TEM characterization of As-deposited and annealed Ni/Al multilayer thin film.

Reactive multilayer thin films that undergo highly exothermic reactions are attractive choices for applications in ignition, propulsion, and joining systems. Ni/Al reactive multilayer thin films were deposited by dc magnetron sputtering with a period of 14 nm. The microstructure of the as-deposited and heat-treated Ni/Al multilayers was studied by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) in plan view and in cross section. The cross-section samples for TEM and STEM were prepared by focused ion beam lift-out technique. TEM analysis indicates that the as-deposited samples were composed of Ni and Al. High-resolution TEM images reveal the presence of NiAl in small localized regions. Microstructural characterization shows that heat treating at 450 and 700°C transforms the Ni/Al multilayered structure into equiaxed NiAl fine grains.

Effects of using mixed wine yeast cultures in the production of Chardonnay wines.

The effect of using mixed cultures of non-Saccharomyces and Saccharomyces cerevisiae yeasts in the physicochemical and sensory qualities of the wines were analyzed in this study. Based on growth curves, sugar consumption and glycerol production in synthetic must, Candida membranifaciens L1805 was selected from a group of four Candidas spp. isolates from Chile and Argentina. This yeast was subsequently used in combination with S. cerevisiae in Chardonnay must. A monoculture of S. cerevisiae was used as control. The wines fermented with mixed cultures had lower volatile acidity and ethanol concentration than the control. Furthermore, the chromatographic analysis showed that the wines from mixed cultures presented differences in the concentration of esters and propanol. These characteristics positively influenced the sensory qualities of the wines produced with mixed cultures, which was reflected in the preference for these wines by a panel of enologists. This study shows that the use of non-Saccharomyces yeasts could be a strategy to obtain distinctive wines using the native microorganisms from each winemaking area.

A comparative proteomic analysis of Gluconacetobacter diazotrophicus PAL5 at exponential and stationary phases of cultures in the presence of high and low levels of inorganic nitrogen compound.

A proteomic view of G. diazotrophicus PAL5 at the exponential (E) and stationary phases (S) of cultures in the presence of low (L) and high levels (H) of combined nitrogen is presented. The proteomes analyzed on 2D-gels showed 131 proteins (42E+32S+29H+28L) differentially expressed by G. diazotrophicus, from which 46 were identified by combining mass spectrometry and bioinformatics tools. Proteins related to cofactor, energy and DNA metabolisms and cytoplasmic pH homeostasis were differentially expressed in E growth phase, under L and H conditions, in line with the high metabolic rate of the cells and the low pH of the media. Proteins most abundant in S-phase cells were stress associated and transporters plus transferases in agreement with the general phenomenon that binding protein-dependent systems are induced under nutrient limitation as part of hunger response. Cells grown in L condition produced nitrogen-fixation accessory proteins with roles in biosynthesis and stabilization of the nitrogenase complex plus proteins for protection of the nitrogenases from O(2)-induced inactivation. Proteins of the cell wall biogenesis apparatus were also expressed under nitrogen limitation and might function in the reshaping of the nitrogen-fixing G. diazotrophicus cells previously described. Genes whose protein products were detected in our analysis were mapped onto the chromosome and, based on the tendency of functionally related bacterial genes to cluster, we identified genes of particular pathways that could be organized in operons and are co-regulated. These results showed the great potential of proteomics to describe events in G. diazotrophicus cells by looking at proteins expressed under distinct growth conditions.

First Report of Dry Rot Caused by Fusarium oxysporum on Rose (Rosa spp.) in Brazil.

Roses are a high-value niche crop in the higher altitudes of northeastern Brazil. From July of 2007 and throughout 2008, severe stem rot and wilting of rose seedlings were observed in commercial fields in the São Benedito District, Ceará State, Brazil. Although economic losses due to the disease are unknown, it poses a threat to the growing rose industry in that region. Symptoms included leaf yellowing and abscission followed by plant collapse. Symptoms appeared earlier when grafted seedlings were produced during periods of high relative humidity (80 to 98%) and warm temperatures (20 to 31°C). In the laboratory, symptomatic seedlings were rinsed with distilled water, surface sterilized with 0.5% NaOCl, and incubated on PDA at 26 ± 2°C. Fusarium oxysporum was consistently isolated from infected scions and rootstocks. Identification of F. oxysporum was based on colony and conidia morphology obtained from single-spore colonies. Five 4-week-old rose ('Carola') seedlings were inoculated with a culture of fungus by spraying the needle-wounded scion with a spore suspension (1 × 105 CFU/ml). The spore suspension was obtained from a 1-week-old PDA culture incubated at 26 ± 2°C. Control seedlings were sprayed with sterile water. Inoculated seedlings were incubated for the first 48 h in a saturated humidity chamber. After 20 days at room temperature, the scion tissue of inoculated seedlings turned necrotic. Two symptomatic seedlings were placed in a saturated humidity chamber for 24 h to determine if fungal sporulation could be observed on the surface of the tissue. After 5 to 7 days, a white mycelium was observed over the necrotic tissue. Seedlings sprayed with sterile water remained symptomless. F. oxysporum was reisolated from symptomatic tissue. An isolate of F. oxyporum (No. 1484) was deposited in the Mycology Collection of Lavras (Minas Gerais State, Brazil). To our knowledge, this is the first report of F. oxysporum causing a disease on rose seedlings in Brazil.

First Report of a Bacterial Leaf and Fruit Spot of Cashew Nut (Anacardium occidentale) Caused by Xanthomonas campestris pv. mangiferaeindicae in Brazil.

In 2003 and 2004, leaves and young fruits of cashew nut plants showing an undescribed disease symptom were observed on plants of an early-dwarf clone in a commercial orchard in Ceará and Piauí states in northeastern Brazil. Initial symptoms consisted of angular, water-soaked, dark-to-black spots on the leaf and at the mid-rib vein surrounding the leaf veins. Eventually, lesions also extended from the mid-rib to the secondary veins, delineating the vein system of the leaf. In young, green fruits, symptoms were large, dark, oily spots surrounded by conspicuous water-soaked areas. A yellow-pigmented colony was consistently recovered from the lesions on nutrient yeast-extract dextrose agar medium (3 g of meat extract, 5 g of peptone, 10 g of dextrose, 5 g of yeast extract, and 18 g of agar per liter). Physiological tests revealed colonies that were gram negative, strictly aerobic, oxidase negative, catalase positive, lacking fluorescent pigmentation on King's B medium, urea hydrolase negative, and able to grow on yeast dextrose calcium carbonate medium yielding yellow colonies. These tests indicated that the bacterium belonged to the genus Xanthomonas. PCR amplification of bacterial DNA using RST2 (1) and Xcv3R (3) primers resulted in identical band patterns to mango isolates Xanthomonas campestris pv. mangiferaeindicae. Restriction fragment length polymorphism analysis of PCR-amplified products of six isolates of X. campestris pv. mangiferaeindicae was conducted with HaeIII and showed different profile patterns on agarose gel, indicating genetic variability among these isolates. Pathogenicity was demonstrated by gently piercing and misting cashew leaves with a bacterial suspension adjusted to 106 CFU/ml. Inoculated plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (29 ± 1°C). Control plants were misted with sterile water and treated the same way. After 8 days, foliar symptoms similar to those observed in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be X. campestris pv. mangiferaeindicae. Control plants remained symptomless. To our knowledge, this is the first description of commercially grown cashew plants as host to X. campestris pv. mangiferaeindicae in Brazil. This disease may pose a serious problem to the cashew-growing industry in Brazil. This bacterial pathogen has been reported on mangoes (Mangifera indica) and cashew in India (2) under the former name of Pseudomonas mangiferae-indicae. References: (1) R. P. Leite, Jr. et al. Appl. Environ. Microbiol. 60:1068, 1994. (2) M. K. Patel et al. Curr. Sci. 17:189, 1948. (3) L. C. Trindade et al. Summa Phytopathol. 33:16, 2007.

TRPA1 Channels Mediate Human Gingival Fibroblast Response to Phenytoin.

Drug-induced gingival enlargement (GE) is a frequent adverse effect observed in patients treated with anticonvulsant, immunosuppressant, and some antihypertensive medications-the antiepileptic phenytoin being the main drug associated with GE due to its high incidence (around 50%). The molecular mechanisms behind drug-induced gingival overgrowth are still unknown. By reverse transcription polymerase chain reaction, we demonstrate that the calcium-permeable ion channels TRPA1, TRPV1, and its capsaicin-insensitive isoform TRPV1b are expressed in human gingival fibroblasts (HGFs), the most abundant cellular type in periodontal tissue. Cultured HGFs responded with intracellular calcium elevations to phenytoin and to the canonical TRPA1 agonist allyl isothiocyanate. Application of phenytoin activated a nonselective cationic current in HGFs with a typical signature for TRPA1 channels. Moreover, this activation was blocked by HC030031, a specific TRPA1 blocker. Similarly, the use of shRNAs against hTRPA1 in HGFs reduced TRPA1 expression and activation by phenytoin. In addition, we show that phenytoin increased intracellular calcium levels in cells transfected with mouse or human TRPA1 channels. Responses to phenytoin were not observed in untransfected cells or cells expressing TRPM8 or TRPV1. The activation of HGFs by phenytoin was markedly reduced in the presence of antioxidant vitamins: ascorbic acid, folic acid, and α-tocopherol. By performing cell proliferation assays, we found that phenytoin did not augment the proliferation rate of HGFs. In contrast, alcian blue and picrosirius red staining of long-term HGFs cultures indicated that phenytoin induces extracellular matrix accumulation of collagen. Collectively, these findings support an important role of TRPA1 channels in phenytoin-induced GE, provide insight into the pathophysiologic mechanism, and offer novel therapeutic opportunities for its treatment.

Color stability and lipid oxidation of broiler breast meat from animals raised on organic versus non-organic production systems.

The aim of the present research was to evaluate the influence of organic and non-organic production systems on color stability and lipid oxidation of broiler meat Pectoralis major (PM) stored under refrigeration (4°C) for 9 days. PM samples from organic (ORG) and non-organic (NORG) production systems were compared based on physicochemical analyses (instrumental color, myoglobin concentration, metmyoglobin reducing activity (MRA), pH, and lipid oxidation) performed in 4 different trials (n = 4). In general, NORG broilers demonstrated higher (P < 0.05) b* and lipid oxidation values than ORG, whereas ORG samples exhibited increased (P < 0.05) MRA, ratio of reflectance at 630 per 580 nanometers (R 630/580), and a* values. The lower color stability observed in NORG samples can be partly due to lipid oxidation. Therefore, the production system can affect color and lipid stability of broiler breast meat during storage.

Efficacy and safety of combined piroxicam, dexamethasone, orphenadrine, and cyanocobalamin treatment in mandibular molar surgery.

Third molar extraction is a common procedure frequently accompanied by moderate or severe pain, and involves sufficient numbers of patients to make studies relatively easy to perform. The aim of the present study was to determine the efficacy and safety of the therapeutic combination of 10 mg piroxicam, 1 mg dexamethasone, 35 mg orphenadrine citrate, and 2.5 mg cyanocobalamin (Rheumazin) when compared with 20 mg piroxicam alone (Feldene) in mandibular third molar surgery. Eighty patients scheduled for removal of the third molar were included in this randomized and double-blind study. They received (vo) Rheumazin or Feldene 30 min after tooth extraction and once daily for 4 consecutive days. Pain was determined by a visual analogue scale and by the need for escape analgesia (paracetamol). Facial swelling was evaluated with a measuring tape and adverse effects and patient satisfaction were recorded. There was no statistically significant difference in facial swelling between Rheumazin and Feldene (control group). Both drugs were equally effective in the control of pain, with Rheumazin displaying less adverse effects than Feldene. Therefore, Rheumazin appears to provide a better risk/benefit ratio in the mandibular molar surgery. Since the side effects resulting from nonsteroidal anti-inflammatory drug administration are a severe limitation to the routine use of these drugs in clinical practice, our results suggest that Rheumazin can be a good choice for third molar removal treatment.

Quality of ham pâté containing bovine globin and plasma as fat replacers.

The effect of the incorporation of globin (10%), plasma (10%) and both combined (5% each) as fat replacers on the quality of ham paté was investigated. The chemical composition, the sensorial analysis (color, flavor and consistency) and the instrumental analysis of the texture (hardness, adhesiveness, elasticity, cohesiveness and tackiness) were evaluated. The results showed an increase of moisture and protein contents after the fat replacement, while the fat reduction of 25-35% led to the preparation of light products. No change was observed for the aroma, taste and the consistency of fat replacing products, but an intensification of the cohesivity and a reduction of color, hardness and tackiness were detected in some samples.

Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular endothelium.

Microfluorometric measurements in Fura-2-loaded single cultured human vascular endothelial cells were used to characterize the intracellular calcium [Ca2+]i responses triggered by extracellular application of adenosine 5'-triphosphate (ATP) and other nucleotides. Application of ATP or uridine 5'-triphosphate (UTP) gave rise to dose-dependent elevations of [Ca2+]i in all the cells tested. At saturating concentrations of agonist, the [Ca2+]i response was biphasic, with an early peak and a sustained plateau. Unlike peak responses, the sustained Ca2+ plateau was sensitive to removal of Ca2+ from the external medium. Mn2+ quenching revealed the presence of Ca2+ influx during the agonist-induced calcium plateau. The agonist-evoked calcium plateau was inhibited in a dose-dependent manner by the Cl-channel blocker NPPB, by the divalent cation Ni2+ and by the imidazole antimycotic econazole. Previously, these compounds have been shown to block store-operated Ca2+ entry. The two phases of the agonist-evoked [Ca2+]i response were blocked by the specific phospholipase C inhibitor U-73122 and by intracellular injection of low molecular weight heparin, suggesting the involvement of IP3-sensitive intracellular Ca2+ stores. The pharmacological profile of the response, using different nucleotides and analogues, ATP = UTP > ADP = UDP, and no responses to P2X1 and P2Y1 agonists, suggested the involvement of P2Y2 receptors. The expression of mRNA for the P2Y2 receptor was detected by RT-PCR analysis. These results indicate that P2Y2 receptors linked to intracellular Ca2+ mobilization are present in human vascular endothelial cells. The initial [Ca2+]i mobilization is followed by a phase of elevated [Ca2+]i influx.

Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones.

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.

Calcium-activated potassium channels in cultured human endothelial cells are not directly modulated by nitric oxide.

Nitric oxide has been proposed to directly activated large conductance Ca(2+)-dependent K+ channels (BKCa) [Bolotina V.M., Najibi S., Palacino J.J., Pagano P.J., Cohen R.A. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994; 368: 850-853]. The nitric oxide (NO) donor S-nitrosocysteine (SNOC) was used to evaluate a possible direct modulation of BKCa by NO in EAhy926 (EA cells), a cultured human umbilical vein derived endothelial cell line, using the whole-cell, cell-attached and inside-out configuration of the patch-clamp technique, together with simultaneous amperometric measurement of NO and the concentration of free intracellular calcium [Ca2+]i. BKCa channels with a large conductance of approximately 190 pS, voltage-dependent activation and a reversal potential close to -80 mV have been identified in EA cells. Exposure of EA cells in the experimental chamber to 1 mM SNOC delivered approximately 5 microM NO, as recorded by an amperometric probe in situ. SNOC produced a modest increases in [Ca2+]i that was insufficient to activate BKCa channels. NO alone neither activated BKCa channels directly nor modulated preactivated BKCa channels in EA cells. These results do not support a direct modulatory effect of NO on large conductance BKCa channels in cultured endothelial cells.

Postnatal changes in rat hypoglossal motoneuron membrane properties.

Intracellular recording techniques were used to characterize changes that take place in rat hypoglossal motoneuronal excitability from early postnatal stages to adulthood. This study focused primarily on the first two weeks of postnatal life, when major changes in the maturation of the neuromuscular system take place. Neonatal hypoglossal motoneurons were identified by their location within the hypoglossal nucleus and by their characteristic electrophysiology. These criteria were supported by antidromic activation and intracellular staining of retrogradely labeled hypoglossal motoneurons. Action potential duration decreased progressively during postnatal development. The reduction was primarily due to a more rapid repolarization, suggesting developmental changes in voltage-dependent potassium conductances. The duration of the calcium-dependent afterhyperpolarization decreased by half during the first two weeks of postnatal life. Changes in subthreshold responses included a decrease in input resistance and an increase in the degree of hyperpolarizing sag and inward rectification with age. Rheobase current was negatively correlated with input resistance, and increased progressively during postnatal development. Membrane time constant decreased almost four-fold over the first two postnatal weeks, suggesting that membrane resistivity is not constant. This decrease in membrane resistivity could account for a large fraction of the change in input resistance and rheobase with age. Thus, the early postnatal development of the rat includes systematic changes in the electrophysiological properties of motoneurons innervating tongue muscles. Some of these modifications are not easily explained by a mere change in neuronal surface area but likely involve changes in the density of expressed ion channels.

Double- and triple-labeling of functionally characterized central neurons projecting to peripheral targets studied in vitro.

The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysiological properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties. The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells.

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.

Thyrotropin-releasing hormone causes excitation of rat hypoglossal motoneurons in vitro.

A brainstem slice preparation and conventional intracellular recording techniques were used to study the effects of the neuropeptide thyrotropin-releasing hormone (TRH) on adult rat hypoglossal motoneurons (HMs) in vitro. We found that TRH (0.1-10 microM) had two effects: 1) it caused depolarization of HMs and 2) decreased their input conductance. We tested the effects of TRH on HMs using a sinusoidal current injection paradigm to approximate the rhythmic activity of HMs in vivo. Waves of injected current that elicited only subthreshold behavior in control conditions caused repetitive firing in the presence of TRH. Compensating for the TRH-induced depolarization by hyperpolarizing direct current injection revealed the consequences of decreased input conductance in isolation. Under these conditions, the spike-firing of HMs at the peak of the sinusoid was still enhanced. In addition, the maximal hyperpolarization at the nadir of the current wave was also increased. This suggests that in the presence of TRH, the effects of both excitatory and inhibitory synaptic inputs on HMs are enhanced and the contrast between excitation and inhibition is sharpened.

Development of hypoglossal motoneurons.

Hypoglossal motoneurons (HMs) are brain stem motoneurons that innervate tongue muscles. Their function is critical in the control of the upper airway. Results from in vitro studies of rat HMs have shown that properties of HMs change during the postnatal period. For example, these studies have uncovered changes in HM morphology and electrical properties (both in ion channels and firing properties) as well as changes in chemical synaptic transmission to HMs during the postnatal period. Morphologically, a marked reduction in complexity of the dendritic tree takes place over the first 2 wk postnatal. In terms of electrical properties, a substantial and progressive fall in motoneuronal input resistance occurs during the first month of life, due to a decrease in specific membrane resistivity. This is primarily responsible for the progressive increase in rheobase and consequent reduction in cell excitability. In addition, the densities of at least two types of membrane ion channels are altered in early postnatal life, contributing to changes in their electroresponsive properties. On the one hand, the depolarizing mixed cationic current that is activated by membrane hyperpolarization was found to be approximately 10-fold larger in adult than in neonatal HMs. By contrast, neonatal HMs possess a transient low-voltage-activated T-type Ca2+ channel with a low single-channel conductance (approximately 7 pS), the density of which rapidly declines during the early postnatal period. The functional relevance of these and other changes occurring during the postnatal period is discussed.

Effects of thyrotropin-releasing hormone on rat motoneurons are mediated by G proteins.

Numerous transmitter receptors are linked via GTP-binding proteins (G proteins) to membrane phosphoinositide metabolism by phospholipase C (PLC) and generation of second messengers such as activated protein kinase C (PKC), inositol trisphosphate (IP3) and/or elevations in intracellular calcium. In many cases, these same receptors also inhibit a resting ('leak') potassium current (IK(L)), thereby depolarizing neurons. It is unclear if activation of this PLC pathway mediates inhibition of IK(L) by neurotransmitter receptors. Therefore, we tested the contribution of this pathway to the TRH-induced inhibition of IK(L) in rat hypoglossal motoneurons (HMs) using conventional intracellular recording in brainstem slices. When HMs were recorded with electrodes containing 3 M KCl or 30 mM GTP (in KCl), TRH induced a depolarization that recovered quickly (within 8-10 min) and could be repeated with only modest tachyphylaxis (< 20%). However, with electrodes containing the non-hydrolyzable G protein activator, GTP gamma S (10 mM), the TRH-induced depolarization was long lasting (up to 1 h); with electrodes containing the G protein inhibitor, GDP beta S (20 mM) the tachyphylaxis with repeated TRH application was exaggerated (approximately 60%). Activation of PKC by phorbol dibutyrate (10 microM in perfusate) neither mimicked nor occluded the effects of TRH. There were no effects on membrane potential, input resistance (RN) or the response to TRH in HMs during long recordings with electrodes containing high concentrations of IP3 (60 mM).(ABSTRACT TRUNCATED AT 250 WORDS)