LIBS analysis of artificial calcified tissues matrices.

In most laser-based analytical methods, the reproducibility of quantitative measurements strongly depends on maintaining uniform and stable experimental conditions. For LIBS analysis this means that for accurate estimation of elemental concentration, using the calibration curves obtained from reference samples, the plasma parameters have to be kept as constant as possible. In addition, calcified tissues such as bone are normally less "tough" in their texture than many samples, especially metals. Thus, the ablation process could change the sample morphological features rapidly, and result in poor reproducibility statistics. In the present work, three artificial reference sample sets have been fabricated. These samples represent three different calcium based matrices, CaCO3 matrix, bone ash matrix and Ca hydroxyapatite matrix. A comparative study of UV (266 nm) and IR (1064 nm) LIBS for these three sets of samples has been performed under similar experimental conditions for the two systems (laser energy, spot size, repetition rate, irradiance, etc.) to examine the wavelength effect. The analytical results demonstrated that UV-LIBS has improved reproducibility, precision, stable plasma conditions, better linear fitting, and the reduction of matrix effects. Bone ash could be used as a suitable standard reference material for calcified tissue calibration using LIBS with a 266 nm excitation wavelength.

Nanosecond and femtosecond laser ablation of brass: particulate and ICPMS measurements.

Femtosecond and nanosecond lasers were compared for ablating brass alloys. All operating parameters from both lasers were equal except for the pulse duration. The ablated aerosol vapor was collected on silicon substrates for particle size measurements or sent into an inductively coupled plasma mass spectrometer. The diameters and size distribution of particulates were measured from scanning electron microscope (SEM) images of the collected ablated aerosol. SEM measurements showed that particles ablated using nanosecond pulses were single spherical entities ranging in diameter from several micrometers to several hundred nanometers. Primary particles ablated using femtosecond ablation were approximately 100 nm in diameter but formed large agglomerates. ICPMS showed enhanced signal intensity and stability using femtosecond compared to nanosecond laser ablation.

Laser plasma spectroscopy of Al-Cu-Fe quasicrystal an d alloy.

Electron number density and temperature were determined from laser-induced plasmas produced by irradiating Al-Cu-Fe targets of a quasicrystal and of an alloy of similar composition. The Al(I) atomic emission spectra of the two systems were measured as a function of the distance from the target and of the time delay after laser irradiation. Differences of plasma characteristics were observed for laser ablation of quasicrystal and alloy targets, and the results were interpreted on the basis of different plasma formation mechanisms for the two systems.

Existence of phase explosion during laser ablation and its effects on inductively coupled plasma-mass spectroscopy.

A sudden increase in crater depth was observed during high irradiance (> 10(10) W/cm2) laser ablation of silicon, and it is attributed to the phenomenon of phase explosion. The threshold irradiance for phase explosion showed a dependence on two laser parameters: laser beam spot size and wavelength. For a larger beam size and longer incident wavelength, a higher laser irradiance was required to generate phase explosion. The rapid increase of crater depth above the phase explosion threshold irradiance correlated with a significant increase in the ICPMS signal intensity. The ratio of crater volume to ICPMS intensity, which represents entrainment efficiency, remained the lowest at laser irradiances slightly above the phase explosion threshold. However, this ratio increased at irradiances well above the threshold (> 10(11) W/cm2). Chemical analysis using laser ablation at irradiance above 10(11) W/cm2 provides increased sensitivity via improved entrainment and transport efficiency and increased ablation rate.

Dorsal root potential produced by a TTX-insensitive micro-circuitry in the turtle spinal cord.

1, The mechanisms underlying the dorsal root potential (DRP) were studied in transverse slices of turtle spinal cord. DRPs were evoked by stimulating one filament in a dorsal root and were recorded from another such filament. 2. The DRP evoked at supramaximal stimulus intensity was reduced but not eliminated after blockade of GABAA receptors. The remaining component was eliminated by blocking NMDA and AMPA receptors. 3. The DRP was reduced but not eliminated after blockade of AMPA receptors. The early component of the remaining DRP was dependent on GABAA receptors and the residual component on NMDA receptors. 4. The DRP was reduced but not eliminated by TTX. GABAA, NMDA and AMPA receptors contributed to the generation of the TTX-insensitive DRP. The early component of the DRP in the presence of TTX depended on GABAA receptor activation, and the late component mainly on the activation of NMDA receptors. 5. Our results show that part of the DRP is generated by a TTX-resistant, probably non-spiking micro-circuit with separate components mediated by GABA and glutamate.

Dynamics of intrinsic electrophysiological properties in spinal cord neurones.

The spinal cord is engaged in a wide variety of functions including generation of motor acts, coding of sensory information and autonomic control. The intrinsic electrophysiological properties of spinal neurones represent a fundamental building block of the spinal circuits executing these tasks. The intrinsic response properties of spinal neurones--determined by the particular set and distribution of voltage sensitive channels and their dynamic non-linear interactions--show a high degree of functional specialisation as reflected by the differences of intrinsic response patterns in different cell types. Specialised, cell specific electrophysiological phenotypes gradually differentiate during development and are continuously adjusted in the adult animal by metabotropic synaptic interactions and activity-dependent plasticity to meet a broad range of functional demands.

Inhibitory control of plateau properties in dorsal horn neurones in the turtle spinal cord in vitro.

1. The role of inhibition in control of plateau-generating neurones in the dorsal horn was studied in an in vitro preparation of the spinal cord of the turtle. Ionotropic and metabotropic inhibition was found to condition the expression of plateau potentials. 2. Blockade of gamma-aminobutyric acid (GABAA) and glycine receptors by their selective antagonists bicuculline (10-50 microM) and strychnine (5-20 microM) enhanced the excitatory response to stimulation of the dorsal root and facilitated the expression of plateau potentials. 3. Bicuculline and strychnine also facilitated the generation of plateau potentials in response to depolarizing current pulses, suggesting the presence of tonic ionotropic inhibitory mechanisms in turtle spinal cord slices. 4. Activation of GABAB receptors also inhibited plateau-generating neurones. The selective agonist baclofen (5-50 microM) inhibited wind-up of the response to repeated depolarizations induced synaptically or by intracellular current pulses. 5. Baclofen reduced afferent synaptic input. This effect was not affected by bicuculline or strychnine and was blocked by the selective GABAB receptor antagonist 2-hydroxysaclofen (2-OH-saclofen, 100-400 microM). 6. Postsynaptically, baclofen inhibited plateau properties. Activation of GABAB receptors produced a hyperpolarization (7.0 +/- 0.5 mV, mean +/- S.E.M., n = 29) with an associated decrease in input resistance (22.7 +/- 3.1%, n = 24). These effects were blocked by extracellular Ba2+ (1-2 mM). 7. When the baclofen-induced hyperpolarization and shunt were compensated for by adjusting the bias current and the strength of the stimulus, baclofen still inhibited generation of plateau potentials. Wind-up and after-discharges were also inhibited by baclofen. These effects remained in the presence of tetrodotoxin (1 microM) and were antagonized by 2-OH-saclofen. 8. The inhibition of plateau properties was observed even when the baclofen-induced hyperpolarization and shunt were blocked by Ba2+ and when potassium channels were blocked by Ba2+ (3 mM), tetraethylammonium (TEA, 15 mM) and apamin (0.25-0.5 microM). The baclofen-sensitive component of the plateau potential was reduced by nifedipine (10 microM), suggesting a modulation of postsynaptic L-type Ca2+ channels. 9. We suggest that inhibitory regulation of plateau properties plays a role in somatosensory processing in the dorsal horn. The inhibitory control of wind-up and after-discharges may be particularly significant in physiological and therapeutic control of central sensitization to pain.

Transient isotachophoretic-electrophoretic separations of lanthanides with indirect laser-induced fluorescence detection.

Indirect laser-induced fluorescence was used for the detection of several lanthanide species separated by capillary electrophoresis. Quinine sulfate was the fluorescent component of the background electrolyte, and α-hydroxyisobutyric acid was added as a complexing agent to enable the separation of analyte ions that have similar mobilities. The UV lines (333-364 nm) of an argon ion laser were used as the excitation source with a diode array detector for monitoring the fluorescent emission at 442 nm. Electrokinetic injections and transient isotachophoresis were implemented to stack the analyte ions into more concentrated zones. On-line preconcentration factors were determined to be ∼700 and resulted in limits of detection for La(3+), Ce(3+), Pr(3+), Nd(3+), Sm(3+), and Eu(3+) in the low-ppb range (6-11 nM).

The cerebral hemisphere of the turtle in vitro. An experimental model with spontaneous interictal-like spikes for the study of epilepsy.

Slice in vitro preparations have been useful to study the cellular basis of some epilepsy related phenomena. However, the cellular mechanisms that generate ictal activity remain poorly understood. Therefore, an experimental in vitro model capable of generating seizure-like activity might contribute to the study of the cellular basis of seizures. The outstanding resistance to hypoxia of turtles enabled us to develop an in vitro preparation that keeps all the cortical neural circuitry intact. A whole cerebral hemisphere of the turtle Chrysemys d'orbigny was isolated (n = 45) and simultaneous electrographic and intracellular recordings were performed in the medial cortex. The electrographic activity was composed by a non-rhythmic, low-voltage (10-20 microV) activity interrupted by spontaneous large (50-700 microV) sharp waves (LSWs). The cellular counterpart of the LSWs was often a burst of action potentials that resembled the paroxysmal depolarisation shift (PDS). Bicuculline (20-40 microM, n = 20) increased the interictal-like activity and in some preparations (3 out of 20) provoked seizure-like events. Complex bursting activity and a slow afterhyperpolarisation were cellular events observed during seizures. We propose that this model might be a valuable tool for the study the cellular mechanisms involved in the transition from the interictal to the ictal activities.

Modulation of plateau properties in dorsal horn neurones in a slice preparation of the turtle spinal cord.

1. Modulation of plateau properties in dorsal horn neurones was studied in a transverse slice preparation of the spinal cord of the turtle. In plateau-generating neurones high frequency stimulation of the ipsilateral dorsal root (10-20 Hz, 0.5-2 min) produced a slow depolarization (2.9 +/- 0.6 mV, mean +/- S.E.M.; n = 6) and enhanced the properties mediated by dihydropyridine-sensitive Ca2+ channels. The tetanic stimulus facilitated wind-up and after-discharges even when fast synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 microM), (+/-)-2-amino-5-phosphonopentanoic acid (AP5, 100 microM), bicuculline (10-20 microM) and strychnine (5-20 microM). 2. Application of cis-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10-50 microM) produced a slow depolarization (5.9 +/- 0.5 mV, n = 21) accompanied by an increase in input resistance (28.8 +/- 5.1%, n = 12). 3. ACPD increased the excitability by facilitating the plateau properties. In the presence of tetrodotoxin (TTX, 1 microM) a lower threshold and a slower decay of the plateau potential were observed. These effects resulted in facilitation of wind-up and prolonged after-discharges. 4. All ACPD-induced effects were blocked by alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM), a selective antagonist of metabotropic glutamate receptors. The selective agonist for the type I metabotropic glutamate receptor ((RS)-3,5-dihydrophenylglycine (DHPG, 50 microM)) reproduced all the effects of ACPD. 5. Application of a supposed neuromodulator, substance P (1-2 microM) produced a transient depolarization (4 +/- 0.6 mV) lasting 4-6 min during continued application of substance P. Variable effects on the input resistance were observed, a slight increase (12 +/- 2%) being the most frequent. In 61% of the cells, substance P induced a clear increase in excitability with no detectable change in input resistance or membrane potential. 6. The effects of substance P on plateau properties were indistinguishable from those produced by ACPD. Unlike the transient depolarization, the facilitation of the plateau properties persisted in the presence of the agonist. 7. The substance P-induced facilitation of the plateau potential was blocked by GR 82334 (5-10 microM), a selective NK-1 tachykinin-receptor antagonist, and was not affected by MEN 10376 (2 microM), a selective NK-2 antagonist. 8. The facilitation of plateau properties produced by dorsal root stimulation was also reduced by antagonists of metabotropic glutamate receptors and NK-1 tachykinin receptors. 9. We propose that modulation of postsynaptic plateau properties in dorsal horn neurones by activation of type I metabotropic glutamate receptors and NK-1 tachykinin receptors is involved in processing nociceptive information.

Analysis of fission products using capillary electrophoresis with on-line radioactivity detection.

Capillary electrophoresis has been used to separate metal ions characteristically associated with nuclear fission. Indirect UV absorbance and on-line radioactivity detection were used simultaneously to monitor the analytes. The radioactivity detector consists of conical plastic scintillating material with the capillary passing through the center to provide a 4π detection geometry. The wide end of the cone is optically coupled to a photomultiplier tube. Transient isotachophoretic techniques were employed to stack large volumes of samples which had low specific activities. Radioactivity detection of (152)Eu and (137)Cs was achieved at the nanocurie level for 80-100 nL injections. The detector is approximately 80% efficient, enabling samples resident in the detector window for 0.1 min to be reliably assayed. The separation of (137)Cs and (137m)Ba isotopes, which are in secular equilibrium, was modeled to demonstrate the effects of the rapid decay of (137m)Ba.

Burst-generating neurones in the dorsal horn in an in vitro preparation of the turtle spinal cord.

1. In transverse slices of the spinal cord of the turtle, intracellular recordings were used to characterize and analyse the responses to injected current and activation of primary afferents in dorsal horn neurones. 2. A subpopulation of neurones, with cell bodies located centrally in the dorsal horn, was distinguished by the ability to generate a burst response following a hyperpolarization from rest or during a depolarization from a hyperpolarized holding potential. The burst response was inactivated at the resting membrane potential. 3. The burst response was mediated by a low threshold Ca2+ spike assumed to be mediated by T-type Ca2+ channels since it resisted tetrodotoxin and was blocked by 3 mM Co2+ or 100-300 microM Ni2+ and resembled the low threshold spike (LTS) described elsewhere. 4. Some burst-generating cells also displayed plateau potentials mediated by L-type Ca2+ channels. In these cells the burst following a hyperpolarizing current pulse, applied from the resting membrane potential, facilitated the activation of the plateau potential. Wind-up of the plateau potential was produced when the hyperpolarizing pulse generating the burst was repeated at 0.1-0.3 Hz or faster. 5. The burst response and the underlying low threshold Ca2+ spike were activated synaptically by primary afferent stimuli in a voltage range hyperpolarized from the resting membrane potential. 6. Cells with bursts were morphologically distinguishable from cells with bursts and plateau properties. 7. Our findings in this and the preceding paper show that the intrinsic response properties of particular subtypes of neurones in the dorsal horn have a profound influence on the amplitude and time course of the responses mediated by primary afferent fibres. We predict that these postsynaptic properties are probable targets for synaptic modulation.

Plateau-generating neurones in the dorsal horn in an in vitro preparation of the turtle spinal cord.

1. In transverse slices of the spinal cord of the turtle, intracellular recordings were used to characterize and analyse the responses to injected current and activation of primary afferents in dorsal horn neurones. 2. A subpopulation of neurones, with cell bodies located laterally in the deep dorsal horn and dendrites radiating towards the pial surface, was distinguished by the ability to generate plateau potentials. Activation of the plateau potential by a suprathreshold depolarizing current pulse produced an increasing firing frequency during the first few seconds and a sustained after-discharge. 3. The plateau potential was assumed to be mediated by L-type Ca2+ channels since it was blocked by Co2+ (3 mM) and nifedipine (10 microM) and enhanced by Bay K 8644 (0.5-2 microM). 4. The threshold for activating the plateau potential declined during the first few seconds of depolarization. The decline in threshold gradually subsided over 3-10 s after repolarization. 5. Frequency potentiation of the plateau potential contributed to wind-up of the response to depolarizing current pulses and primary afferent stimuli repeated at frequencies higher than 0.1-0.3 Hz. 6. The sustained after-discharge mediated by the plateau potential was curtailed by a slow after-hyperpolarization (sAHP) evoked by strong depolarizations. The relative strength of the plateau potential and sAHP varied among cells. In some cells the plateau potential and sAHP interacted to produce damped oscillations upon depolarization. The sAHP was mediated by both apamin and tetraethylammonium (TEA)-sensitive K+ channels. 7. Our findings suggest that basic properties of sensory integration may reside with the specialized intrinsic response properties of particular subtypes of neurones in the dorsal horn.

Monosynaptic connections between primary afferents and giant neurons in the turtle spinal dorsal horn.

This paper reports the occurrence of monosynaptic connections between dorsal root afferents and a distinct cell type-the giant neuron-deep in the dorsal horn of the turtle spinal cord. Light microscope studies combining Nissl stain and transganglionic HRP-labeling of the primary afferents have revealed the occurrence of axosomatic and axodendritic contacts between labeled boutons and giant neurons. The synaptic nature of these contacts has been confirmed by use of electron microscope procedures involving the partial three-dimensional reconstruction of identified giant neurons. Intracellular recording in spinal cord slices provided functional evidence indicating the monosynaptic connections between dorsal root afferents and giant neurons. The recorded neurons were morphologically identified by means of biocytin injection and with avidin conjugates. Electrical stimulation of the ipsilateral dorsal roots evoked synaptic responses with short, fixed latencies (1.6-5.6 ms), which remained unchanged at high frequencies (10 Hz). Excitatory polysynaptic potentials were also observed. By means of pharmacological procedures the short-latency response was dissected in two components: one insensitive to tetrodotoxin, the other abolished by the drug. The toxin-resistant component was presumed to be sustained by small-diameter C fibers. The synaptic response was mainly mediated by the glutamate-AMPA receptor subtype; however, a small component mediated by NMDA receptor was also present.

Short-term plasticity in turtle dorsal horn neurons mediated by L-type Ca2+ channels.

Windup--the gradual increase of the response--of dorsal horn neurons to repeated activation of primary afferents is an elementary form of short-term plasticity that may mediate central sensitization to pain. In deep dorsal horn neurons of the turtle spinal cord in vitro we report windup of the response to repeated depolarizing current pulses as well as to repeated stimulation of the ipsilateral dorsal root. We found both forms of windup to be mediated by a depolarizing potential produced by increasing activation of postsynaptic L-type Ca2+ channels. These results suggest a central role for intrinsic postsynaptic properties in nociceptive plasticity and for L-type Ca2+ channels as a promising target for therapeutic intervention.

Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex.

The electroencephalogram of the in vitro brain of the turtle Chrysemys d' orbigny shows spontaneous random large sharp waves (LSWs) which may be compared to interictal spikes. In order to evaluate the role of excitatory amino acids (EAAs)--in particular through the N-methyl-D-aspartate (NMDA) receptor--in the generation of LSWs, the bath application of NMDA and its antagonists 3-((+/-)-2-carboxypiperazin-4y)-propyl-1-phosphonic acid (CPP) and DL-2-amino-5-phosphonovaleric acid (APV), was performed in the whole open hemisphere (WOH) in vitro. Field recordings in WOH showed that both CPP and APV unexpectedly increased LSW amplitude. Consistently, NMDA in the bath suppressed the LSWs. Iontophoretically applied glutamate, kainate and NMDA produced a hyperpolarization of intracellularly recorded medial cortex pyramidal cells both in WOH and in slices. The EAA-induced hyperpolarization was tetrodotoxin (TTX) and bicuculline sensitive and reversed close to -70 mV. It would therefore seem to be due to the activation of gamma-aminobutyric acid (GABA) interneurons. The NMDA could also produce an excitation of pyramidal cells--always following a previous inhibitory phase. In some cases rhythmic bursting discharges or plateau potentials were observed. These NMDA effects were mainly elicited by a direct effect on pyramidal cells. A long-lasting hyperpolarizing response following the NMDA excitatory phase was also observed. This long-lasting response was an intrinsic property of pyramidal cells since it was TTX resistant. This study demonstrates that GABAergic interneurons from the turtle medial cortex can be activated by EAAs, a mechanism that can account for the effects of NMDA antagonists on LSWs.

Electroencephalogram in vitro and cortical transmembrane potentials in the turtle Chrysemys d'orbigny.

An experimental stable model of an in vitro turtle brain (Chrysemys d'orbigny) was developed in order to compare electrographic activity (EEG) with transmembrane potentials. Two preparations were used: a whole intact hemisphere and a whole open hemisphere. The latter permitted easier impalement of cortical neurons through the ependymal surface. The EEG characteristics were similar to those described in turtles in vivo. The EEG was nonrhythmic (rhythmicity coefficient less than 0.40). The power spectrum presented a high energy band between 1 and 3 Hz, decreasing progressively towards the higher frequencies. Total power of the EEG was one order of magnitude greater than the system noise. Random large amplitude sharp waves (22-300 microV, 500-1,900 ms) were recorded spontaneously. Hypoxia produced an increase in frequency and amplitude of the large sharp waves, without modification of either EEG background activity or membrane potentials. Physostigmine provoked the disappearance of the large sharp waves, an effect reversed by atropine. The addition of TTX to the medium provoked the abolition of the EEG, although spikes and plateaus determined by Ca2+ conductances persisted. The power spectra band of maximum relative potency was 0.8-2.5 Hz for both EEG and slow membrane potentials.

Collinear photothermal deflection spectroscopy with light-scattering samples.

An analytical model that incorporates effects of light scattering was developed for dual-beam photothermal deflection spectroscopy. Thermal gradients induced by a modulated excitation beam deflect an optical probe beam which was treated as being of finite dimensions. Mechanisms by which thermal gradients produce refractive index gradients are discussed, with an explicit expression for dn/dT being derived. Experimental studies with suspensions of small latex particles in Nd(3+) solutions demonstrated that the model accurately predicts both the shape of the deflection signal and the attenuation of the signal due to light scattering. The absolute magnitude of the observed signal is approximately predicted by theory.