Biomedical subjects

M Nedergaard

Publications and source records attributed to M Nedergaard.

At least 19 recordsLinked to original sources

PubMed2001

Expression and function of astrocytic gap junctions in aging.

Astrocytic gap junctions have been implicated in a variety of signaling pathways essential to normal brain function. However, no information exists on the prevalence of gap junction channels and their function in the aging brain. Here we have compared the expression of the two most abundant astrocytic gap junction proteins in young and senescent brains and quantified the extent of functional gap junction coupling. The expression level of Cx43 peaked in 7-month-old mice. The relative numbers of Cx43 immunoreactive plaques were 596+/-61, 734+/-62, and 755+/-114 in 3-, 7-, and 21-month-old mice, whereas plaques size averaged 0.9+/-0.1 microm(2) (3 months), 1.3+/-0.1 microm(2) (7 months), and 0.7+/-0.1 microm(2) (21 months). The expression level of Cx30 was also highest in 7-month-old animals (315+/-49 plaques, size 0.8+/-0.07 microm(2) vs. 585+/-51 plaques, size 0.9+/-0.1 microm(2) in 3- and 7-month-old mice, respectively), but only 262+/-63 plaques (size 0.4+/-0.04 microm(2)) in 21-month-old mice. Western blot analysis revealed that the content of both Cx43 and Cx30 remained relatively constant at 3, 7, and 21 months. The fluorescence recovery of photobleach technique (FRAP) was used to evaluate coupling in freshly prepared hippocampal slices. Gap junction coupling did not change significantly as a function of aging, but a tendency towards reduced coupling was observed as the animals aged. Average fluorescence recovery after 2 min was 63+/-6% in younger animals, 59+/-5% in adult animals, and 54+/-4% in old brain. These observations indicate that although astrocytic gap junction proteins are maintained at high levels through the entire lifespan of mice, aging is associated with changes in the number and size of both Cx30 and Cx43 gap junction plaques.

Aging↗

PubMed2001

A kainate receptor increases the efficacy of GABAergic synapses.

Brain functions are based on the dynamic interaction of excitatory and inhibitory inputs. Spillover of glutamate from excitatory synapses may diffuse to and modulate nearby inhibitory synapses. By recording unitary inhibitory postsynaptic currents (uIPSCs) from cell pairs in CA1 of the hippocampus, we demonstrated that low concentrations of Kainate receptor (KAR) agonists increased the success rate (P(s)) of uIPSCs, whereas high concentrations of KAR agonists depressed GABAergic synapses. Ambient glutamate released by basal activities or stimulation of the stratum radiatum increases the efficacy of GABAergic synapses by activating presynaptic KARs, which facilitate Ca(2+)-dependent GABA release. The results suggest that glutamate released from excitatory synapses may also function as an intermediary between excitatory and inhibitory synapses to protect overexcitation of local circuits.

2-Amino-5-phosphonovalerate↗

PubMed2001

Glutamate release promotes growth of malignant gliomas.

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.

Amino Acid Transport System X-AG↗

PubMed2000

Tamoxifen-induced enhancement of calcium signaling in glioma and MCF-7 breast cancer cells.

The antiestrogen tamoxifen is commonly used to treat breast cancer, but it also has therapeutic activity in several other types of cancer. Many of these tumors, including malignant gliomas, are estrogen receptor negative. Nonetheless, high concentrations of tamoxifen can directly reduce cell proliferation in some of these tumors and induce apoptosis. In this study, the role of tamoxifen in calcium signaling and calcium-induced cell death was studied in both malignant glioma cell lines and MCF-7 breast cancer cells. Tamoxifen potently increased the spatial expansion of calcium waves by 30-150% while significantly enhancing and prolonging agonist-induced calcium elevations. Furthermore, tamoxifen pretreatment accelerated calcium ionophore-induced death by more than 20 min, suggesting that tamoxifen lowered cellular resistance to calcium loads. In contrast to its potentiating of calcium signaling in tumors, tamoxifen had no significant effect on calcium signaling in cultures of primary astrocytes from either human or rat brain. This study demonstrates the existence of calcium signaling in breast cancer and glioma cells and identifies tamoxifen as a potential modulator of tumor-associated calcium signaling.

Adenosine Triphosphate↗

PubMed2000

ATP-mediated glia signaling.

Glia calcium signaling has recently been identified as a potent modulator of synaptic transmission. We show here that the spatial expansion of calcium waves is mediated by ATP and subsequent activation of purinergic receptors. Ectopic expression of gap junction proteins, connexins (Cxs), leads to an increase in both ATP release and the radius of calcium wave propagation. Cx expression was also associated with a phenotypic transformation, and cortical neurons extended longer neurites when co-cultured with Cx-expressing than with Cx-deficient cells. Purinergic receptor activation mediated both these effects, because treatment with receptor antagonists restored the glia phenotype and slowed neurite outgrowth. These results identify a key role of ATP in both short-term calcium signaling events and in long-term differentiation regulated by glia.

Adenosine Triphosphate↗

PubMed2000

Paired-pulse modulation at individual GABAergic synapses in rat hippocampus.

1. Unitary inhibitory postsynaptic currents (uIPSCs) were recorded in synaptically coupled pairs of CA1 hippocampal interneurons and pyramidal neurons in rat brain slices by using dual patch-clamp techniques. Paired-pulse modulation of uIPSCs at individual GABAergic synapses was tested. 2. GABAergic synapses could be divided into two subgroups, high and low failure, depending on their failure rate. 3. The external Ca2+ levels modulate the failure rate of uIPSCs. In 0.51 mM Ca2+, low-failure pairs had a high-failure characteristic, whereas high-failure pairs had a low-failure characteristic in 8 mM Ca2+. The results suggest that uIPSC failures result from the Ca2+-dependent release mechanism rather than axon propagation failures. 4. Paired-pulse facilitation (PPF) occurred in high-failure pairs when the interspike interval was 20 ms. Paired-pulse depression (PPD) was not predominant in high-failure pairs. 5. Potency of uIPSCs, the average amplitude of non-failure events, was enhanced by PPF, suggesting that multiple synapses connect each pair. Differing numbers of activated synapses contributed to the variable amplitude of uIPSCs from a given pair. 6. PPD occurred in low-failure pairs at the tested range of interspike intervals (20-200 ms). The uIPSC2 after a large uIPSC1 was smaller than the uIPSC2 after a small uIPSC1, suggesting that PPD is use dependent and due to a decrease in the quantal content (m) after the first release. 7. In 8 mM Ca2+, PPD occurred in high-failure pairs and was larger in low-failure pairs, suggesting that the occurrence of PPF or PPD depends on the baseline release probability. 8. The GABAB receptor antagonist CGP 55845A (5 microM) decreased the baseline release probability of inhibitory synapses and attenuated PPD indirectly, rather than by blocking presynaptic GABAB autoreceptors.

Animals↗

PubMed2000

Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone.

Adult humans, like their nonhuman mammalian counterparts, harbor persistent neural progenitor cells in the forebrain ventricular lining. In the absence of adequate surface markers, however, these cells have proven difficult to isolate for study. We have previously identified and selected neural progenitor cells from both the fetal and adult rodent ventricular zone (VZ), by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1:hGFP). We have now extended this approach by purifying both P/Talpha1:hGFP tubulin-defined neuronal progenitors, as well as potentially less committed E/nestin:hGFP-defined neural progenitor cells, from the adult human VZ. The ventricular wall of the temporal horn of the lateral ventricle was dissected from temporal lobes obtained from four adult patients undergoing therapeutic lobectomy. These samples were dissociated, and the cultured cells transduced with either P/Talpha1:hGFP or E/nestin:EGFP plasmid DNA. A week later, the cells were redissociated, selected via fluorescence-activated cell sorting (FACS) on the basis of neural promoter-driven GFP expression, and replated. The majority of these cells expressed the early neuronal protein betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically evident neurons that coexpressed betaIII-tubulin and microtubule-associated protein (MAP)-2. Many of these neurons had incorporated bromodeoxyuridine in vitro in the days before FACS, indicating their mitogenesis in vitro. Thus, the use of fluorescent transgenes under the control of early neural promoters permits the enrichment of neuronal progenitor cells from the adult human ventricular zone. The specific acquisition, in both purity and number, of residual neural progenitor cells from the adult human brain may now permit hitherto unfeasible studies of both their biology and practical application.

Adult↗

PubMed2000

Perspectives on spreading depression.

Spreading depression (SD) consists of a transient suppression of all neuronal activity that spreads slowly across regions of gray matter. The paper is divided into three parts. Martins-Ferreira describes 30 years of research on SD in the isolated retina. Much of this work has relied on the prominent intrinsic optical signals that accompany SD in the retina. By inducing SD to propagate in circles with a velocity of 3.7 mm min(-1), it is possible to investigate the finely balanced electrochemical equilibrium that maintains the traveling wave. SD is accompanied by a slow negative extracellular voltage and ion movements that are greatest in the inner plexiform layer of the retina. Nedergaard discusses the role of astrocytes in SD propagation. Astrocytes mediate slowly moving waves of intracellular Ca(2+) increase, for which gap junctions are essential. SD is accompanied by entry of Ca(2+) into cells and fails when gap junctions are blocked. SD, however, is blocked by glutamate receptor antagonists but glial Ca(2+) waves are not. Astrocytic Ca(2+) waves are probably involved in the initiation of SD but other factors, including K(+), glutamate and purinergic receptors, are necessary for sustained propagation. Nicholson describes studies on the different preparations that helped clarify the role of extracellular space in SD. It has long been known that extracellular K(+) reaches levels of 50 mM or more during SD. Studies with ion-selective microelectrodes showed that extracellular Na(+) and Cl(-) fall by as much as 100 mM during SD, and water leaves the extracellular space. Further work showed that extracellular Ca(2+) falls 10-fold during SD and significant changes in extracellular pH and ascorbate occur. These studies imply that large perturbations of the extracellular milieu occur during SD and are an essential part of the interlocking cascade of events that produce this still mysterious phenomenon.

Animals↗

PubMed2000

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Adult↗

PubMed2000

Meningeal cells can communicate with astrocytes by calcium signaling.

Mechanical stimulation of adult human and rat pia-arachnoid cell cultures (loaded with calcium indicator dye) produced an increase in calcium in the stimulated cell. This change then propagated rapidly among neighboring cells, producing a calcium wave with a maximum distance of propagation and velocity resembling calcium waves in astrocytes. The pia-arachnoid waves were blocked by either octanol or apyrase, suggesting that propagation might occur either by gap junction communication or extracellular movement of ATP. Calcium waves in pia-arachnoid cells could invade contiguous astrocytes, and vice versa. Gap junction coupling between pia-arachnoid cells and astrocytes was shown by dye transfer experiments, in conjunction with immunostaining for connexin43. We infer that calcium signals from cells in the cortical parenchyma may be transmitted to the pia-arachnoid and might then serve in the induction of neurovascular changes, including those postulated to be responsible for the pain of migraine headache.

Astrocytes↗

PubMed1999

Direct gap junction communication between malignant glioma cells and astrocytes.

Gap junctions are intercellular channels that connect the interiors of coupled cells. We sought to determine the extent to which malignant glioma cells form gap junction channels with astrocytes from either adult human brain or rat forebrain. The astrocytic gap junction protein, connexin 43 (Cx43), was identified in immunoreactive plaques at areas of cell-to-cell contact between cocultured glioma cells and astrocytes. These gap junction plaques were composed of functional channels, because extensive dye coupling was evident between the glioma cells and astrocytes from both human and rat brain. Calcium signaling was also readily transmitted from glioma cells to astrocytes and vice versa. In live rat brain, injection of glioma cells prelabeled with the gap junction tracer, dicarboxy-dichlorofluorescein, revealed extensive dye transfer to host cells, demonstrating that malignant glioma cells directly couple with normal brain cells. These observations suggest that intercellular communication via gap junctions may play a role in regulating cellular interactions during tumor invasion. In fact, the presence of gap junctions between astrocytes and glioma cells was sufficient to induce a transformation of astrocytic phenotype. Astrocytes cocultured with C6 glioma cells overexpressing Cx43 were significantly smaller and expressed a lower level of glial fibrillary acidic protein than astrocytes cocultured with otherwise identical mock-transfected, gap junction-deficient C6 cells. Thus, direct cellular coupling with glioma cells result in a phenotypic transformation of astrocytes that may contribute to the susceptibility of surrounding tissue to glioma invasion.

Adult↗

PubMed1999

Glucocorticoids-potent modulators of astrocytic calcium signaling.

Glucocorticoids are the first line of choice in the treatment of cerebral edema associated with brain tumors. High-dose glucocorticoids reduce the extent of edema within hours, often relieving critical increases in intracranial pressure, but the mechanisms by which glucocorticoids modulate brain water content are not well-understood. A possible target of action may be glucocorticoid receptor-expressing astrocytes, which are the primary regulators of interstitial ion homeostasis in brain. In this study, we demonstrate that two glucocorticoids, methylprednisolone and dexamethasone, potentiate astrocytic signaling, via long-range calcium waves. Glucocorticoid treatment increased both resting cytosolic calcium (Ca2+i) level and the extent and amplitude of Ca2+ wave propagation two-fold, compared to matched controls. RU-486, a potent steroid receptor antagonist, inhibited the effects of methylprednisolone. The glucocorticoid-associated potentiation of Ca2+ signaling may result from upregulation of the cellular ability to mobilize Ca2+ and release ATP, because both agonist-induced Ca2+i increments (via ATP and bradykinin) and ATP release were proportionally enhanced by glucocorticoids. In contrast, neither gap junction expression (as manifested connexin 43 immunoreactivity) nor functional coupling was significantly affected by methylprednisolone. Confocal microscopy revealed both the expression of glucocorticoid receptors and nuclear translocation of these receptors when exposed to methylprednisolone. We postulate that the edemolytic effects of glucocorticoids may result from enhanced astrocytic calcium signaling.

Adenosine Triphosphate↗

PubMed1999

Estrogen promotes the initial migration and inception of NgCAM-dependent calcium-signaling by new neurons of the adult songbird brain.

The adult avian forebrain continues to generate neurons from ventricular zone (VZ) precursor cells, whose neuronal progeny then migrate into the brain parenchyma. Migrating neurons respond to the Ig-family adhesion molecule NgCAM with increments in cytosolic calcium, and migration is disrupted by anti-NgCAM Ig. The calcium response to NgCAM is developmentally restricted to bipolar migrants during a period spanning 6 to 9 DIV. This period corresponds to the postmitotic age at which new neurons leave the adult VZ to traverse a subjacent layer of estrogen-receptive "gatekeeper" neurons. Since neuronal passage through this layer occurs concurrently with the onset of NgCAM-dependent calcium signaling, we asked whether acquisition of the calcium response to NgCAM required estrogen exposure. Among neurons arising from explants of the adult finch neostriatal VZ, only those supplemented with estrogen developed calcium responses to NgCAM; neither explants raised in the absence of estrogen, nor those supplemented with testosterone, did so. Neurons in all three groups expressed NgCAM, had equivalent baseline calcium levels, and responded identically to K+-depolarization. Nonetheless, many more neurons migrated from explants of both finch and canary VZ raised in estrogen-supplemented media than from their estrogen-deprived counterparts, even though no effect of estrogen on neuronal survival per se was noted. These findings suggest that estrogen encourages the initial departure and assumption of signal competence by neurons arising from the adult avian VZ, thereby promoting their parenchymal recruitment and migration success.

Animals↗

PubMed1998

Connexins regulate calcium signaling by controlling ATP release.

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl- channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl- channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.

Adenosine Triphosphatases↗

PubMed1998

Cytoskeletal assembly and ATP release regulate astrocytic calcium signaling.

We have studied the role of actin fiber assembly on calcium signaling in astrocytes. We found that (1) after astrocytes have been placed in culture, it takes several hours for organization of the definitive actin cytoskeleton. Actin organization and the number of cells engaged in calcium signaling increased in parallel. (2) Disruption of the actin cytoskeleton attenuated the calcium wave propagation; cytochalasin D treatment reduced the number of astrocytes engaged in calcium signaling. (3) Propagation of calcium waves depends on cytoskeletal function; inhibition of myosin light chain kinase suppressed wave activity. (4) Astrocytic calcium signaling is mediated by release of ATP and purinergic receptor stimulation, because agents that interfere with this cascade attenuated or reduced calcium signaling. Because purinergic receptors are fully functional shortly after plating and not affected by cytochalasin D, these observations indicate that cytoskeleton organization is a prerequisite for interastrocytic calcium signaling mediated by release of ATP.

Actins↗

PubMed1998

Astrocytic gap junctions remain open during ischemic conditions.

Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca2+ and inositol triphosphate (IP3) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.

Animals↗

PubMed1998

Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells.

The adult mammalian forebrain harbors neuronal precursor cells in the subependymal zone (SZ). Neuronal progenitors also persist in the adult human SZ and have been cultured from epileptic temporal lobe. In the present study, we sought to identify these neural progenitors in situ, and to direct their expansion and neuronal differentiation in vitro. We prepared explants of adult human SZ, obtained from temporal lobe resections of refractory epileptics. The resultant cultures were treated with fibroblast growth factor-2 (FGF-2) for a week, with concurrent exposure to [3H]thymidine, then switched to media containing brain-derived neurotrophic factor (BDNF) for up to 2 months. Sporadic neuronal outgrowth, verified antigenically and physiologically, was observed from SZ cultures regardless of FGF-2/BDNF treatment; however, only FGF-2/BDNF-treated cultures exhibited profuse outgrowth, and these displayed neuronal survival as long as 9 weeks in vitro. In addition, cortical cultures derived from two brains generated microtubule-associated protein-2+ neurons, which incorporated [3H]thymidine and exhibited significant calcium increments to depolarization. In histological sections of the subependyma, both uncommitted and restricted progenitors, defined respectively by musashi and Hu protein expression, were identified. Thus, the adult human subependyma harbors neural progenitors, which are able to give rise to neurons whose numbers can be supported for prolonged periods in vitro.

Adult↗

PubMed1998

Gap-junction-mediated propagation and amplification of cell injury.

Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.

Animals↗