The synapse specificity of long-term potentiation (LTP) ensures that no interference arises from inputs irrelevant to the memory to be encoded. In hippocampi of aged (21-28 months) mice, LTP was ...relayed to unstimulated synapses, blemishing its synapse specificity. Diminished levels of the K(+)/Cl(-) cotransporter KCC2 and a depolarizing GABAA receptor-mediated synaptic component following LTP were the most likely causes for the spreading of potentiation, unveiling mechanisms hindering information storage in the aged brain and identifying KCC2 as a potential target for intervention.
Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant ...huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K(+) channel functional expression, leading to elevated in vivo striatal extracellular K(+), which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K(+), ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K(+) homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is ...necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.
Transcranial ultrasound stimulation (tUS) shows potential as a noninvasive brain stimulation (NIBS) technique, offering increased spatial precision compared to other NIBS techniques. However, its ...reported effects on primary motor cortex (M1) are limited. We aimed to better understand tUS effects in human M1 by performing tUS of the hand area of M1 (M1hand) during tonic muscle contraction of the index finger. Stimulation during muscle contraction was chosen because of the transcranial magnetic stimulation-induced phenomenon known as cortical silent period (cSP), in which transcranial magnetic stimulation (TMS) of M1hand involuntarily suppresses voluntary motor activity. Since cSP is widely considered an inhibitory phenomenon, it presents an ideal parallel for tUS, which has often been proposed to preferentially influence inhibitory interneurons. Recording electromyography (EMG) of the first dorsal interosseous (FDI) muscle, we investigated effects on muscle activity both during and after tUS. We found no change in FDI EMG activity concurrent with tUS stimulation. Using single-pulse TMS, we found no difference in M1 excitability before versus after sparsely repetitive tUS exposure. Using acoustic simulations in models made from structural MRI of the participants that matched the experimental setups, we estimated in-brain pressures and generated an estimate of cumulative tUS exposure experienced by M1hand for each subject. We were unable to find any correlation between cumulative M1hand exposure and M1 excitability change. We also present data that suggest a TMS-induced MEP always preceded a near-threshold cSP.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many forms of signal transduction occur when Ca(2+) enters the cytoplasm of a cell. It has been generally thought that there is a fast buffer that rapidly reduces the free Ca(2+) level and that it is ...this buffered level of Ca(2+) that triggers downstream biochemical processes, notably the activation of calmodulin (CaM) and the resulting activation of CaM-dependent enzymes. Given the importance of these transduction processes, it is crucial to understand exactly how Ca(2+) activates CaM. We have determined the rate at which Ca(2+) binds to CaM and found that Ca(2+) binds more rapidly to CaM than to other Ca(2+)-binding proteins. This property of CaM and its high concentration support a new view of signal transduction: CaM directly intercepts incoming Ca(2+) and sets the free Ca(2+) level (that is, it strongly contributes to fast Ca(2+) buffering) rather than responding to the lower Ca(2+) level set by other buffers. This property is crucial for making CaM an efficient transducer. Our results also suggest that other Ca(2+) binding proteins have a previously undescribed role in regulating the lifetime of Ca(2+) bound to CaM and thereby setting the gain of signal transduction.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most ...genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.
Objective
The objective of this study is to characterize the effects of the sleep‐wake cycle on neurovascular and behavioral characteristics of cortical spreading depression (CSD).
Background
There ...is an important bi‐directional relationship between migraine and the sleep–wake cycle, but the basic mechanisms of this relationship are poorly understood.
Methods
We have developed a minimally invasive microchip system to continuously monitor cerebral blood volume (CBV) with optical intrinsic signal (OIS), head movement, and multiple other physiological and behavioral parameters in freely behaving mice over weeks. Behavior is also monitored with simultaneous video recording. This system can also be used to intermittently trigger and record CSD and accompanying neurovascular and behavioral responses. CSD was triggered optically in different stages of the sleep–wake cycle.
Results
The optical stimulus threshold to trigger CSD was significantly higher in the wake state compared to sleep (stimulation duration = 16.4 ± 9.7 s vs. 10.8 ± 5.8 s, p = 0.037, n = 6 mice). CSD evoked in the wake versus sleep state produced changes in CBV that were smaller (largest relative change –4.5 ± 5.0% ∆OIS vs. –14.3 ± 8.5% ∆OIS, p = 0.001) and shorter in duration (33:22 ± 6:37 vs. 49:42 ± 8:05 min:s, p = 0.012, n = 6 mice). The threshold for CSD and kinetics of associated CBV changes were correlated with the time since falling asleep or awakening (n = 47 CSDs in 6 mice). CSD triggered in the wake state was associated with a transient freezing behavior. CSD triggered during sleep typically caused a transient awakening and behavioral response. This was followed by a return to sleep until recovery from the sustained phase of decreased CBV that occurred 30–60 min later, at which time there was consistent awakening with behaviors similar to those that occurred at CSD onset. CSD triggered in the wake state evoked a transient decrease in heart rate (from 11.9 ± 0.8 to 9.6 ± 0.8 Hz, p = 0.002, n = 5), whereas when triggered in the sleep state there was a transient increase in HR (from 7.5 ± 0.4 Hz to 9.3 ± 1.1 Hz, p = 0.016, n = 5).
Conclusions
The sleep–wake cycle has significant effects on CSD that may have relevance to the clinical presentations of migraine and brain injury.
A minimally invasive, microchip‐based approach enables continuous long‐term recording of brain neurovascular activity, heart rate, and head movement in freely behaving rodents. This approach can also ...be used for transcranial optical triggering of cortical activity in mice expressing channelrhodopsin. The system uses optical intrinsic signal recording to measure cerebral blood volume, which under baseline conditions is correlated with spontaneous neuronal activity. The arterial pulse and breathing can be quantified as a component of the optical intrinsic signal. Multi‐directional head movement is measured simultaneously with a movement sensor. A separate movement tracking element through a camera enables precise mapping of overall movement within an enclosure. Data is processed by a dedicated single board computer, and streamed from multiple enclosures to a central server, enabling simultaneous remote monitoring and triggering in many subjects. One application of this system described here is the characterization of changes in of cerebral blood volume, heart rate and behaviour that occur with the sleep‐wake cycle over weeks. Another application is optical triggering and recording of cortical spreading depression (CSD), the slowly propagated wave of neurovascular activity that occurs in the setting of brain injury and migraine aura. The neurovascular features of CSD are remarkably different in the awake vs. anaesthetized state in the same mouse. With its capacity to continuously and synchronously record multiple types of physiological and behavioural data over extended time periods in combination with intermittent triggering of brain activity, this inexpensive method has the potential for widespread practical application in rodent research.
Key points
Recording and triggering of brain activity in mice and rats has typically required breaching the skull, and experiments are often performed under anaesthesia
A minimally invasive microchip system enables continuous recording and triggering of neurovascular activity, and analysis of heart rate and behaviour in freely behaving rodents over weeks
This system can be used to characterize physiological and behavioural changes associated with the sleep‐wake cycle over extended time periods
This approach can also be used with mice expressing channelrhodopsin to trigger and record cortical spreading depression (CSD) in freely behaving subjects. The neurovascular responses to CSD are remarkably different under anaesthesia compared with the awake state.
The method is inexpensive and straightforward to employ at a relatively large scale. It enables translational investigation of a wide range of physiological and pathological conditions in rodent models of neurological and systemic diseases.
figure legend Permanent head‐fixed microelectronics to record and stimulate inter‐cranial physiology
Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand ...binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-mus rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions ( approximately 100 nM) to approximately 1 muM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Calcium-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. They are the fastest players within the Ca2+ toolkit responding within microseconds to Ca2+ changes. The CBPs ...compete for Ca2+ which plays a direct role in modulating Ca2+ transients and the resulting biochemical message. The kinetic properties of the CBPs have to be known to have a good understanding of Ca2+ signaling.
Most techniques used to measure binding kinetics are too slow to accurately determine the fast kinetics of most CBP. Furthermore, many CBPs bind Ca2+ in a cooperative way, which should be incorporated in the kinetic modeling. Here we will review a new ultra-fast in vitro technique for measuring Ca2+ binding properties of CBPs following flash photolysis of caged Ca2+. Compartmental modeling is used to resolve the kinetics of fast cooperative Ca2+ binding to CBPs.
Currently this technique has only been used to quantify the kinetics of three CBPs (calbindin, calretinin and calmodulin), but has already provided remarkable insights into the specific role that these kinetics in Ca2+ signaling.
The potential to gain novel insights into Ca2+ signaling by quantifying kinetics of other CBPs using this technique is very promising. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.
► Kinetics of Ca2+ binding proteins (CBPs) are too fast to gauge with normal methods. ► The fast kinetics of CBPs can be determined using flash photolysis. ► Cooperativity should be included when quantifying binding kinetics of CBPs. ► The binding kinetics of CBPs play a key role in calcium signaling.
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