Focused ultrasound transiently increases membrane conductance in isolated crayfish axon.

We report a novel phenomenon produced by focused ultrasound (US) that may be important for understanding its effects on cell membranes. When a US burst (2.1 MHz, 1-mm focal diameter, 0.1-1 MPa) was focused on a motor axon of the crayfish neuromuscular junction, it consistently produced a fast hyperpolarization, which was followed or superseded by subthreshold depolarizations or action potentials in a stochastic manner. The depolarization persisted in the presence of voltage-gated channel blockers [1 µM TTX ( INa), 50 µM ZD7288 ( Ih), and 200 µM 4-aminopyridine ( IK)] and typically started shortly after the onset of a 5-ms US burst, with a mean latency of 3.35 ± 0.53 ms (SE). The duration and amplitude of depolarizations averaged 2.13 ± 0.87 s and 10.1 ± 2.09 mV, with a maximum of 200 s and 60 mV, respectively. The US-induced depolarization was always associated with a decrease in membrane resistance. By measuring membrane potential and resistance during the US-induced depolarization, the reversal potential of US-induced conductance ( gus) was estimated to be -8.4 ± 2.3 mV, suggesting a nonselective conductance. The increase in gus was 10-100 times larger than the leak conductance; thus it could significantly influence neuronal activity. This change in conductance may be due to stimulation of mechanoreceptors. Alternatively, US may perturb the lateral motion of phospholipids and produce nanopores, which then increase gus. These results may be important for understanding mechanisms underlying US-mediated modulation of neuronal activity and brain function. NEW & NOTEWORTHY We report a specific increase in membrane conductance produced by ultrasound (US) on neuronal membrane. When a 5-ms US tone burst was focused on a crayfish motor axon, it stochastically triggered either depolarization or a spike train. The depolarization was up to 60 mV in amplitude and 200 s in duration and therefore could significantly influence neuronal activity. Depolarization was still evoked by US burst in the presence of Na+ and Ca2+ channel blockers and had a reversal potential of -8.4 ± 2.3 mV, suggesting a nonselective permeability. US can be applied noninvasively in the form of a focused beam to deep brain areas through the skull and has been shown to modulate brain activity. Understanding the depolarization reported here should be helpful for improving the use of US for noninvasive modulation and stimulation in brain-related disease.

Ultrasound-Induced Membrane Hyperpolarization in Motor Axons and Muscle Fibers of the Crayfish Neuromuscular Junction.

OBJECTIVE: Focused ultrasound (FUS) can modulate neuronal activity by depolarization or hyperpolarization. Although FUS-evoked depolarization has been studied extensively, the mechanisms underlying FUS-evoked hyperpolarization (FUSH) have received little attention. In the study described here, we developed a procedure using FUS to selectively hyperpolarize motor axons in crayfish. As a previous study had reported that these axons express mechano- and thermosensitive two-pore domain potassium (K2P) channels, we tested the hypothesis that K2P channels underlie FUSH. METHODS: Intracellular recordings from a motor axon and a muscle fiber were obtained simultaneously from the crayfish opener neuromuscular preparation. FUSH was examined while K2P channel activities were modulated by varying temperature or by K2P channel blockers. RESULTS: FUSH in the axons did not exhibit a coherent temperature dependence, consistent with predicted K2P channel behavior, although changes in the resting membrane potential of the same axons indicated well-behaved K2P channel temperature dependence. The same conclusion was supported by pharmacological data; namely, FUSH was not suppressed by K2P channel blockers. Comparison between the FUS-evoked responses recorded in motor axons and muscle fibers revealed that the latter exhibited very little FUSH, indicating that the FUSH was specific to the axons. CONCLUSION: It is not likely that K2P channels are the underlying mechanism for FUSH in motor axons. Alternative mechanisms such as sonophore and axon-specific potassium channels were considered. Although the sonophore hypothesis could account for electrophysiological features of axonal recordings, it is not consistent with the lack of FUSH in muscle fibers. An axon-specific and mechanosensitive potassium channel is also a possible explanation.

Effects of Osmolarity on Ultrasound-Induced Membrane Depolarization in Isolated Crayfish Motor Axon.

We have previously identified a novel non-selective membrane conductance (gUS) opened by focused ultrasound (FUS) in crayfish motor axons. In the work described here, we studied gUS properties further by comparing FUS-evoked depolarization (FUSD) in control and hypotonic saline with 75% of control osmolarity. The FUS was a train of 20 FUS bursts (2.1 MHz and 50 µs per burst) delivered at 1 kHz. The amplitude, onset latency, frequency of occurrence and duration of FUSD were compared in a 15-min time window before and after switching to hypotonic saline. Significant increases were observed for amplitude (p < 0.001) and frequency of occurrence (p < 0.01) while the onset latency exhibited a significant decrease (p < 0.001). FUSD duration did not significantly differ. These results support predictions based on our hypothesis that gUS is mediated by opening of nanopores in the lipid bilayer and that stretching of axonal membrane caused by swelling at low osmolarity should increase the probability of nanopore formation under FUS. The FUSD parameters, in addition, exhibited time-dependent trends when the window of observation was expanded to 45 min in each saline. The statistical significance of amplitude and duration differed between 15- and 45-min time windows, indicating the presence of adaptive responses of axonal membrane to osmotic manipulation.

Arachidonic acid potently inhibits both postsynaptic-type Kv4.2 and presynaptic-type Kv1.4 IA potassium channels.

Arachidonic acid (AA) is a free fatty acid membrane-permeable second messenger that is liberated from cell membranes via receptor- and Ca(2+)-dependent events. We have shown previously that extremely low [AA](i) (1 pm) inhibits the postsynaptic voltage-gated K(+) current (I(A)) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic I(A) is mediated by Kv4.2 gene products, whereas presynaptic I(A) is mediated by Kv1.4 channel subunits. To address the interaction of AA with these alpha-subunits we studied the modulation of A-currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole-cell voltage-clamp recording. For both currents 1 pm [AA](i) inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by -9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co-application of Trolox C (10 microm), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4-mediated I(A) by -32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on I(A) amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4-mediated I(A). Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic I(A) in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel alpha-subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity.

Development of epileptiform excitability in the deep entorhinal cortex after status epilepticus.

Epileptiform neuronal activity during seizures is observed in many brain areas, but its origins following status epilepticus (SE) are unclear. We have used the Li low-dose pilocarpine rat model of temporal lobe epilepsy to examine early development of epileptiform activity in the deep entorhinal cortex (EC). We show that during the 3-week latent period that follows SE, an increasing percentage of neurons in EC layer 5 respond to a single synaptic stimulus with polysynaptic burst depolarizations. This change is paralleled by a progressive depolarizing shift of the inhibitory postsynaptic potential reversal potential in layer 5 neurons, apparently caused by upregulation of the Cl(-) inward transporter NKCC1 and concurrent downregulation of the Cl(-) outward transporter KCC2, both changes favoring intracellular Cl(-) accumulation. Inhibiting Cl(-) uptake in the latent period restored more negative GABAergic reversal potentials and eliminated polysynaptic bursts. The changes in the Cl(-) transporters were highly specific to the deep EC. They did not occur in layers 1-3, perirhinal cortex, subiculum or dentate gyrus during this period. We propose that the changes in Cl(-) homeostasis facilitate hyperexcitability in the deep entorhinal cortex leading to epileptiform discharge there, which subsequently affects downstream cortical regions.

High intracranial pressure effects on cerebral cortical microvascular flow in rats.

To manage patients with high intracranial pressure (ICP), clinicians need to know the critical cerebral perfusion pressure (CPP) required to maintain cerebral blood flow (CBF). Historically, the critical CPP obtained by decreasing mean arterial pressure (MAP) to lower CPP was 60 mm Hg, which fell to 30 mm Hg when CPP was reduced by increasing ICP. We examined whether this decrease in critical CPP was due to a pathological shift from capillary (CAP) to high-velocity microvessel flow or thoroughfare channel (TFC) shunt flow. Cortical microvessel red blood cell velocity and NADH fluorescence were measured by in vivo two-photon laser scanning microscopy in rats at CPP of 70, 50, and 30 mm Hg by increasing ICP or decreasing MAP. Water content was measured by wet/dry weight, and cortical perfusion by laser Doppler flux. Reduction of CPP by raising ICP increased TFC shunt flow from 30.4±2.3% to 51.2±5.2% (mean±SEM, p<0.001), NADH increased by 20.3±6.8% and 58.1±8.2% (p<0.01), and brain water content from 72.9±0.47% to 77.8±2.42% (p<0.01). Decreasing CPP by MAP decreased TFC shunt flow with a smaller rise in NADH and no edema. Doppler flux decreased less with increasing ICP than decreasing MAP. The decrease seen in the critical CPP with increased ICP is likely due to a redistribution of microvascular flow from capillary to microvascular shunt flow or TFC shunt flow, resulting in a pathologically elevated CBF associated with tissue hypoxia and brain edema, characteristic of non-nutritive shunt flow.

Differential changes of glutathione levels in astrocytes and neurons in ischemic brains by two-photon imaging.

Using two-photon imaging techniques with monochlorobimane as a glutathione (GSH) probe, we investigated GSH levels in both core and penumbra regions of an ischemic brain after middle cerebral artery occlusion. We found that the GSH level significantly decreased in the ischemic core, but increased significantly in the penumbra. Furthermore, we observed a differential change of the GSH levels in neurons and astrocytes in the penumbra. The GSH level in neurons increased significantly whereas it decreased slightly in astrocytes in the penumbra. These findings reveal critical region and cell type-dependent changes of the GSH level in an ischemic brain.