Axonal and glial currents activated during the post-tetanic hyperpolarization in non-myelinated nerve

 Changes in membrane potential and potassium concentration in the extracellular space ([K⁺]_e) of rabbit vagus nerve were measured simultaneously during electrical activity and during the period of recovery using a modified sucrose-gap method and potassium-sensitive microelectrodes. After stimulation for 15 s at 15 Hz the main activity-induced increase in [K⁺]_e reached 16.9 mM. This increase in [K⁺]_e was paralleled by a depolarization of the preparation. The period of activity was followed by a post-tetanic hyperpolarization (PTH) lasting tens of seconds, generated by the axonal electrogenic Na⁺-K⁺ pump and to a lesser extent by the pump of the surrounding Schwann cells. The amplitude of the PTH dramatically increased in experiments in which inward currents were blocked by removal of Cl^– or after application of Cs⁺ or Ba²⁺, indicating that under normal conditions the current generated by the Na⁺-K⁺ pump is strongly short-circuited. A pharmacological and kinetic study showed that these currents are: (1) the hyperpolarization-activated current I _h, and (2) the inwardly rectifying I _KIR current. The results show that the latter originates from Schwann cells. Our data indicate that in non-myelinated nerves there is a subtle association of inward ionic channels which (1) helps the cell to maintain an optimal membrane potential after a period of activity, and (2) contributes to the removal of excess K⁺ from the extracellular space. Received: 7 August 1997 / Received after revision 6 April 1998 / Accepted: 15 April 1998     

Membrane properties of rat locus coeruleus neurones

Intracellular recordings were made from neurones in the locus coeruleus contained within a slice cut from rat pons and maintained in vitro. Most neurones fired action potentials spontaneously at frequencies of between 1 and 5 Hz; this did not arise from spontaneous synaptic input but appeared to result from endogenous properties of the membrane conductances. Under voltage clamp at potentials near threshold for action potential generation (? 55 mV) there was a persistent inward calcium current. This current became less with membrane hyperpolarization and was abolished at about ?70 mV. Two potassium currents were observed. The first had properties similar to that generally described as the “fast” potassium current (I_K,A); it flowed transiently (for about 200 ms) when the membrane potential passed from about ?65 to ?45 mV, and was blocked by 4-aminopyridine. The second was a calcium-activated potassium current (I_K,Ca); it flowed for several seconds following a burst of calcium action potentials. Spontaneous and evoked action potentials had both tetrodotoxin-sensitive and tetrodotoxin-resistant components. The latter was apparently due to calcium entry. The potential changes occurring during the spontaneous firing of locus coeruleus neurones could be reconstructed qualitatively from the ionic conductances observed. The membrane properties of the locus coeruleus neurones were remarkably uniform; however, about 5% of cells impaled within the region of the locus coeruleus were electrophysiologically distinct. These atypical cells had short duration action potentials, did not fire spontaneously and had large spontaneous depolarizing synaptic potentials.     

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery

1. The aim of the present study was to identify the sources of Ca²⁺ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release.
2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 μM indomethacin, 300 μM N^G-nitro-L-arginine or 10 μM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF.
3. In the presence of extracellular Ca²⁺, 1 μM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca²⁺-free medium, ACh produced only transient hyperpolarization.
4. Pretreatment with 100 nM thapsigargin and 3 μM cyclopiazonic acid, endoplasmic reticulum Ca²⁺-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 μM ryanodine.
5. In the presence of 5 mM Ni²⁺ or 3 mM Mn²⁺, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 μM nifedipine had no effect on ACh-induced hyperpolarization.
6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 μM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization.
7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca²⁺ release from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca²⁺ influx via a Ni²⁺- and Mn²⁺-sensitive pathway distinct from L-type Ca²⁺ channels. The Ca²⁺-influx mechanism seems to be activated following IP₃-induced depletion of the pool.

     

Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium-dependent relaxation to bradykinin in human small resistance-like coronary arteries

1. The effects of the nitric oxide (NO) synthase inhibitor, N^G-nitro-L-arginine (L-NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K⁺ upon endothelium-dependent relaxation to bradykinin were investigated in human isolated small coronary arteries.
2. Endothelium-dependent relaxations to bradykinin were compared in vessels contracted to ∼50% of their maximum contraction to 124 mM KCl Krebs solution, regardless of treatments, with the thromboxane A₂ mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force.
3. L-NOARG (100 μM) caused a small but significant, 12% (P<0.01), decrease in the maximum relaxation (R_max: 91.5±5.4%) to bradykinin but did not significantly affect the sensitivity (pEC₅₀: 8.08±0.17). Increasing the concentration of L-NOARG to 300 μM had no further effect on the pEC₅₀ or R_max to bradykinin. HbO (20 μM) and a combination of HbO (20 μM) and L-NOARG (100 μM) reduced R_max to bradykinin by 58% (P<0.05) and 54% (P<0.05), respectively. HbO (20 μM) and L-NOARG (100 μM, combined but not HbO (20 μM) alone, caused a significant 11 fold (P<0.05) decrease in sensitivitiy to bradykinin. HbO (20 μM) decreased the sensitivity to the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), approximately 17 fold (P<0.05).
4. Raising the extracellular concentration of K⁺ isotonically to 30 mM, reduced the R_max to bradykinin from 96.6±3.1% to 43.9±10.1% (P<0.01) with no significant change in sensitivity. A combination of HbO, L-NOARG and high K⁺ (30 mM) abolished the response to bradykinin. High K⁺ did not change either the sensitivity or maximum relaxation to SNAP.
5. In conclusion, L-NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non-NO -dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K⁺-sensitive vasodilator mechanism, possibly endothelium-derived hyperpolarizing factor (EDHF).

     

A continuous‐flow,high‐throughput,high‐pressure parahydrogen converter for hyperpolarization in a clinical setting

Pure parahydrogen (pH₂) is the prerequisite for optimal pH₂‐based hyperpolarization experiments, promising approaches to access the hidden orders of magnitude of MR signals. pH₂ production on‐site in medical research centers is vital for the proliferation of these technologies in the life sciences. However, previously suggested designs do not meet our requirements for safety or production performance (flow rate, pressure or enrichment). In this article, we present the safety concept, design and installation of a pH₂ converter, operated in a clinical setting. The apparatus produces a continuous flow of four standard liters per minute of ≈98% enriched pH₂ at a pressure maximum of 50 bar. The entire production cycle, including cleaning and cooling to 25 K, takes less than 5 h, only ≈45 min of which are required for actual pH₂ conversion. A fast and simple quantification procedure is described. The lifetimes of pH₂ in a glass vial and aluminum storage cylinder are measured to be T_1C(glass vial) = 822 ± 29 min and T_1C(Al cylinder) = 129 ± 36 days, thus providing sufficiently long storage intervals and allowing the application of pH₂ on demand. A dependence of line width on pH₂ enrichment is observed. As examples, ¹H hyperpolarization of pyridine and ¹³C hyperpolarization of hydroxyethylpropionate are presented. Copyright © 2012 John Wiley & Sons, Ltd.     

Hyperpolarized 13C magnetic resonance evaluation of renal ischemia reperfusion injury in a murine model

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) ¹³C dehydroascorbate (DHA) and ¹³C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham‐operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP ¹³C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP ¹³C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP ¹³C lactate/pyruvate ratio at day 7 compared with baseline, although the ¹³C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham‐operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP ¹³C MRS for the non‐invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.     

Automated transfer and injection of hyperpolarized molecules with polarization measurement prior to in vivo NMR

Hyperpolarized magnetic resonance via dissolution dynamic nuclear polarization necessitates the transfer of the hyperpolarized molecules from the polarizer to the imager prior to in vivo measurements. This process leads to unavoidable losses in nuclear polarization, which are difficult to evaluate once the solution has been injected into an animal. We propose a method to measure the polarization of the hyperpolarized molecules inside the imager bore, 3 s following dissolution, at the time of the injection, using a precise quantification of the infusate concentration. This in situ quantification allows for distinguishing between signal modulations related to variations in the nuclear polarization at the time of the injection and signal modulations related to physiological processes such as tissue perfusion. In addition, our method includes a radical scavenging process that leads to a minor reduction in sample concentration and takes place within a couple of seconds following the dissolution in order to minimize the losses due to the presence of paramagnetic polarizing agent in the infusate. We showed that proton exchange between vitamin C, the scavenging molecule and the deuterated solvent shortens the long carboxyl ¹³C longitudinal relaxation time in [1‐¹³C]acetate. This additional source of dipolar relaxation can be avoided by using deuterated ascorbate. Overall, the method allows for a substantial gain in polarization and also leads to an extension of the time window available for in vivo measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

Intrahippocampal infusion of the Ih blocker ZD7288 slows evoked theta rhythm and produces anxiolytic‐like effects in the elevated plus maze

Hippocampal theta rhythm has been associated with a number of behavioral processes, including learning and memory, spatial behavior, sensorimotor integration and affective responses. Suppression of hippocampal theta frequency has been shown to be a reliable neurophysiological signature of anxiolytic drug action in tests using known anxiolytic drugs (i.e., correlational evidence), but only one study to date (Yeung et al. ( 2012 ) Neuropharmacology 62:155–160) has shown that a drug with no known effect on either hippocampal theta or anxiety can in fact separately suppress hippocampal theta and anxiety in behavioral tests (i.e., prima facie evidence). Here, we attempt a further critical test of the hippocampal theta model by performing intrahippocampal administrations of the I_h blocker ZD7288, which is known to disrupt theta frequency subthreshold oscillations and resonance at the membrane level but is not known to have anxiolytic action. Intrahippocampal microinfusions of ZD7288 at high (15 µg), but not low (1 µg) doses slowed brainstem‐evoked hippocampal theta responses in the urethane anesthetized rat, and more importantly, promoted anxiolytic action in freely behaving rats in the elevated plus maze. Taken together with our previous demonstration, these data provide converging, prima facie evidence of the validity of the theta suppression model. © 2012 Wiley Periodicals, Inc.