Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus.

BACKGROUND: Optogenetic tools allow precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Currently available optogenetic inhibitors are not suitable for prolonged use due to short-lasting photocurrents, tissue heating, and unintended changes in ion distributions, which may interfere with normal neuron physiology. To overcome these limitations, a novel potassium channel-based optogenetic silencer, named PACK, was recently developed. The PACK tool has two components: a photoactivated adenylyl cyclase from Beggiatoa (bPAC) and a cAMP-dependent potassium channel, SthK, which carries a large, long-lasting potassium current in mammalian cells. Previously, it has been shown that activating the PACK silencer with short light pulses led to a significant reduction of neuronal firing in various in vitro and acute in vivo settings. Here, we examined the viability of performing long-term studies in vivo by looking at the inhibitory action and side effects of PACK and its components in healthy and epileptic adult male mice. RESULTS: We targeted hippocampal cornu ammonis (CA1) pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-min intermittent (0.1 Hz) illumination, especially in the gamma frequency range. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Light activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. Furthermore, we applied bPAC and PACK in the contralateral hippocampus of chronically epileptic mice following a unilateral injection of intrahippocampal kainate. Unexpectedly, the expression of bPAC in the contralateral CA1 area was sufficient to prevent the spread of spontaneous epileptiform activity from the seizure focus to the contralateral hippocampus. CONCLUSION: Our study highlights the PACK tool as a potent optogenetic inhibitor in vivo. However, further refinement of its light-sensitive domain is required to avoid unexpected physiological changes.

AFP-producing adenocarcinoma of the esophagogastric junction: report of a case with atypical immunohistochemical findings responding to palliative chemotherapy with 5-fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT regime).

AFP-producing adenocarcinoma of the esophagus and esophagogastric junction are rare tumor diseases. These tumors show an aggressive behavior characterized by early occurrence of liver metastases and mimic hepatocellular carcinoma (HCC). A general recommendation for palliative therapy is not established for these special tumors.Here we report about a 61-year-old man with multiple liver metastases and high serum alpha-fetoprotein (AFP) level. First, HCC was suspected, but further evaluation showed an AFP-producing adenocarcinoma of the esophagogastric junction with unusual findings on further immunohistochemical analysis. Palliative chemotherapy with FLOT (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel) regime showed a 9 month duration of partial response.

Pregnancy and renal failure: the case for application of dosage guidelines.

Pregnancies in women with renal disease, undergoing dialysis treatment or with kidney transplants are increasingly observed. Serious problems with drug dose adjustment may arise in pregnant women with renal impairment. This review gives a practical overview on the risks of drug use during gestation, the recommended drugs of choice (e.g. methyldopa, cyclosporin), and provides some proposals for dosage adjustments in pregnant women with renal impairment. In normal pregnancy, the glomerular filtration rate and plasma volume increase, whereas plasma protein binding and liver function may be impaired. An increase in dosage is needed for cyclosporin and for methadone because of increased hepatic clearance. The dosage of erythropoetin must be increased because of lower potency in pregnant women. Little more is known on the impact of gestation on drug dose, since pharmacokinetic studies are rarely done in pregnant women. The dosages of magnesium, lithium and morphine must be reduced in renal impairment. Dose adjustment to renal function is critical and is essential for anti-infective agents (e.g. ceftazidime, ganciclovir). Basing drug dose on estimated creatinine clearance might be the most practical solution in pregnant women with renal impairment.

A remark on the high-conductance calcium-activated potassium channel in human endothelial cells.

The patch-clamp technique was used to examine the presence of large conductance calcium-activated potassium channels (BKCa) in human endothelial cells and to characterize their properties in terms of voltage dependence, ion conduction and blockade by iberiotoxin (IbTX). Experiments were performed using cell-attached and outside-out configurations on human umbilical vein endothelial cells (HUVEC). For the experiments HUVECs, which were passaged 6-19 times, were used. In early passages channel activities were absent suggesting the appearance of BKCa depending on cell culture time. The inverse logarithmic voltage sensitivity was 10.17 mV (median) for cell-attached recordings and 12.10 mV (median) for outside-out patches (membrane voltage range: 60-120 mV, symmetrical 140 mM K+ solutions). The I/V relationship was quasilinear in the range of 0-80 mV and exhibited a nonlinear behaviour under further depolarization suggesting some kind of saturation mechanism. Using a sigmoid function to fit the data, channel conductance was calculated as 172.9 pS (median) for cell-attached patches and as 262.1 pS (median) for outside-out patches. IbTX, known as one of the most selective blockers of BKCa was perfused to outside-out patches. In two out of three experiments there was complete block of the ion channel after 1 min.

Role of the cytoskeleton in the regulation of Cl- channels in human embryonic skeletal muscle cells.

The effects of volume change and cytoskeleton manipulation on the Cl- channels in human embryonic skeletal muscle cells were studied. Trypsination, used for production of myoballs, changes the channel properties only a little. When the external osmolarity was reduced from 300 to 270 mosmol/l, the specific Cl- conductance, gCl, (at -80 mV) of myoballs increased from 5.1 +/- 1.9 to 30.4 +/- 12.2 microS/cm2 (SD; n = 6) within 15 min. Concomitantly, the kinetics of Cl- currents, elicited by clamping the membrane potential from a negative to positive values, changed from activation and subsequent slow inactivation to instantaneous activation with fast inactivation. G protein activation, protein kinase action or [Ca2+]i elevation seemed not to be involved in these effects. Similar changes were produced in the absence of a transmembrane osmotic gradient by 500 nM intracellular cytochalasin D (gCl = 34.3 +/- 10.3 microS/cm2; n = 6) or 12.5 microM colchicine (gCl = 15.4 +/- 1.4 microS/cm2; n = 5). When the external osmolarity was increased to 418 mosmol/l, 1 microM cytochalasin D did not affect gCl. In four of six cell-attached patches the open probability of the intermediate Cl- channel was increased after reduction of the bath osmolarity. In inside-out patches, the drugs increased the open probability of the channels. It is concluded that the Cl- channels are under control of the cytoskeleton.

Chloride channels in cultured human skeletal muscle are regulated by G proteins.

The regulation of Cl- channels in human myoballs by G proteins was studied using whole-cell and inside-out patch recordings. After perfusion of the cell with 0.1 mM GTP[gamma S], the specific Cl- conductance, GCl, at standard resting potential (-85 mV) was increased from 5.9 microS/cm2 to 103 microS/cm2, and the kinetics upon stepping the potential to positive values was changed from an activating current with very slow inactivation to a fast inactivating current with no potential-dependent activation. These effects were not affected by the simultaneous blockade of several signal cascades involving G proteins. Addition of the protein kinase blockers PKI (25 microM), H8 (10 microM), or of the phospholipase-A2-blocking agent quinacrine (10 microM), had not much influence on these GTP[gamma S] effects. Buffering of the intracellular Ca2+ concentration (0.1 microM) or addition of the Ca2+/calmodulin antagonist trifluoperazine (50 microM) was also without effect. Pre-incubation of the cells with pertussis toxin or with cholera toxin did not change GCl. In excised inside-out patches voltage-clamped at -85 mV, application of GTP[gamma S] influenced the "intermediate" Cl- channel, the Cl- channel type having the highest density in these cells, by increasing the number of transitions in a half-conductance state. The probability of the channel being in one of the two conducting states rose from 0.015 to 0.67, and the kinetics of the single-channel currents was changed so that, on average, it was similar to the whole-cell current kinetics seen after application of GTP[gamma S]. It is concluded that a G protein is directly interacting with these channels.