Christine R. Rose

Neurotrophin-evoked rapid excitation through TrkB receptors

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells.

The neurotrophin receptor TrkB is essential for normal function of the mammalian brain. It is expressed in three splice variants. Full-length receptors (TrkBFL) possess an intracellular tyrosine kinase domain and are considered as those TrkB receptors that mediate the crucial effects of brain-derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5). By contrast, truncated receptors (TrkB-T1 and TrkB-T2) lack tyrosine kinase activity and have not been reported to elicit rapid intracellular signalling. Here we show that astrocytes predominately express TrkB-T1 and respond to brief application of BDNF by releasing calcium from intracellular stores. The calcium transients are insensitive to the tyrosine kinase blocker K-252a and persist in mutant mice lacking TrkBFL. By contrast, neurons produce rapid BDNF-evoked signals through TrkBFL and the Nav1.9 channel. Expression of antisense TrkB messenger RNA strongly reduces BDNF-evoked calcium signals in glia. Thus, our results show that, unexpectedly, TrkB-T1 has a direct signalling role in mediating inositol-1,4,5-trisphosphate-dependent calcium release; in addition, they identify a previously unknown mechanism of neurotrophin action in the brain.

pH regulation and proton signalling by glial cells

The regulation of H+ in nervous systems is a function of several processes, including H+ buffering, intracellular H+ sequestering, CO2 diffusion, carbonic anhydrase activity and membrane transport of acid/base equivalents across the cell membrane. Glial cells participate in all these processes and therefore play a prominent role in shaping acid/base shifts in nervous systems. Apart from a homeostatic function of H(+)-regulating mechanisms, pH transients occur in all three compartments of nervous tissue, neurones, glial cells and extracellular spaces (ECS), in response to neuronal stimulation, to neurotransmitters and hormones as well as secondary to metabolic activity and ionic membrane transport. A pivotal role for H+ regulation and shaping these pH transients must be assigned to the electrogenic and reversible Na(+)-HCO3-membrane cotransport, which appears to be unique to glial cells in nervous systems. Activation of this cotransporter results in the release and uptake of base equivalents by glial cells, processes which are dependent on the glial membrane potential. Na+/H+ and Cl-/HCO3-exchange, and possibly other membrane carriers, accomplish the set of tools in both glial cells and neurones to regulate their intracellular pH. Due to the pH dependence of a great variety of processes, including ion channel gating and conductances, synaptic transmission, intercellular communication via gap junctions, metabolite exchange and neuronal excitability, rapid and local pH transients may have signalling character for the information processing in nervous tissue. The impact of H+ signalling under both physiological and pathophysiological conditions will be discussed for a variety of nervous system functions.

Stores Not Just for Storage: Intracellular Calcium Release and Synaptic Plasticity

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.

INTRACELLULAR SODIUM HOMEOSTASIS IN RAT HIPPOCAMPAL ASTROCYTES

1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI‐AM (acetoxymethylester of sodium‐binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the ‘Na+’ signal. 2. Baseline [Na+]i was 14.6 +/‐ 4.9 mM (mean +/‐ S.D.) in CO2/HCO3(‐)‐containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/‐ 0.32 mM min‐1) followed by a slower phase (0.15 +/‐ 0.09 mM min‐1). 3. Changing from CO2/HCO3(‐)‐free to CO2/HCO3(‐)‐buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)‐HCO3‐ cotransport by CO2/HCO3‐. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)‐K(+)‐2Cl‐ cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage‐gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)‐ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min‐1, respectively. This indicated that Na+, K(+)‐ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o‐induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/‐ 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)‐HCO3‐ cotransport, Na(+)‐K(+)‐2Cl‐ cotransport and Na+, K(+)‐ATPase, and that both Na+, K(+)‐ATPase and inward Na(+)‐K(+)‐2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.

Polybrominated diphenyl ethers induce developmental neurotoxicity in a human in vitro model: evidence for endocrine disruption.

Background Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic. Objective Because there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs. Methods We used the two PBDE congeners BDE-47 and BDE-99 (0.1–10 μM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation. Results PBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect. Conclusion PBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Calbindin in Cerebellar Purkinje Cells Is a Critical Determinant of the Precision of Motor Coordination

Long-term depression (LTD) of Purkinje cell–parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor–IP3–calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.

Regulation of intracellular sodium in cultured rat hippocampal neurones.

1. We studied regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium‐binding benzofuran isophthalate). 2. In standard CO2/HCO3(‐)‐buffered saline with 3 mM K+, neurones had a baseline [Na+]i of 8.9 +/‐ 3.8 mM (mean +/‐ S.D.). Spontaneous, transient [Na+]i increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+]i increases were often synchronized among nearby neurones and were blocked reversibly by 1 microM tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage‐gated Na+ channels by application of 50 microM veratridine caused a TTX‐sensitive [Na+]i increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+]i to values close to zero within 10 min. Inhibition of Na+,K(+)‐ATPase by removal of extracellular K+ or ouabain application evoked a [Na+]i increase of 5 mM min‐1. Baseline [Na+]i was similar in the presence or absence of CO2/HCO3‐; switching from CO2/HCO3(‐)‐free to CO2/HCO3(‐)‐buffered saline, however, increased [Na+]i transiently by 3 mM, indicating activation of Na(+)‐dependent Cl(‐)‐HCO3‐ exchange. Inhibition of Na(+)‐K(+)‐2Cl‐ cotransport by bumetanide had no effect on [Na+]i. 4. Brief, small changes in extracellular K+ concentration ([K+]o) influenced neuronal [Na+]i only weakly. Virtually no change in [Na+]i was observed with elevation or reduction of [K+]o by 1 mM. Only 30% of cells reacted to 3 min [K+]o elevations of up to 5 mM. In contrast, long [K+]o alterations (> or = 10 min) to 6 mM or greater slowly changed steady‐state [Na+]i in the majority of cells. 5. Our results indicate several differences between [Na+]i regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+]i is lower in neurones compared with astrocytes and is mainly determined by Na+,K(+)‐ATPase, whereas Na(+)‐dependent Cl(‐)‐HCO3‐ exchange, Na(+)‐HCO3‐ cotransport or Na(+)‐K(+)‐2Cl‐ cotransport do not play a significant role. In contrast to glial cells, [Na+]i of neurones changes only weakly with small alterations in bath [K+]o, suggesting that activity‐induced [K+]o changes in the brain might not significantly influence neuronal Na+,K(+)‐ATPase activity.

Developmental profile and properties of sulforhodamine 101—Labeled glial cells in acute brain slices of rat hippocampus

The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31-7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24-27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na+ currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.

Functional Reconstitution of Vascular Smooth Muscle Cells With cGMP-Dependent Protein Kinase I Isoforms

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKI&agr; and cGKI&bgr;, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca2+ concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKI&agr; and cGKI&bgr;. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca2+ transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKI&agr; and cGKI&bgr;. The defective Ca2+ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKI&agr; and enhanced green fluorescent protein (EGFP) but not by a cGKI&bgr;-EGFP construct. Fluorescence imaging indicated that the cGKI&agr;-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKI&bgr;-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKI&agr; isoform, which decreases the noradrenaline-stimulated cytosolic Ca2+ level.