The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block

Summary The time course and extent of local anaesthetic blocks within the spinal cord of cats were evaluated. A monopolar stimulation electrode with the tip lowered into the dorsal columns (DC) 1000 m below cord surface was used to activate antidromically DC fibers at the T13 level and evoke cord dorsum potentials at the level of the lumbar spinal cord. The amplitude of the negative deflection, the N-wave, was determined for various stimulation intensities (stimulation-response-function, SRF). Lidocaine (1%) was microinjected in volumes of 0.5 or 1.0 l into the DC from a glass micropipette 1 mm caudal to the stimulation site. Conduction block was characterized by a reversible shift of the SRFs to higher stimulation intensities. The diameter of the blocked area in the transverse plane was evaluated from threshold intensities and was found to be 0.9±0.1 mm 4 to 30 min after the injection of 0.5 l lidocaine and 1.6±0.36 mm 10 to 45 min after the injection of 1.0 l lidocaine. In the sagittal plane, the diameter of the blocked area following 1.0 l lidocaine was found to be up to 2.8 mm. The DC-block was reversible within 92 min following injection of 1.0 l and 69 min after the injection of 0.5 l lidocaine. The application of the present findings for blocks in other CNS structures is discussed.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at -molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10–100 ms.In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1–3 m internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had to be found in which channels open at a relatively low frequency.     

Afferent control of human stance and gait: evidence for blocking of group I afferents during gait

Summary The cerebral potentials (c.p.) evoked by electrical stimulation of the tibial nerve during stance and in the various phases of gait of normal subjects were compared with the c.p. and leg muscle e.m.g. responses evoked by perturbations of stance and gait. Over the whole step cycle of gait the c.p. evoked by an electrical stimulus were of smaller amplitude (3 V and 9 V, respectively) than that seen in the stance condition, and appeared with a longer latency (mean times to first positive peak: 63 and 43 ms, respectively). When the electrical stimulus was applied during stance after ischaemic blockade of group I afferents, the c.p. were similar to those evoked during gait. The c.p. evoked by perturbations were larger in amplitude than those produced by the electrical stimulus, but similar in latencies in both gait and stance (mean 26 V and 40 V; 65 ms and 42 ms, respectively) and configurations. The large gastrocnemius e.m.g. responses evoked by the stance and gait perturbations arose with a latency of 65 to 70 ms. Only in the stance condition was a smaller, shorter latency (40 ms) response seen. It is concluded that during gait the signals of group I afferents are blocked at both segmental and supraspinal levels which was tested by tibial nerve stimulation. It is suggested that the e.m.g. responses induced in the leg by gait perturbations are evoked by group II afferents and mediated via a spinal pathway. The c.p. evoked during gait most probably reflect the processing of this group II input by supraspinal motor centres for the coordination of widespread arm and trunk muscle activation, necessary to restablish body equilibrium.     

The paratrigeminal nucleus. I. Neurons and synaptic organization

Summary The paratrigeminal nucleus, a diffuse collection of neurons on the lateral medullary surface, lies embedded in the fibres of the restiform body, ascending spinocerebellar tract and the spinal tract of the trigeminal nerve. Its rostrocaudal extent is approximately 1500–2000m in the rat, 2800–3500 m in the rhesus monkey, and 5000–6000/m in the human, where it is a well-defined nucleus. In Golgi preparations the neuronal somata are generally fusiform, ranging from 8–15 m in width and 15–25/m in length. Electron microscope studies show that their polar dendritic processes are thick and long and they intertwine in bundles among islands of cells or myelinated fibres. The perikarya have a high nucleus to cytoplasm ratio and the scanty cytoplasm is conspicuous for its paucity of organelles and its plethora of polysomal arrays. The neuropil is complex and contains a heterogeneous population of axonal varicosities displaying markedly different axoplasmic structures. Many different types of large granular vesicle-containing axons prevail. These axons engage in axo—somatic, axo—dendritic, axo—spinous and axo—axonic synapses with the processes of the cells within the nucleus.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Cytochalasin B: Evidence for a membrane-directed action in B-cells from rat pancreatic islets

Summary In the presence of cytochalasin B (CCB) concentrations from 50 to 200 g/ml there is a dose-dependant inhibition of insulin release from isolated rat pancreatic islets. Inhibition is unspecific with respect to glucose, leucine, tolbutamide or theophylline and is reversible. Production of¹⁴CO₂ from uniformly labeled D-glucose is decreased. Islets pretreated with an high (200 g/ml) or low (10 g/ml) CCB dose release more insulin in response to a subsequent glucose or leucine stimulus in a CCB free medium compared with controls. The data are compatible with a membrane-directed action of CCB.     

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or the leukotrienes (LT) C₄ and E₄ (1 M) but not D₄ (1 M) appliedin vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 M) or verapamil (20 M) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Forskolin inhibition of hexose transport in cardiomyocytes

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75×10^–2 nmol/mg cell protein/second in the absence of insulin, and 8.25×10^–2 nmol/mg cell protein/second in the presence of 12.3 M insulin. Forskolin at 5 M nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparentK _i for hexose transport inhibition was about 0.3 M forskolin. Epinephrine and isoproterenol at 50 M increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We coclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.Supported by NIH-HL 07094     

The use of microelectrodes for measurement of local H+ activity in the cortical subarachnoidal space of cats

Summary pH microelectrodes with pointed tip (Hinketype) were constructed for the continuous measurement of the local pH in the perivascular space of pial arteries in the feline cerebral cortex. The sensitive tip had a length of 20–60 and a base diameter of 10–25 . As reference electrode, a micropipette (tip diameter 2 ), filled with 150 mM KCl was used. Calibration curves were linear and showed a sensitivity of 54.5–57.5 mV/pH unit at 38°C. Advantages of such electrodes are the easy penetration of the subarachnoid membrane, the long life span, the quick response, and a minimal drift.—The electrodes were tested in vivo during hyper- and hypoventilation and during local perivascular injection of mock spinal fluid at varying pH. A close correlation was observed between the change in perivascular pH and the corresponding change in pial arterial diameter.A preliminary report of these investigations was presented at the autumn meeting of the German and Austrian Physiological Society, Vienna, Sept. 23–26, 1975, Pflügers Arch., Suppl. R148 (1975)Supported by the Deutsche Forschungsgemeinschaft     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Dynamics of the Golgi method: a time-lapse study of the early stages of impregnation in single sections

Summary This paper describes the early stages of impregnation by the Golgi method. Sections of aldehyde-fixed and potassium dichromate-treated cerebral cortex were mounted on glass slides and cover slipped. The dichromate solution was replaced by silver nitrate solution and events in the section were followed and recorded by time lapse microphotography and video recording until stopped by replacement of silver nitrate solution by glycerol. The sections were subsequently prepared for electron microscopy (EM) study.In sections about 2×2 mm and 100 m thick a fine, dark granular precipitate formed at the edges within the first minutes of exposure to silver nitrate and a wave of brownish colouration spread to a depth of about 0.3 mm. After approximately 7 min, shrub-like focal precipitates (nucleation centres) appeared in the sections. From these nucleation centres (but also from the section edges) thread-like outgrowths, usually identified as dendrites, spread into somata. Sometimes impregnation began within the soma and spread into dendrites. The rate of impregnation (i.e., of silver chromate deposition within dendrites) was typically 1–7 m min^s-1, faster in the earlier stages (up to 3 m s^s-1) and very slow after 30 min, by which time many neurons were more or less fully impregnated.The dimensions of the section, the width of an agar frame around the sections, and the frequency with which the silver nitrate in the sections was replenished all affected the extent and time course of the impregnation.By EM the earliest intracellular deposits consisted of tubulolamellar formations which did not cross plasma or endocellular membrane boundaries and which contained irregularly shaped and scattered granules, initially about 10 nm in diameter. The latter progressively enlarged and coalesced as the tubulolamellar formations extended, eventually to fill the cross-sectional area of neuronal processes and cell bodies.These observations shed light on why so few neurons become impregnated with the Golgi method. Impregnation occurred only in those cells a part of which was within a nucleation centre.     

Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K+ channels

A swelling-activated, background K⁺ current in the corneal epithelium is characteristically activated by fenamates and inhibited by diltiazem. Fatty acids also stimulate this current, indicating that its origin is a lipid-sensitive mechano-gated 2P domain K⁺ channel. In the present study, modulation of TREK-1, TREK-2, and TRAAK channels by fenamates and diltiazem was examined. TREK-1, TREK-2, and TRAAK currents transiently expressed in COS-7 cells were recorded by the perforated-patch configuration. As previously reported, arachidonic acid (20 M) stimulated all of these channels, and a volatile anesthetic, halothane (1 mM) augmented TREK-1 and TREK-2 but not TRAAK. Flufenamic acid (FA, 100 M), niflumic acid (NA, 100 M), and mefenamic acid (MA, 100 M) markedly stimulated TREK-1, TREK-2, and TRAAK. The potency sequence for the activation of TREK-1 and TREK-2 was FA > NA = MA, and the potency sequence for the activation of TRAAK was FA = NA > MA. Diltiazem (1 mM) inhibited TREK-1 and TREK-2, but not TRAAK. In conclusion, fenamates are openers of the lipid-sensitive mechano-gated 2P domain K⁺ channels, and diltiazem may be a specific blocker for TREK. These novel findings could help to further understand channel functions of the mechano-gated 2P domain K⁺ channels.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

A quasi-totally shielded,low-capacitance glass-microelectrode with suitable amplifiers for high-frequency intracellular potential and impedance measurements

The fabrication of a new glass microelectrode for high-speed recording is reported. It consists of a Ling-Gerard microelectrode which is slit into a spacer capillary with the tip slightly protruding. Except for the ultimate tip (of 30 m length) the entire assembly is coated with silver and insulated. After proper insertion into the cell the electrode provides a shield, which extends from the surface of the cell membrane up to the input of the amplifier. This electrode has two advantages: 1. it eliminates all capacitive pickup of bath potentials which is of paramount importance in impedance measurements and 2. if used in conjunction with negative capacitance amplifiers, it allows more faithful high-frequency recordings, than was possible with the conventional techniques, because the capacitance neutralization can be properly set even though the non-ideal transmission properties of the electrode tips are not known. Test experiments using a rapid amplifier circuit of own design show that the electrode assembly allows error-free phaseshift measurements of up to 30 kHz to be obtained with electrodes of 20 M or risetimes (10–90%) of squarewave pulses of 10 to 12 s. The corresponding time constants of 5 to 6 s indicate that the time resolution of the new technique is superior to the apparent time resolution of the conventional techniques.     

Mechanical properties of mammalian single smooth muscle cells I. A low cost large range microforce transducer

Summary A transducer has been developed for measuring the minute forces generated during isometric contractions (1.0–10.0N) of single smooth muscle cells from the pig urinary bladder and the human uterus. In addition to its high sensitivity, resolution and stability (100 mVN^–1, <0.1N and <2.0N h^–1), the transducer features a very wide range (100–140N) with good linearity, enabling measurement of contractions as well as passive force-length characteristics within one uninterrupted measurement session. Since the transducer features an independent and interchangeable force to displacement conversion system, different force ranges can be realized by inserting force conversion systems with different compliances.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.