Effects of macrophage-colony-stimulating factor on cyclophosphamide-injected mouse NK1.1+ cell activity

 We injected cyclophosphamide into mice and examined their natural killer (NK) activity both in vitro and in vivo. Cyclophosphamide injection temporarily abrogated the lung clearance activity of Yac-1 lymphoma cells, which is considered to be an index of NK activity in vivo. However, administration of recombinant human macrophage-colony-stimulating-factor (rhM-CSF) to cyclophosphamide-injected mice restored the lung clearance activity. To clarify whether the administration of rhM-CSF activated NK cells, we purified NK1.1⁺ cells from mice treated with cyclophosphamide and/or rhM-CSF and examined their functions (cytotoxicity, proliferation, and interferon γ production) in vitro. Cyclophosphamide injection decreased the number, but did not suppress the functions of NK1.1⁺ cells. The numbers of NK1.1⁺ cells in cyclophosphamide-injected mice restored by rhM-CSF administration. And the functions of NK1.1⁺ cells from both saline-injected and cyclophosphamide-injected mice were accelerated by rhM-CSF administration. These results suggested that the temporary abrogation of NK activity in vivo caused by cyclophosphamide injection was due to a decrease in the number and not to suppression of the functions of NK1.1⁺ cells. The injection of cyclophosphamide into mice increased the number of tumor (B16 melanoma) nodules formed in the lungs and liver. However, treatment with rhM-CSF recovered the anti-metastatic activity in the lungs of cyclophosphamide-injected mice. These results show that administration of rhM-CSF restores NK activity suppressed by cyclophosphamide injection in vivo. Received: 28 September 1999 / Accepted: 23 December 1999     

Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.     

Interleukin-1β inhibits voltage-gated sodium currents in a time- and dose-dependent manner in cortical neurons

Interleukin-1β (IL-1β) is a multifunctional proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na⁺ channel is essential for the excitability and electrical properties of neurons. However, it is not known whether IL-1β directly affects the central Na⁺ channels. In the present study, we examined the effects of IL-1β on Na⁺ currents in cultured cortical neurons using patch-clamp recording. Our results showed that IL-1β suppressed Na⁺ currents through its receptor in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. PKC and then p38 MAPK were involved in this inhibition. The spike amplitude was also inhibited by IL-1β in the doses that decreased the Na⁺ currents. Our findings revealed the inhibition of chronic IL-1β treatment on voltage-gated Na⁺ channels in the CNS, and showed that the action potential (AP) amplitude was reduced by IL-1β due to a decrease of Na⁺ currents.     

Possible mechanisms underlying effects of N-uronoyl derivatives of amino acids on electrical activity in snail neurons

Using intracellular recording, we studied the effects of N-uronoyl derivatives of an amino acid and peptides (1,2:3,4-di-O-isopropylidene-αa-D-galactopyranuronoyl)-β-alanine (DAGU-Ala), DAGU-glycyl-glycine (DAGU-Gly-Gly), DAGU-glycyl-D,L-glutamic acid (DAGU-Gly-Glu), as well as of 1,2:3,4-di-O-isopropylidene-αa-D-galactopyranosyluronic acid (DAGU itself), β-alanine (β-Ala), D,L-glutamic acid (D,L-Glu), and glycyl-glycine (Gly-Gly), which were added to the extracellular milieu, on the electrical activity of PPa1 and PPa2 neurons and unidentified neurons of Helix albescens Rossm. DAGU-Gly-Gly applied in concentrations of 10⁻⁴ to 10⁻² M hyperpolarized the membrane in a dose-dependent manner and decreased insignificantly the amplitude of action potentials (APs). Applications of DAGU-Ala, β-Ala, DAGU-Gly-Glu, D,L-Glu, and Gly-Gly in the same doses resulted in a shift of the membrane potential toward depolarization and in a drop in the amplitude of APs. Measurements of the first AP derivatives showed that all the above-mentioned substances suppressed in a concentration-dependent manner both inward and outward transmembrane ion currents. In this case, DAGU suppressed both inward and outward currents, while DAGU-Ala, β-Ala, DAGU-Glu, D,L-Glu, and Gly-Gly inhibited predominantly the outward potassium ion current; DAGU-Gly-Gly inhibited inward sodium and potassium ion currents. Results of a comparative analysis of the neurotropic action of the tested amino acids and their N-uronoyl derivatives showed that modification of the molecules of neurotransmitter amino acids leads to a decrease in their neurotoxicity and to an increase in their membranotropic properties. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 416–425, September–December, 2006.     

Developmental change in calcium-activated chloride current during the differentiation of Xenopus spinal neurons in culture.

The duration and ionic dependence of action potentials change during the differentiation of embryonic amphibian spinal neurons both in vivo and in culture. The development of sodium, calcium, and potassium currents has been characterized in these cells and the shortening of the action potential has been shown to depend to a great extent on developmental changes of potassium currents. Previous evidence suggests that a chloride current may also be present in these embryonic neurons. Chloride currents were investigated with intracellular current-clamp and single-electrode and whole-cell voltage-clamp techniques. Most neurons exhibited a calcium-activated chloride current (ICl(Ca] that contributed to the postdepolarization following the action potential recorded in the absence of sodium and potassium currents. This current appeared to decrease in density and its deactivation rate increased during the first day in culture. Its incidence also declined during this period. A much larger Ca(2+)-dependent Cl- current was also observed in a subset of neurons after 24 hr, but was absent at earlier stages of development. The results suggest the presence of two Cl- currents with different developmental fates. The early current probably contributes to the repolarization of long calcium-dependent action potentials at initial stages of neuronal development, when potassium currents are small, and may serve to reduce the extent of repetitive firing.     

Tumor-derived Fas ligand induces toxicity in lymphoid organs and plays an important role in successful chemotherapy

 Recent studies have suggested that Fas ligand (FasL⁺) tumor cells can induce apoptosis in Fas⁺ T cells. However, the effect of growth of FasL⁺ tumors in vivo, on lymphoid tissues of the host is not clear and therefore was the subject of this investigation. Injection of FasL⁺ LSA tumor caused a significant decrease in cellularity of the thymus and spleen, resulting from marked apoptosis, in syngeneic C57BL/6+/+ (wild-type) but not C57BL/6-lpr/lpr (Fas-deficient) mice. The tumor-induced toxicity resulted from tumor-derived rather than host-derived FasL, inasmuch as LSA tumor growth in C57BL/6-gld/gld (FasL-defective) mice, induced marked apoptosis and toxicity in the thymus and spleen. The LSA tumor growth induced a significant decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus of C57BL/6+/+ mice and an increase in the percentage of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T cells. Of the four subpopulations tested, the CD4⁺CD8⁺ T cells showed maximum apoptosis. The LSA (FasL⁺) but not P815(FasL⁻) tumor cell lysates and culture supernatants induced marked apoptosis in Fas⁺ thymocytes, when tested both in vitro and in vivo. The LSA-tumor-induced apoptosis in vitro was inhibited by antibodies against FasL or by caspase and other inhibitors of apoptosis. Chemotherapy of LSA-tumor-bearing C57BL/6+/+ mice at advanced stages of tumor growth failed to cure the mice, whereas, more than 80% of LSA-tumor-bearing C57BL/6-lpr/lpr mice, similarly treated, survived. Together, the current study demonstrates that FasL produced by LSA tumor cells is functional in vivo and can cause severe toxicity in lymphoid organs of the host. Also, Fas/FasL interactions may play an important role in the successful chemotherapy of FasL-bearing tumor. Received: 31 August 1999 / Accepted: 12 November 1999     

Persistent inward currents in cultured Retzius cells of the medicinal leech

Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na⁺-free saline and blocked by Cs⁺, but not Ba²⁺. Voltage clamp experiments revealed a Cs⁺-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs⁺ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca²⁺ influx with Ni²⁺ and suppression of K⁺ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li⁺ as the charge carrier, but were eliminated when Na⁺ was replaced with N-methyl-d-glucamine. A persistent Na⁺ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na⁺ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na⁺ current may underlie Na⁺-dependent bursting recorded in neurons of intact ganglia exposed to Ca²⁺-channel blockers. Accepted: 22 September 1998     

Influences of dopamine and noradrenaline on outward potassium currents through the somatic membrane of neurons of the molluscs (Limnaea) of different ages

Dopamine- and noradrenaline-induced modifications of outward potassium currents were studied in identified neurons of the lesser parietal ganglion of adult (10–12 months) and old (22–24 months) molluscsLimnaea stagnalis. In the neurons of old molluscs, 2·10⁻⁵ M dopamine made activation of potassium channels of delayed current 2.5 times more frequent than in adult molluscs. Noradrenaline (5·10⁻⁵ M) significantly increased delayed outward potassium currents in adult molluscs and did not modify these currents in old molluscs. It is supposed that there are age-related modifications of the ratio between the active and passive components of potassium ion transport in the mechanism responsible for monoamine-induced reactions of a neuron.     

Millimeter Wave Radiation Activates Leech Nociceptors via TRPV1-Like Receptor Sensitization

There is evidence that millimeter waves (MMWs) can have an impact on cellular function, including neurons. Earlier in vitro studies have shown that exposure levels well below the recommended safe limit of 1 mW/cm² cause changes in the action potential (AP) firing rate, resting potential, and AP pulse shape of sensory neurons in leech preparations as well as alter neuronal properties in rat cortical brain slices; these effects differ from changes induced by direct heating. In this article, we compare the responses of thermosensitive primary nociceptors of the medicinal leech under thermal heating and MMW irradiation (80–170 mW/cm² at 60 GHz). The results show that MMW exposure causes an almost twofold decrease in the threshold for activation of the AP compared with thermal heating (3.9 ± 0.4 vs. 8.3 ± 0.4 mV, respectively). Our analysis suggests that MMWs-mediated threshold alterations are not caused by the enhancement of voltage-gated sodium and potassium conductance. We propose that the reduction in AP threshold can be attributed to the sensitization of the transient receptor potential vanilloid 1-like receptor in the leech nociceptor. In silico modeling supported our experimental findings. Our results provide evidence that MMW exposure stimulates specific receptor responses that differ from direct thermal heating, fostering the need for additional studies.     

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca²⁺-resistant transient outward K⁺ current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.     

Response to red and blue lights by electrical currents on the surface of intact leaves

Exposure to red and blue lights caused an increase in electrical currents (0.14 μA cm^-2 for red and 0.05 μA cm^-2 for blue, respectively) flowing on the lower surface of leaves fromCommelina communis. However, no changes were measured in currents from isolated epidermal cells. To determine the influence of the mesophyll on such electrical changes, those cells were infiltrated with photosynthesis inhibitors. Both DCCD treated and control leaf discs showed the same level of response to red light. Epidermal strips were also removed to measure the currents above partially exposed mesophyll cells in order to elucidate the relationship between intact leaves and those mesophyll cells. Changes in current were smaller in the latter type. The partially exposed mesophyll cells of a leaf also showed electrical current changes, but smaller than those of the intact leaf. In DCMU-infiltrated leaf discs, the electrical currents of intact leaves were increased to 0.05 μA cm^-2 in response to red light. For sodium azide-infiltrated leaf discs, however, intact leaves showed no response. Likewise, a measure of photosynthetic efficiency, the Fv/Fm ratio, was reduced to that measured in the control, thereby indicating that photosynthetic activity significantly altered the electrical current for intact leaves. Therefore, these results demonstrate that the current observed from the lower side of intact leaves is related to photosynthetic activity in the mesophyll cells.     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons

Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na⁺ channels that belong to the epithelial Na⁺ channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extracellular pH changes from 7.4 to slightly <7. Such local extracellular pH values may be reached by pH fluctuations due to normal neuronal activity. Furthermore, in the plasma membrane, ASICs are localized in close proximity to voltage-gated Na⁺ and K⁺ channels, providing the conditions necessary for the transduction of local pH changes into electrical signals. cellular excitability; neuronal signaling; pH     

Effect of “Chemical” Hypoxia on the Potassium Conductance of the Membrane of Pheochromocytoma Cells

We studied the effect of “chemical” (induced by the action of sodium thiosulfate, STS) hypoxia on the potassium conductance of the membrane of pheochromocytoma cells. Application of 1 to 10 mM STS decreased in a dose-dependent manner the amplitude of integral potassium current without changes in the voltage dependence of its activation. The concentration dependence of the action of STS on the amplitude of potassium current was estimated using the Boltzmann equation. The value of concentration for 50% inhibition was 2.7 ± 0.2 mM, while the slope coefficient was 0.9 ± 0.2 mM⁻¹. In the presence of 10 mM STS, the decrease in the amplitude of potassium current reached, on average, 55%. Therefore, “chemical” hypoxia influences rather significantly the potassium conductance of the membrane of pheochromocytoma PC12 cells.     

Effect of Calcium on Electrical Energy Transfer by Microtubules

Microtubules (MTs) are important cytoskeletal superstructures implicated in neuronal morphology and function, which are involved in vesicle trafficking, neurite formation and differentiation and other morphological changes. The structural and functional properties of MTs depend on their high intrinsic charge density and functional regulation by the MT depolymerising properties of changes in Ca^2 + concentration. Recently, we reported on remarkable properties of isolated MTs, which behave as biomolecular transistors capable of amplifying electrical signals (Priel et al., Biophys J 90:4639–4643, 2006). Here, we demonstrate that MT-bathing (cytoplasmic) Ca^2 + concentrations modulate the electrodynamic properties of MTs. Electrical amplification by MTs was exponentially dependent on the Ca^2 + concentration between 10^− 7 and 10^− 2 M. However, the electrical connectivity (coupling) of MTs was optimal at a narrower window of Ca^2 + concentrations. We observed that while raising bathing Ca^2 + concentration increased electrical amplification by MTs, energy transfer was highest in the presence of ethylene glycol tetraacetic acid (lowest Ca^2 + concentration). Our data indicate that Ca^2 + is an important modulator of electrical amplification by MTs, supporting the hypothesis that this divalent cation, which adsorbs onto the polymer’s surface, plays an important role as a regulator of the electrical properties of MTs. The Ca^2 +-dependent ability of MTs to modulate and amplify electrical signals may provide a novel means of cell signaling, likely contributing to neuronal function.     

Serotonin, nitric oxide and histamine enhance the excitability of neuron MCC by diverse mechanisms

Serotonin, nitric oxide (NO) and histamine are neuromodulators used in molluscan nervous systems. We have found that each of them depolarizes and increases the excitability of the serotonergic feeding neural circuit modulator neuron, MCC, of Aplysia, but each induces different changes in background ionic currents and uses a different second messenger. Stimulation of neuron C2 in the cerebral ganglion induces a vsEPSP in MCC using NO and histamine. When these neurons are isolated in culture they form synapses that mediate the vsEPSP. The ionic currents induced by these neuromodulators were investigated in isolated cultured MCCs. Histamine reduced a background outward current between -70 and -30 mV that was blocked by cobalt treatment, indicating that it is a calcium activated potassium current. Serotonin reduced a background outward current from -65 mV to -30 mV and enhanced a potassium inward current more negative than -70 mV that was blocked by cesium and barium. This response was mimicked by 8-Br-cAMP. NO donors reduced a cobalt insensitive background outward current between -70 and -30 mV. This response was mimicked by 8-Br-cGMP. These responses show that MCC can produce complex time and state-dependent activity during its modulation of the feeding neural circuit.     

Electrophysiological properties of rainbow trout cardiac myocytes in serum-free primary culture

A low-density primary culture of trout ventricular myocytes in serum-free growth medium was established and maintained for up to 10 days at 17 degrees C. The myocytes retained their normal rod shaped morphology, capacitive surface area of the sarcolemma (SL), and contractile quiescence. However, sarcolemmal cation currents changed significantly, some permanently, some transiently, after 8-10 days of culture. TTX-sensitive sodium current (I(Na)) and Ba(2+)-sensitive background inward rectifier potassium current (I(K1)) were permanently depressed to 24-28% of their control density measured in freshly isolated myocytes. In contrast, L-type calcium current (I(Ca)) was only transiently downregulated; after 2-3 days in culture, the density of the current was 32% of the control and recovered to the control value after 8-10 days in culture. The changes in membrane currents were reflected in the shape of the action potential (AP). After 2-3 days in culture, maximal overshoot potential and resting potential were significantly reduced, and the durations of the AP at 50 and 90% repolarization were significantly increased. These changes became significantly more pronounced after 8-10 days of culture, with the exception of AP duration at 50% repolarization level. The shortening of the early plateau phase may reflect an additional change to an outward current, presumably the rapid component of the delayed rectifier (I(Kr)). Although the present findings indicate that fish cardiac myocytes can be maintained in serum-free primary culture for at least 10 days at 17 degrees C, some but not all of the electrophysiological characteristics of the myocytes change markedly during culture. The changes in ion currents were not due to loss of sarcolemmal membrane and therefore are likely to represent altered expression of cation currents as an adaptive response to culture conditions.