Excitability of Vascular Smooth Muscle

Regulation of pressure and local blood flow occurs at the level of resistance arteries and arterioles. Under physiological conditions, these small vessels exist in a state of partial constriction, termed myogenic tone. Myogenic tone is considered to be an intrinsic property of arteriolar smooth muscle cells, which membranes depolarize in response to increase in the intraluminal pressure. Oscillations of membrane potential in smooth muscles are mediated by the activity of voltage-gated L-type Ca2+ channels, which provide an influx of Ca2+ to activate various voltage-gated and Ca2+-sensitive channels of smooth muscle cells and to initiate endothelial Ca2+ signaling needed for vasodilation. Although a relationship between change in membrane potential and myogenic response is considered to be universal throughout various smooth muscle tissues, it may be regulated differently based on autoregulatory responses and channels expression. Here we review electrophysiological signature of arteriolar smooth muscle in various tissues, with an emphases and specific examples of the excitability of 4th order arterioles isolated from skeletal muscle

Multichannel Silicon Probes for Awake Hippocampal Recordings in Large Animals

Decoding laminar information across deep brain structures and cortical regions is necessary in order to understand the neuronal ensembles that represent cognition and memory. Large animal models are essential for translational research due to their gyrencephalic neuroanatomy and significant white matter composition. A lack of long-length probes with appropriate stiffness allowing penetration to deeper structures with minimal damage to the neural interface is one of the major technical limitations to applying the approaches currently utilized in lower order animals to large animals. We therefore tested the performance of multichannel silicon probes of various solutions and designs that were developed specifically for large animal electrophysiology. Neurophysiological signals from dorsal hippocampus were recorded in chronically implanted awake behaving Yucatan pigs. Single units and local field potentials were analyzed to evaluate performance of given silicon probes over time. EDGE-style probes had the highest yields during intra-hippocampal recordings in pigs, making them the most suitable for chronic implantations and awake behavioral experimentation. In addition, the cross-sectional area of silicon probes was found to be a crucial determinant of silicon probe performance over time, potentially due to reduction of damage to the neural interface. Novel 64-channel EDGE-style probes tested acutely produced an optimal single unit separation and a denser sampling of the laminar structure, identifying these research silicon probes as potential candidates for chronic implantations. This study provides an analysis of multichannel silicon probes designed for large animal electrophysiology of deep laminar brain structures, and suggests that current designs are reaching the physical thresholds necessary for long-term (∼1 month) recordings with single-unit resolution

Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles

<div><p>Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4^th order arterioles in mouse skeletal muscle. Currents through L-type Ca²⁺ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca²⁺-free bathing solution or by addition of L-type Ca²⁺ channel blocker nifedipine (10 μM). Instead, Na⁺ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca²⁺ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na⁺ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca²⁺ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca²⁺ currents (15 pA/pF in 20 mM Ca²⁺), we also found Ca²⁺ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca²⁺). Based on their biophysical properties, these Ca²⁺ currents are likely to be through voltage-gated T-type Ca²⁺ channels. Our results suggest that Na⁺ and at least two types (T- and L-) of Ca²⁺ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na⁺ channels appear to be under a tight control by Ca²⁺ signaling.</p></div

Electrical properties of smooth muscle cells.

<p><b>A</b>, Skeletal muscle arterioles were loaded with 10 μM Fluo-4 and visualized under the microscope while gently pressed to a cover slip by a holding pipette prior to patch-clamp experiments (left panel). Fourth-order arterioles were indentified based on their morphology, with a single layer of smooth muscle cells (middle panel) and endothelial cells (right panel) oriented perpendicular to each other). <b>B</b>, Capacitive transients were measured in response to the voltage step from –70 to –60 mV. The electrically coupled cells (trace a) became uncoupled after treatment with the gap-junction inhibitory peptides during gramicidin-perforated patch clamp experiments (trace b-c) and after establishing the conventional whole-cell approach (trace d); <b>C</b>, Distribution of the resting membrane potential values was fitted by a single Gaussian function peaking at –77 ± 2 mV, n = 81 (smooth line). The average resting potential was V_rest = –68 ± 2 mV, n = 81.</p

TTX affected action potentials in the absence of extracellular Ca²⁺.

<p><b>A)</b> Hyperpolarizing current steps produced only passive voltage responses during gramicidin-perforated patch clamp experiments whereas depolarizing current steps induced action potentials starting from the threshold of about –50 mV. <b>B)</b> Effects of 1 μM TTX (n = 6, blue trace) and 10 μM nifedipine (n = 6, red trace) are shown in panel a at normal physiological conditions (2Ca solution, n = 10); in nominally Ca²⁺-free (0Ca, n = 4) solution, application of 1 μM TTX (0Ca + 1TTX, n = 4) had much greater effect as shown in panel b. <b>C)</b> Averaged effects of TTX and nifedipine on the maximal rate of upstroke (as recorded in Fig 2B). Maximal rates of upstrokes recorded in different solutions were normalized to that recorded in 2Ca. TTX and nifedipine significantly reduced the maximal rate of upstroke in 2Ca solution. The effect of TTX was significantly greater in 0Ca than in 2Ca solutions. Asterisks indicate significance of the difference from control values in the 2Ca solution (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

Tail currents of low-voltage Ca²⁺ channels are slow.

<p><b>A)</b> Test voltage protocol (top) and representative tail currents for pre-pulses to -20 mV (tracings a) and +30 mV (tracings b). <b>B)</b> Averaged time constants of tail currents recorded at different voltages (n = 4). <b>C</b>) The average amplitudes of tail currents recorded at different voltages (n = 4). Tail currents from -20 mV were fitted by a single exponential. Tail currents from 30 mV were fitted by the sum of two exponentials. Kinetics of the slow component elicited after pre-pulse to 30 mV was similar to the tail current elicited after pre-pulse to -20 mV.</p

TTX-sensitive Na⁺ currents.

<p><b>A)</b> Steps to different voltages (indicated) produced inward currents with at least two kinetically distinct components (2Ca solution, n = 6). The fast component was through voltage-gated Na⁺ channels as it was blocked by application of 1 μM TTX (n = 6). It was partially blocked by extracellular Ca²⁺ (10Ca solution, n = 6). The thick lines highlight the maximal currents recorded at 10 mV. <b>B)</b> Averaged peak current–voltage relationships of inward currents recorded in 2Ca (n = 6) and 10Ca (n = 4) bath solutions.</p

Two types of voltage-gated Ca²⁺ channels.

<p><b>A)</b> Whole-cell Ba²⁺ currents were recorded in the presence of 1μM TTX. Two kinetically different components were observed. Voltage steps up to –10 mV produced fast inactivating current (tracings a, n = 6). Further depolarization activated slow inactivating current of greater amplitude (tracings b, n = 6). <b>B)</b> In the presence of 10 μM L-type Ca²⁺ channel blocker nifedipine (n = 6), the rapidly activated component inactivated notably faster with Ba²⁺ (left panel) than with Ca²⁺ (right panel). <b>C)</b> Averaged peak current-voltage relationships determined in the presence of 1 μM TTX and 10 μM nifedipine as indicated. The nifedipine-resistant current peaked at about 0 mV. Its magnitude was nearly the same with Ba²⁺ and with Ca²⁺ (compare open and filled squares).</p

Inactivation of low-voltage activated Ca²⁺ currents.

<p><b>A)</b> 100 ms long pre-pulse to -20 mV inactivates Ca²⁺ current that peaks at –20 mV (tracings a) but not the one that peaks at +30 mV (tracings b). <b>B)</b> Voltage-dependence of inactivation of low- (filled circles) and high- (open circles) voltage- activated Ca²⁺ currents (n = 3). The smooth lines are the best fits by equation .</p

Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing

Summary: A number of mitochondrial diseases arise from single-nucleotide variant (SNV) accumulation in multiple mitochondria. Here, we present a method for identification of variants present at the single-mitochondrion level in individual mouse and human neuronal cells, allowing for extremely high-resolution study of mitochondrial mutation dynamics. We identified extensive heteroplasmy between individual mitochondrion, along with three high-confidence variants in mouse and one in human that were present in multiple mitochondria across cells. The pattern of variation revealed by single-mitochondrion data shows surprisingly pervasive levels of heteroplasmy in inbred mice. Distribution of SNV loci suggests inheritance of variants across generations, resulting in Poisson jackpot lines with large SNV load. Comparison of human and mouse variants suggests that the two species might employ distinct modes of somatic segregation. Single-mitochondrion resolution revealed mitochondria mutational dynamics that we hypothesize to affect risk probabilities for mutations reaching disease thresholds. : Morris et al. use independent sequencing of multiple individual mitochondria from mouse and human brain cells to show high pervasiveness of mutations. The mutations are heteroplasmic within single mitochondria and within and between cells. These findings suggest mechanisms by which mutations accumulate over time, resulting in mitochondrial dysfunction and disease. Keywords: single mitochondrion, single cell, human neuron, mouse neuron, single-nucleotide variatio