Probe-type near-field confocal having feedback for adjusting probe distance

A method and apparatus for achieving optical microscopic images and monitoring metabolic processes of living biological specimens such as cells at a resolution superior to the diffraction limit is disclosed. A primary difficulty in performing near-field scanning optical microscopy of living cells, e.g., determining the separation between the cell surface and the illuminating probe tip, is overcome by using a photon-density feedback method in which a fluorescent dye signal strength is monitored in the cell as the tip is brought to the cell surface, and registering a maximum value, at which point the tip begins to dimple the cell surface and can get no closer to the dye. Thereafter the tip is either maintained in contact with the membrane for point measurements of metabolic processes or is withdrawn a selected distance from the surface as measured against a corresponding decrease in the fluorescent dye signal strength The signal strength serves as a photon-density feedback for maintaining the probe tip at a constant elevation above the cell surface as scanning is performed or time-series measurements of metabolism are recorded. Advantageously, the apparatus also combines confocal means in the form of a pin-hole or the like for high-fidelity light detection in three dimensions from the cell surface in the near-field of the probe tip

Wakefulness affects synaptic and network activity by increasing extracellular astrocyte-derived adenosine

Loss of sleep causes an increase in sleep drive and deficits in hippocampal-dependent memory. Both of these responses are thought to require activation of adenosine A1 receptors (adorA1Rs) and release of transmitter molecules including ATP, which is rapidly converted to adenosine in the extracellular space, from astrocytes in a process termed gliotransmission. Although it is increasingly clear that astrocyte-derived adenosine plays an important role in driving the homeostatic sleep response and the effects of sleep loss on memory (Halassa et al., 2009; Florian et al., 2011), previous studies have not determined whether the concentration of this signaling molecule increases in response to wakefulness. Here, we show that the level of adorA1R activation increases in response to wakefulness in mice (Mus musculus). We found that this increase affected synaptic transmission in the hippocampus and modulated network activity in the cortex. Direct biosensor-based measurement of adenosine showed that the net extracellular concentration of this transmitter increased in response to normal wakefulness and sleep deprivation. Genetic inhibition of gliotransmission prevented this increase and attenuated the wakefulness-dependent changes in synaptic and network regulation by adorA1R. Consequently, we conclude that wakefulness increases the level of extracellular adenosine in the hippocampus and that this increase requires the release of transmitters from astroctyes

Gliotransmission modulates baseline mechanical nociception

Pain is a physiological and adaptive process which occurs to protect organisms from tissue damage and extended injury. Pain sensation beyond injury, however, is a pathological process which is poorly understood. Experimental models of neuropathic pain demonstrate that reactive astrocytes contribute to reduced nociceptive thresholds. Astrocytes release "gliotransmitters" such as D-serine, glutamate, and ATP, which is extracellularly hydrolyzed to adenosine. Adenosine 1 receptor activation in the spinal cord has anti-nociceptive effects on baseline pain threshold, but the source of the endogenous ligand (adenosine) in the spinal cord is unknown. In this study we used a transgenic mouse model in which SNARE-mediated gliotransmission was selectively attenuated (called dnSNARE mice) to investigate the role of astrocytes in mediating baseline nociception and the development of neuropathic pain. Under baseline conditions, immunostaining in the dorsal horn of the spinal cord showed astrocyte-specific transgene expression in dnSNARE mice, and no difference in expression levels of the astrocyte marker GFAP and the microglia marker Iba1 relative to wild-type mice. The Von Frey filament test was used to probe sensitivity to baseline mechanical pain thresholds and allodynia following the spared nerve injury model of neuropathic pain. DnSNARE mice exhibit a reduced nociceptive threshold in response to mechanical stimulation compared to wild-type mice under baseline conditions, but nociceptive thresholds following spared nerve injury were similar between dnSNARE and wild-types. This study is the first to provide evidence that gliotransmission contributes to basal mechanical nociception

Gliotransmission modulates baseline mechanical nociception

Abstract Pain is a physiological and adaptive process which occurs to protect organisms from tissue damage and extended injury. Pain sensation beyond injury, however, is a pathological process which is poorly understood. Experimental models of neuropathic pain demonstrate that reactive astrocytes contribute to reduced nociceptive thresholds. Astrocytes release "gliotransmitters" such as D-serine, glutamate, and ATP, which is extracellularly hydrolyzed to adenosine. Adenosine 1 receptor activation in the spinal cord has anti-nociceptive effects on baseline pain threshold, but the source of the endogenous ligand (adenosine) in the spinal cord is unknown. In this study we used a transgenic mouse model in which SNARE-mediated gliotransmission was selectively attenuated (called dnSNARE mice) to investigate the role of astrocytes in mediating baseline nociception and the development of neuropathic pain. Under baseline conditions, immunostaining in the dorsal horn of the spinal cord showed astrocyte-specific transgene expression in dnSNARE mice, and no difference in expression levels of the astrocyte marker GFAP and the microglia marker Iba1 relative to wild-type mice. The Von Frey filament test was used to probe sensitivity to baseline mechanical pain thresholds and allodynia following the spared nerve injury model of neuropathic pain. DnSNARE mice exhibit a reduced nociceptive threshold in response to mechanical stimulation compared to wild-type mice under baseline conditions, but nociceptive thresholds following spared nerve injury were similar between dnSNARE and wild-types. This study is the first to provide evidence that gliotransmission contributes to basal mechanical nociception. Keywords: Adenosine, Astrocyte, Gliotransmission, Pain Findings Pain sensation is an adaptive response to impending tissue damage that protects an organism from extended injury. Pain perception involves a series of cellular interactions and responses from immune cells, glia and neurons. Signals from glial cells trigger neuronal responses, and vice versa, initiating a complex cascade of cell-cell interactions and feedback mechanisms Acute pain stimuli excite primary nociceptive neurons, which synapse and release glutamate and substance-P onto postsynaptic neurons in the dorsal horn of the spinal cord. Under chronic pain conditions, this synapse exhibits an LTP-like state where increased responses from dorsal horn neurons are elicited by afferent stimulatio

Enhanced Astrocytic Ca\u3csup\u3e2+\u3c/sup\u3e Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus.We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOSTM clones with insert sizes ~20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter b-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain's computational power and behavioural output. These more active functions are endowed by the Ca2+-based excitability displayed by astrocytes. An increase in cytosolic Ca2+ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca2+ dynamics, Ca2+-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca2+ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca2+ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy

Integrating Hebbian and homeostatic plasticity: the current state of the field and future research directions

We summarize here the results presented and subsequent discussion from the meeting on Integrating Hebbian and Homeostatic Plasticity at the Royal Society in April 2016. We first outline the major themes and results presented at the meeting. We next provide a synopsis of the outstanding questions that emerged from the discussion at the end of the meeting and finally suggest potential directions of research that we believe are most promising to develop an understanding of how these two forms of plasticity interact to facilitate functional changes in the brain.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'

Population dynamics of a pathogen: the conundrum of vivax malaria

Building a mathematical model of population dynamics of pathogens within their host involves considerations of factors similar to those in ecology, as pathogens can prey on cells in the host. But within the multicellular host, attacked cell types are integrated with other cellular systems, which in turn intervene in the infection. For example, immune responses attempt to sense and then eliminate or contain pathogens, and homeostatic mechanisms try to compensate for cell loss. This review focuses on modeling applied to malarias, diseases caused by single-cell eukaryote parasites that infect red blood cells, with special concern given to vivax malaria, a disease often thought to be benign (if sometimes incapacitating) because the parasite only attacks a small proportion of red blood cells, the very youngest ones. However, I will use mathematical modeling to argue that depletion of this pool of red blood cells can be disastrous to the host if growth of the parasite is not vigorously check by host immune responses. Also, modeling can elucidate aspects of new field observations that indicate that vivax malaria is more dangerous than previously thought