. Intracellular labeling with Lucifer Yellow revealed that these cells had a radial glia‐like morphology, with cell bodies lined up along the base of the brain and long processes traversing the nucleus; they were not dye‐coupled. Parallel immunocytochemical labelings showed that they expressed strong GABAA receptor and glial fibrillary acidic protein (GFAP) immunoreactivities. In addition, our electrophysiological and morphological analyses revealed another population of astrocytes in this nucleus, located next to the subarachnoid space. They were less numerous than the radial type, had a round morphology and few processes, and were dye‐coupled. Unlike the radial astrocytes, they showed little immunoreactivity for GABAA receptor or GFAP. Moreover, they did not respond to GABA but to glutamate, a response that was partially mimicked by aspartate, indicating glutamate transporter expression. Taken together, our observations add to growing evidence illustrating heterogeneity of astrocytes in the adult brain, a heterogeneity that reflects striking differences in form and function of astrocytic populations in regions as discrete as the SON of the hypothalamus.