Rise Time Constants Research Articles

Calcium concentration changes during sensory transduction in spider mechanoreceptor neurons

Most mechanoreceptor neurons encode mechanical signals into action potential trains within the same cell. Evidence suggests that intracellular calcium ion concentration, [Ca2+], increases during mechanotransduction, either by direct entry through mechanically activated channels or indirectly through voltage-activated calcium channels. However, little is known about the amounts of calcium involved or its roles in mechanotransduction. We estimated [Ca2+] in mechanoreceptor neurons of the spider, Cupiennius salei, during mechanical stimulation using Oregon Green BAPTA-1, and a single-compartment model of [Ca2+] as a function of action potential firing rate. Resting [Ca2+] was approximately 400 nM and increased to up to 2 microM at 30 action potentials/s. Similar levels of resting and stimulated [Ca2+] were obtained in the cell soma, axon and two parts of the sensory dendrite, including the region immediately adjacent to the site of sensory transduction. The time constant of rise and fall of [Ca2+] was 1-5 s in the dendrite and axon, but up to 15 s in the soma. Calcium elevation was dependent on action potentials and could not be induced by the receptor potential alone. Blockade of voltage-activated calcium channels by nickel ions prevented calcium increase, but thapsigargin, which empties intracellular calcium stores, had no effect. Estimates of calcium entry per action potential from fluorescence changes agreed approximately with estimates based on action potential voltage-time profile and previous reports of calcium channel properties. This first report of calcium levels during transduction in spiking mechanoreceptors suggests that calcium signaling plays important roles in primary somatosensory neurons.

Transient Voltage-Dependent Potassium Currents Are Reduced in NTS Neurons Isolated From Renal Wrap Hypertensive Rats

Whole cell patch-clamp measurements were made in neurons enzymatically dispersed from the nucleus of the solitary tract (NTS) to determine if alterations occur in voltage-dependent potassium channels from rats made hypertensive (HT) by unilateral nephrectomy/renal wrap for 4 wk. Some rats had the fluorescent tracer DiA applied to the aortic nerve before the experiment to identify NTS neurons receiving monosynaptic baroreceptor afferent inputs. Mean arterial pressure (MAP) was greater in 4-wk HT (165 +/- 5 mmHg, n = 26, P < 0.001) rats compared with normotensive (NT) rats (109 +/- 3 mmHg measured in 10 of 69 rats). Transient outward currents (TOCs) were observed in 67-82% of NTS neurons from NT and HT rats. At activation voltages from -10 to +10 mV, TOCs were significantly less in HT neurons compared with those observed in NT neurons (P < 0.001). There were no differences in the voltage-dependent activation kinetics, the voltage dependence of steady-state inactivation, and the rise and decay time constants of the TOCs comparing neurons isolated from NT and HT rats. The 4-aminopyridine-sensitive component of the TOC was significantly less in neurons from HT compared with NT rats (P < 0.001), whereas steady-state outward currents, whether or not sensitive to 4-aminopyridine or tetraethylammonium, were not different. Delayed excitation, studied under current clamp, was observed in 60-80% of NTS neurons from NT and HT rats and was not different comparing neurons from NT and HT rats. However, examination of the subset of NTS neurons exhibiting somatic DiA fluorescence revealed that DiA-labeled neurons from HT rats had a significantly shorter duration delayed excitation (n = 8 cells, P = 0.022) than DiA-labeled neurons from NT rats (n = 7 cells). Neurons with delayed excitation from HT rats had a significantly broader first action potential (AP) and a slower maximal downstroke velocity of repolarization compared with NT neurons with delayed excitation (P = 0.016 and P = 0.014, respectively). The number of APs in the first 200 ms of a sustained depolarization was greater in HT than NT neurons (P = 0.012). These results suggest that HT of 4-wk duration reduces TOCs in NTS neurons, and this contributes to reduced delayed excitation and increased AP responses to depolarizing inputs. Such changes could alter baroreflex function in hypertension.

Vibrational energy relaxation of azulene studied by the transient grating method. I. Supercritical fluids

The vibrational energy dissipation process of the ground-state azulene in supercritical xenon, carbon dioxide, and ethane has been studied by the transient grating spectroscopy. In this method, azulene in these fluids was photoexcited by two counterpropagating subpicosecond laser pulses at 570 nm, which created a sinusoidal pattern of vibrationally hot ground-state azulene inside the fluids. The photoacoustic signal produced by the temperature rise of the solvent due to the vibrational energy relaxation of azulene was monitored by the diffraction of a probe pulse. The temperature-rise time constants of the solvents were determined at 383 and 298 K from 0.7 to 2.4 in rho(r), where rho(r) is the reduced density by the critical density of the fluids, by the fitting of the acoustic signal based on a theoretical model equation. In xenon, the temperature-rise time constant was almost similar to the vibrational energy-relaxation time constant of the photoexcited solute determined by the transient absorption measurement [D. Schwarzer, J. Troe, M. Votsmeier, and M. Zerezke, J. Chem. Phys. 105, 3121 (1996)] at the same reduced density irrespective of the solvent temperature. On the other hand, the temperature-rise time constants in ethane were larger than the vibrational energy-relaxation time constants by a factor of about 2. In carbon dioxide, the difference was small. From these results, the larger time constants of the solvent temperature rise than those of the vibrational energy relaxation in ethane and carbon dioxide were interpreted in terms of the vibrational-vibrational (V-V) energy transfer between azulene and solvent molecules and the vibrational-translational (V-T) energy transfer between solvent molecules. The contribution of the V-V energy transfer process against the V-T energy transfer process has been discussed.

Vibrational energy relaxation of azulene studied by the transient grating method. II. Liquid solvents

The vibrational energy dissipation process of the ground-state azulene in various liquids has been studied by the transient grating spectroscopy. The acoustic signal produced by the temperature rise of the solvent due to the vibrational energy relaxation of azulene was monitored. The temperature rise-time constant of the solvent has been determined both by the fitting of the acoustic signal to a theoretical model equation and by the analysis of the acoustic peak shift. We found that the temperature rise-time constants determined by the transient grating method in various solvents are larger than the vibrational energy relaxation time constants determined by the transient absorption measurement [D. Schwarzer, J. Troe, M. Votsmeier, and M. Zerezke, J. Chem. Phys. 105, 3121 (1996)]. The difference is explained by different energy dissipation pathways from azulene to solvent; vibrational-vibrational (V-V) energy transfer and vibrational-translational (V-T) energy transfer. The contribution of the V-V energy transfer is estimated in various liquid solvents from the difference between the temperature rise time and vibrational energy relaxation time, and the solvent V-T relaxation time.

Femtosecond Fluorescence Dynamics Imaging Using a Fluorescence Up-Conversion Microscope

Femtosecond fluorescence dynamics imaging microscopy was performed. Femtosecond fluorescence dynamics images were constructed based on the "mean" fluorescence decay or rise time constants that were evaluated by the time-resolved intensity sampling using a fluorescence up-conversion microscope. This dynamics imaging microscopy was carried out for the organic microcrystals alpha-perylene and tetracene-doped anthracene microcrystal, and ultrafast dynamics in the organic microcrystals were clearly imaged in the two-dimensional manner. For the alpha-perylene microcrystal, the obtained dynamics images showed that the crystal edges exhibited relatively shorter free exciton and the Y-state lifetimes compared to the crystal center, reflecting the higher concentration of defects. For the tetracene-doped anthracene microcrystal, the image was constructed based on the time constant of excitation energy transfer from anthracene to tetracene. By experiments changing the doping ratio of tetracene in anthracene, it was concluded that the inhomogeneity observed in the dynamics image arises from the difference in the local concentration of tetracene in the mixed crystal.

Multiple gated-integrator as an effective tool for processing nuclear radiation detector signals

In a previous paper the double gated-integrator with exponential prefilter was investigated in detail [J. Gál, Nucl. Instr. and Meth. A 462 (2001) 506]. It was shown that it has very good noise characteristics. Its noise figure of demerit relating to the cusp shaping is 1.016. However, a more detailed analysis of the output of the double gated-integrator shows that the ballistic deficit of this signal, due to its sharp peaking, is not negligible, if the rise time constant of the detector pulse is comparable with the shaping time constant. In the present paper the theoretical study of the so-called multiple gated-integrator is presented. It will be shown that using multiple gated-integration and performing linear combination of the multiple integrated signals several pulse shapes can be realized. For example, by combining the outputs of the single and the double gated-integrators ramp or trapezoidal shaping can be implemented. The effect of the ballistic deficit on the spectral resolution is also discussed. The calculations confirm the well known fact that trapezoidal shaping is a good compromise when both the ballistic deficit and the system noise have comparable effect on the spectral resolution. At the same time, the multiple integrated signals can be used to derive such quantities which characterize the shape of the input signal. This makes it possible to realize pulse shape discrimination in order to identify particle type or to eliminate unwanted pulses appearing e.g., due to slow charge collection or due to pile-up. In the present paper the characteristics of a shape parameter deduced from the single and double gated-integrator outputs are investigated and the figure of merit of the pulse shape discrimination using this shape parameter is calculated.

Enhanced phase noise modeling of fractional-N frequency synthesizers

Mathematical models for the behavior of fractional-N phase-locked-loop frequency synthesizers (Frac-N) are presented. The models are intended for calculating rms phase error and determining spurs in the output of Frac-N. The models describe noise contributions due to the charge pump (CP), the phase frequency detector (PFD), the loop filter, the voltage control oscillator, and the delta-sigma modulator. Models are presented for the effects of static CP gain mismatch, CP dynamic mismatch and PFD reset delay mismatch. A simple analytic expression shows the level of /spl Delta//spl Sigma/ sequence noise caused by static CP current mismatch. We further show that un-equal rise time and fall time constants of the CP result in dynamic mismatch noise. Reset delay mismatch in PFD is shown to also contribute significantly to close-in phase noise. The model takes into account the reduction in CP thermal and flicker noise due to the changing duty cycle of Frac-N CP. Our model is therefore useful in characterizing the noise performance of Frac-N at the system-level, simplifying the design of fractional-N synthesizers and transmitters. Analytical and simulated results are compared and show good agreement with prior published data on Frac-N realizations.

Ultrafast Dynamics of Pyrene Excimer Formation in Y Zeolites

Pyrene excimer emission in alkali-metal-exchanged zeolites is readily observed to grow in using an ultrafast streak camera with 3.3 ps time-resolution. Rise-time constants range from 7 to 14 ps depending on the composition of the zeolite. These results show that two pyrene molecules in doubly occupied cavities must move from their pre-excitation orientation into the face-to-face orientation required for the excimer.

Ultrafast Dynamics of Pyrene Excimer Formation in Y Zeolites

Pyrene excimer emission in alkali-metal-exchanged zeolites is readily observed to grow in using an ultrafast streak camera with 3.3 ps time-resolution. Rise-time constants range from 7 to 14 ps depending on the composition of the zeolite. These results show that two pyrene molecules in doubly occupied cavities must move from their pre-excitation orientation into the face-to-face orientation required for the excimer.

Depth-Penetrating Temperature Measurements of Thermal Barrier Coatings Incorporating Thermographic Phosphors

Thermographic phosphors have been previously demonstrated to provide effective non-contact, emissivity-independent surface temperature measurements. Due to the translucent nature of thermal barrier coatings (TBCs), thermographic-phosphor-based temperature measurements can be extended beyond the surface to provide depth-selective temperature measurements by incorporating the thermographic phosphor layer at the depth where the temperature measurement is desired. In this paper, thermographic phosphor (Y2O3:Eu) fluorescence decay time measurements are demonstrated to provide through-the-coating-thickness temperature readings up to 1100 °C with the phosphor layer residing beneath a 100-µm-thick TBC (plasmasprayed 8 wt.% yttria-stabilized zirconia). With an appropriately chosen excitation wavelength and detection configuration, it is shown that sufficient phosphor emission is generated to provide effective temperature measurements, despite the attenuation of both the excitation and emission intensities by the overlying TBC. This depth-penetrating temperature measurement capability should prove particularly useful for TBC diagnostics where a large thermal gradient is typically present across the TBC thickness. The fluorescence decay from the Y2O3:Eu layer exhibited both an initial short-term exponential rise and a longer-term exponential decay. The rise time constant was demonstrated to provide better temperature indication below 500 °C while the decay time constant was a better indicator at higher temperatures.

Depth-penetrating temperature measurements of thermal barrier coatings incorporating thermographic phosphors

Thermographic phosphors have been previously demonstrated to provide effective non-contact, emissivity-independent surface temperature measurements. Because of the translucent nature of thermal barrier coatings (TBCs), thermographic phosphor-based temperature measurements can be extended beyond the surface to provide depth-selective temperature measurements by incorporating the thermographic phosphor layer at the depth where the temperature measurement is desired. In this paper, thermographic phosphor (Y2O3:Eu) fluorescence decay time measurements are demonstrated to provide through-the-coating thickness temperature readings up to 1100 C with the phosphor layer residing beneath a 100 micron thick TBC (plasma-sprayed 8wt% yttria-stabilized zirconia). With an appropriately chosen excitation wavelength and detection configuration, it is shown that sufficient phosphor emission is generated to provide effective temperature measurements, despite the attenuation of both the excitation and emission intensities by the overlying TBC. This depth-penetrating temperature measurement capability should prove particularly useful for TBC diagnostics where a large thermal gradient is typically present across the TBC thickness. The fluorescence decay from the Y2O3:Eu layer exhibited both an initial short-term exponential rise and a longer-term exponential decay. The rise time constant was demonstrated to provide better temperature indication below 500 C while the decay time constant was a better indicator at higher temperatures.

Brain-derived neurotrophic factor attenuates mouse cerebellar granule cell GABA(A) receptor-mediated responses via postsynaptic mechanisms.

In addition to exerting long-term neurotrophic influences on developmental process such as neuronal survival and neuritic outgrowth, brain-derived neurotrophic factor (BDNF) has been reported to modulate synaptic transmission in the short-term. Considerable evidence indicates that BDNF acutely modulates NMDA receptor-mediated synaptic activity. However, whether BDNF modulates inhibitory synaptic transmission remains to be firmly established. In the present study, we examined the effect of acute BDNF exposure on GABA-evoked whole-cell responses as well as GABAergic synaptic activity in cultured mouse cerebellar granule cells. GABA-evoked responses were reduced by 39.5 +/- 4.7 % upon acute and focal application of BDNF (100 ng ml-1). The reduction of the GABA response recovered only partially even minutes after removal of BDNF. TrkB-IgG and K252a, but not K252b, prevented the BDNF-induced attenuation of the GABA response. BDNF exposure shifted the cumulative peak amplitude distribution leftward for both spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) without affecting the rise time and decay time constants. Acute exposure to BDNF also resulted in internalization of GABAA receptors in cultured cerebellar granule cells, as reflected by diminished immunostaining with an antibody against the GABAA receptor beta2/3 subunit. Although the BDNF-induced GABAA receptor internalization was sensitive to K252a, it did not become manifest until 5 min after exposure to BDNF. Therefore, receptor internalization alone cannot account for the prompt BDNF-induced attenuation of GABA-mediated activity. We conclude that BDNF modulates GABAA receptor-mediated activity through TrkB receptor signalling that triggers a kinase-dependent short latency effect and a delayed longer latency effect hallmarked by receptor internalization.

Molecular site of action of the antiarrhythmic drug propafenone at the voltage-operated potassium channel Kv2.1.

The effects of the antiarrhythmic drug propafenone at Kv2.1 channels were studied with wild-type and mutated channels expressed in Xenopus laevis oocytes. Propafenone decreased the Kv2.1 currents in a time- and voltage-dependent manner (decrease of the time constants of current rise, increase of block with the duration of voltage steps starting from a block of less than 19%, increase of block with the amplitude of depolarization yielding a fractional electrical distance delta of 0.11 to 0.16). Block of Kv2.1 appeared with application to the intracellular, but not the extracellular, side of membrane patches. In mutagenesis experiments, all parts of the Kv2.1 channel were successively exchanged with those of the Kv1.2 channel, which is much more sensitive to propafenone. The intracellular amino and carboxyl terminus and the intracellular linker S4-S5 reduced the blocking effect of propafenone, whereas the linker S5-S6, as well as the segment S6 of the Kv1.2 channel, abolished it to the value of the Kv1.2 channel. In the linker S5-S6, this effect could be narrowed down to two groups of amino acids (groups 372 to 374 and 383 to 384), which also affected the sensitivity to tetraethylammonium. In segment S6, several amino acids in the intracellularly directed part of the helix significantly reduced propafenone sensitivity. The results suggest that propafenone blocks the Kv2.1 channel in the open state from the intracellular side by entering the inner vestibule of the channel. These results are consistent with a direct interaction of propafenone with the lower part of the pore helix and/or residues of segment S6.

Current rise time constants in switch-on process of SiC thyristors

An analytical approach to analysis of the switch-on process in silicon carbide (SiC) thyristors is taken. Expressions for the current rise time are obtained for both low, τ rL, and high injection levels, τ rH, in the blocking p 0-base of a thyristor, with account taken of the fact that the injection coefficient of the SiC p +n-junction is less than unity. It is shown that in 4H-SiC thyristors the τ rL/ τ rH ratio may be as high as 25–30. The results obtained are in agreement with the available experimental data and computer simulations.

火災時における無被覆鋼材の温度上昇簡易予測式

A simple formula has been developed to calculate the temperature rise of unprotected steel elements during fire. The formula includes the effect of steel heat capacity through section factor H_S/A_S, where H_s is the heated perimeter length of cross section, A_s is the cross sectional area. To derive the formula, a series of fire resistance tests were conducted for H-sectioned columns, box columns and H-sectioned beams of various cross section sizes. The measured steel temperature histories were fitted with exponential curves to obtain the steel temperature rise coefficient (inverse of time constants of temperature rise). Good correlation was obtained between the section factors and the steel temperature rise coefficients for respective categories of steel shapes.

Sensitivity of synaptic GABA(A) receptors to allosteric modulators in hippocampal oriens-alveus interneurons.

GABA(A) receptors are heteropentamers that are heterogeneously distributed at different synapses in the central nervous system. Although the modulation of GABA(A) receptors received much attention in hippocampal pyramidal cells, information is scarce regarding the pharmacology of these receptors in inhibitory interneurons. We investigated the pharmacological properties of GABA(A)-mediated miniature inhibitory postsynaptic currents (mIPSCs) using whole-cell voltage clamp recordings in two morphologically identified types of hippocampal CA1 interneurons, horizontal and vertical cells of stratum oriens-alveus. The negative modulators zinc (200 microM) and furosemide (600 microM) significantly decreased the amplitude of mIPSCs. Benzodiazepine agonists also produced significant effects: 10 microM zolpidem increased the amplitude, rise time, and decay time constant (decay tau) of mIPSCs, whereas 10 microM flunitrazepam affected similarly the amplitude and decay tau, but not the rise time. The neurosteroid allopregnanolone (10 microM) prolonged the decay tau of mIPSCs. Since these modulators act on different GABA(A) receptor subunits, this pharmacological profile suggests that GABA(A) receptors at spontaneously active inhibitory synapses onto vertical and horizontal interneurons are heterogeneous and formed by co-assembly of different combinations of subunits (alpha(1-5)beta(1-3)gamma(1-3)). Furthermore, these synaptic GABA(A) receptors appear in large part pharmacologically similar to those of pyramidal cells.

Investigation of carrier relaxation dynamics in single CdSe/ZnSe self-organized quantum dot by time-resolved micro-photoluminescence

We have investigated carrier relaxation dynamics in single CdSe/ZnSe quantum dot (QD) by time-resolved micro-photoluminescence (PL). The discrete sharp lines, originated from individual QD states, exhibit various rise and decay time constants. The decay times of the sharp lines from ground states and excited states are estimated to be 700≈800 psec and 400≈500 psec, respectively, and the rise times of the ground states become longer compared with those of the excited states. There results are in contrast to successive change of the rise and decay times observed in time-resolved macro-PL with varying the detection wave-length. The quasi-continuum higher states with much shorter decay times are clearly observed over the discrete states of the QDs.

Unitary synaptic currents between lacunosum-moleculare interneurones and pyramidal cells in rat hippocampus.

1. Unitary inhibitory postsynaptic currents (uIPSCs) were characterised between 23 synaptically coupled interneurones at the border of stratum radiatum and lacunosum-moleculare (LM) and CA1 pyramidal cells (PYR) using dual whole-cell recordings and morphological identification in rat hippocampal slices. 2. LM interneurones presented a morphology typical of stellate cells, with a fusiform soma as well as dendritic and axonal arborisations in stratum radiatum and lacunosum-moleculare. 3. Single spikes in interneurones triggered uIPSCs in pyramidal cells that were blocked by the GABA(A) antagonist bicuculline and mediated by a chloride conductance. The latency, rise time, duration and decay time constant of uIPSCs were a function of amplitude in all pairs, suggesting a homogeneity in the population sampled. 4. During paired pulse stimulation, individual LM-PYR connections exhibited facilitation or depression. The paired pulse ratio was inversely related to the amplitude of the first response. The transition from facilitation to depression occurred at 26 % of the maximal amplitude of the first uIPSC. Paired pulse depression was not modified by CGP 55845 and thus was GABA(B) receptor independent. 5. CGP 55845 failed to modify the amplitude of uIPSCs, suggesting an absence of tonic presynaptic GABA(B) inhibition at LM-PYR connections. 6. Increasing GABA release by repetitive activation of interneurones failed to induce GABA(B) IPSCs. With extracellular minimal stimulation, increasing stimulation intensity above threshold, or repetitive activation, evoked GABA(B) IPSCs, probably as a result of coactivation of several GABAergic fibres. 7. Thus, dendritic inhibition by LM interneurones involves GABA(A) uIPSCs with kinetics dependent on response amplitude and subject to GABA(B)-independent paired pulse plasticity.

Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei.

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.

Influence of starting voltage waveform on time sequence to restart DC transmission lines

Based on experiments and calculations, this paper describes the influence of the time constant of the dc starting voltage on the no-voltage time required for successful restarting and the time required to complete the restarting. (1) The 300-kV-class dc tension insulator assembly tested in this study was composed of disk-type insulator units used for ac transmission lines. The insulation recovery characteristics of the dc tension insulator assembly after dc arc extinctions of 400 and 4000 A were obtained for the switching impulse voltage and simulated dc starting voltage. The results show that both methods obtained the same minimum values for the required no-voltage time. Thus, we verified this method's validity in calculating the minimum value of the no-voltage time required for successful restarting based on the insulation recovery characteristics obtained by the switching impulse voltage. (2) Based on the insulation recovery characteristics obtained by the switching impulse voltage after arc extinction, we calculated the relationships between the required no-voltage time and restarting completion time versus the time constant of the dc starting voltage. As the rising time constant of the dc starting voltage increased, the required no-voltage time decreased and the restarting completion time increased. It was shown that reducing the rising time constant of the dc starting voltage was an effective measure to shorten the restarting completion time after the arc extinction. © 2000 Scripta Technica, Electr Eng Jpn, 133(4): 63–70, 2000