A lifetime’s adventure in extracellular K+ regulation: the Scottish connection

In a career that has spanned 45 years and shows no signs of slowing down, Dr Bruce Ransom has devoted considerable time and energy to studying regulation of interstitial K+. When Bruce commenced his studies in 1969 virtually nothing was known of the functions of glial cells, but Bruce’s research contributed to the physiological assignation of function to mammalian astrocytes, namely interstitial K+ buffering. The experiments that I describe in this review concern the response of the membrane potential (Em) of in vivo cat cortical astrocytes to changes in [K+]o, an experimental manoeuvre that was achieved in two different ways. The first involved recording the Em of an astrocyte while the initial aCSF was switched to one with different K+, whereas in the second series of experiments the cortex was stimulated and the response of the astrocyte Em to the K+ released from neighbouring neurons was recorded. The astrocytes responded in a qualitatively predictable manner, but quantitatively the changes were not as predicted by the Nernst equation. Elevations in interstitial K+ are not sustained and K+ returns to baseline rapidly due to the buffering capacity of astrocytes, a phenomenon studied by Bruce, and his son Chris, published 27 years after Bruce’s initial publications. Thus, a lifetime spent investigating K+ buffering has seen enormous advances in glial research, from the time cells were identified as ‘presumed’ glial cells or ‘silent cells’, to the present day, where glial cells are recognised as contributing to every important physiological brain function

Synaptically evoked glutamate transporter currents in Spinal Dorsal Horn Astrocytes

<p>Abstract</p> <p>Background</p> <p>Removing and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na⁺ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents.</p> <p>Results</p> <p>Whole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG) area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd²⁺. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 – 1 Hz) repetitive stimulation.</p> <p>Conclusion</p> <p>These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.</p

Enhancement Effects of Martentoxin on Glioma BK Channel and BK Channel (α+β1) Subtypes

BACKGROUND: BK channels are usually activated by membrane depolarization and cytoplasmic Ca(2+). Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca(2+)-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca(2+) sensitivity than other known BK channel subtypes. METHODOLOGY AND PRINCIPAL FINDINGS: The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca(2+) imaging. In the presence of cytoplasmic Ca(2+), martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC(50) of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitative change of cytoplasmic Ca(2+) concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca(2+). The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca(2+), the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn't be affected by the toxin. CONCLUSIONS AND SIGNIFICANCE: Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca(2+)-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin

Functional Diffusion Tensor Imaging: Measuring Task-Related Fractional Anisotropy Changes in the Human Brain along White Matter Tracts

Functional neural networks in the human brain can be studied from correlations between activated gray matter regions measured with fMRI. However, while providing important information on gray matter activation, no information is gathered on the co-activity along white matter tracts in neural networks.We report on a functional diffusion tensor imaging (fDTI) method that measures task-related changes in fractional anisotropy (FA) along white matter tracts. We hypothesize that these fractional anisotropy changes relate to morphological changes of glial cells induced by axonal activity although the exact physiological underpinnings of the measured FA changes remain to be elucidated. As expected, these changes are very small as compared to the physiological noise and a reliable detection of the signal change would require a large number of measurements. However, a substantial increase in signal-to-noise ratio was achieved by pooling the signal over the complete fiber tract. Adopting such a tract-based statistics enabled us to measure the signal within a practically feasible time period. Activation in the sensory thalamocortical tract and optic radiation in eight healthy human subjects was found during tactile and visual stimulation, respectively.The results of our experiments indicate that these FA changes may serve as a functional contrast mechanism for white matter. This noninvasive fDTI method may provide a new approach to study functional neural networks in the human brain

Glial Hsp70 Protects K+ Homeostasis in the Drosophila Brain during Repetitive Anoxic Depolarization

Neural tissue is particularly vulnerable to metabolic stress and loss of ion homeostasis. Repetitive stress generally leads to more permanent dysfunction but the mechanisms underlying this progression are poorly understood. We investigated the effects of energetic compromise in Drosophila by targeting the Na+/K+-ATPase. Acute ouabain treatment of intact flies resulted in subsequent repetitive comas that led to death and were associated with transient loss of K+ homeostasis in the brain. Heat shock pre-conditioned flies were resistant to ouabain treatment. To control the timing of repeated loss of ion homeostasis we subjected flies to repetitive anoxia while recording extracellular [K+] in the brain. We show that targeted expression of the chaperone protein Hsp70 in glial cells delays a permanent loss of ion homeostasis associated with repetitive anoxic stress and suggest that this is a useful model for investigating molecular mechanisms of neuroprotection

Astrocytes convert network excitation to tonic inhibition of neurons

<p>Abstract</p> <p>Background</p> <p>Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.</p> <p>Results</p> <p>Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na⁺concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg²⁺] <it>in vitro </it>model of epilepsy. Under <it>in vivo </it>conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.</p> <p>Conclusions</p> <p>The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamat<it>ergic </it>excitation into GABA<it>ergic </it>inhibition providing an adjustable, <it>in situ </it>negative feedback on the excitability of neurons.</p

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words