Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis.

At the large excitatory calyx of Held synapse, the quantal size during an evoked EPSC and the number of active zones contributing to transmission are not known. We developed a nonstationary variant of EPSC fluctuation analysis to determine these quantal parameters. AMPA receptor-mediated EPSCs were recorded in slices of young (postnatal 8-10 d) rats after afferent fiber stimulation, delivered in trains to induce synaptic depression. The means and the variances of EPSC amplitudes were calculated across trains for each stimulus number. During 10 Hz trains at 2 mm Ca(2+) concentration ([Ca(2+)]), we found linear EPSC variance-mean relationships, with a slope that was in good agreement with the quantal size obtained from amplitude distributions of spontaneous miniature EPSCs. At high release probability with 10 or 15 mm [Ca(2+)], competitive antagonists were used to partially block EPSCs. Under these conditions, the EPSC variance-mean plots could be fitted with parabolas, giving estimates of quantal size and of the binomial parameter N. With the rapidly dissociating antagonist kynurenic acid, quantal sizes were larger than with a slowly dissociating antagonist, suggesting that the effective glutamate concentration was increased at high release probability. Considering the possibility of multivesicular release and moderate saturation of postsynaptic AMPA receptors, we conclude that the binomial parameter N (637 +/- 117; mean +/- SEM) represents an upper limit estimate of the number of functional active zones. We estimate that during normal synaptic transmission, the probability of vesicle fusion at single active zones is in the range of 0.25-0.4.

Intracellular calcium dependence of transmitter release rates at a fast central synapse.

Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central nervous system, however, there are no experimental estimates of this local [Ca2+]i signal. Here we show, by using calcium ion uncaging in the large synaptic terminals of the calyx of Held, that step-like elevations to only 10 microM [Ca2+]i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3 ms. Kinetic analysis of transmitter release rates after [Ca2+]i steps revealed the rate constants for calcium binding and vesicle fusion. These show that transient (around 0.5 ms) local elevations of [Ca2+]i to peak values as low as 25 microM can account for transmitter release during single presynaptic action potentials. The calcium sensors for vesicle fusion are far from saturation at normal release probability. This non-saturation, and the high intracellular calcium cooperativity in triggering vesicle fusion, make fast synaptic transmission very sensitive to modulation by changes in local [Ca2+]i.

Properties of a model of Ca++-dependent vesicle pool dynamics and short term synaptic depression.

We explore the properties of models of synaptic vesicle dynamics, in which synaptic depression is attributed to depletion of a pool of release-ready vesicles. Two alternative formulations of the model allow for either recruitment of vesicles from an unlimited reserve pool (vesicle state model) or for recovery of a fixed number of release sites to a release-ready state (release-site model). It is assumed that, following transmitter release, the recovery of the release-ready pool of vesicles is regulated by the intracellular free Ca(++) concentration, [Ca(++)](i). Considering the kinetics of [Ca(++)](i) after single presynaptic action potentials, we show that pool recovery can be described by two distinct kinetic components. With such a model, complex kinetic and steady-state properties of synaptic depression as found in several types of synapses can be accurately described. However, the specific assumption that enhanced recovery is proportional to [Ca(++)](i), as measured with Ca(++) indicator dyes, is not confirmed by experiments at the calyx of Held, in which [Ca(++)](i)-homeostasis was altered by adding low concentrations of the exogenous Ca(++) buffer, fura-2, to the presynaptic terminal. We conclude that synaptic depression at the calyx of Held is governed by localized, near membrane [Ca(++)](i) signals not visible to the indicator dye, or else by an altogether different mechanism. We demonstrate that, in models in which a Ca(++)-dependent process is linearly related to [Ca(++)](i), the addition of buffers has only transient but not steady-state consequences.

Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse.

The size of a pool of readily releasable vesicles at a giant brainstem synapse, the calyx of Held, was probed with three independent approaches. Using simultaneous pre- and postsynaptic whole-cell recordings, two forms of presynaptic Ca2+ stimuli were applied in rapid succession: uncaging of Ca2+ by flash photolysis and the opening of voltage-gated Ca2+ channels. The ensuing transmitter release showed a nearly complete cross-inhibition between the two stimuli, indicating the depletion of a limited pool of about 700 transmitter quanta. The pool size was confirmed in experiments using enhanced extracellular Ca2+ concentrations, as well as short, high-frequency stimulus trains. The results reveal a surprisingly large pool of functionally available vesicles, of which a fraction of about 0.2 is released by a single presynaptic action potential under physiological conditions.

The epithelial inward rectifier channel Kir7.1 displays unusual K+ permeation properties.

Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.

Altered voltage dependence of fractional Ca2+ current in N-methyl-D-aspartate channel pore mutants with a decreased Ca2+ permeability.

The Ca2+ permeability properties of an N-methyl-D-aspartate (NMDA) channel pore mutant (NR1E603K-NR2A) were studied using whole-cell patch-clamp recordings in human embryonic kidney cells. Measurements of reversal potential shifts indicated that the relative permeability of Ca2+ over monovalent ions, P(Ca)/P(M), was 1.6, a value reduced by a factor of approximately 2 with respect to the wild-type channel. The ratio of Ca2+ current over total current (fractional Ca2+ current), however, was 19.7 +/- 1% at -50 mV and 2 mM external Ca2+ concentration, a value similar to that of the wild-type channel, but 2.3-fold larger than that predicted by simple permeation models for the corresponding P(Ca)/P(M) value. The deviation from predicted values gradually disappeared with membrane depolarization. Similar results were obtained for two cysteine mutations at asparagine residues of the NR1 and NR2A subunits. When interpreted in terms of a two-barrier one-site model for ion permeation, the results indicate that changes in the relative Ca2+ permeability occur close to an internal energy barrier limiting ion permeation.

Presynaptic depression at a calyx synapse: the small contribution of metabotropic glutamate receptors.

Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at approximately 1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to approximately 30% and approximately 10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1-5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD and L-AP4 and found them to be reversed in a dose-dependent manner by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 microM, CPPG completely reversed the effects of L-AP4 and (1S, 3S)-ACPD, but reduced 5-10 Hz elicited depression by only approximately 6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5-10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.

Coupling of permeation and gating in an NMDA-channel pore mutant.

We report a strong coupling between permeation and gating in a mutant NMDA channel (NR1 N598Q-NR2A). The channel opens to two states that differ by their conductance and, surprisingly, by their selectivity for two permeant monovalent cations, Na+ and Cs+. The two open states are linked to the closed state via a cyclic gating reaction that proceeds preferentially in one direction under biionic conditions, indicating that the gating mechanism is not at equilibrium. The direction and the magnitude of this gating asymmetry can be accounted for by assuming that ions bound to a site in the permeation pathway influence the gating of this mutant channel, and that in the closed state, the channel site is accessible to internal cations.

Simultaneous measurement of Ca2+ influx and reversal potentials in recombinant N-methyl-D-aspartate receptor channels.

The Ca(2+) permeability of N-methyl-D-aspartate receptor (NMDA-R) channels was studied in human embryonic kidney cells transfected with the NR1-NR2A subunit combination. To determine the fractional Ca(2+) current (P(f)), measurements of fura-2-based Ca(2+) influx and whole-cell currents were made in symmetrical monovalent ion concentrations at membrane potentials between -50 mV and the reversal potential. The ratios of Ca(2+) flux over net whole-cell charge at 2, 5, and 10 mM external Ca(2+) concentrations ([Ca](o)) were identical at a membrane potential close to the reversal potential of the monovalent current component. Assuming unity of P(f) at this potential, the percentage of current carried by Ca(2+) was found to be 18.5 +/- 1.3% at 2 mM [Ca](o) and -50 mV. This value, which is higher than the ones reported previously, was confirmed in independent experiments in which a pure flux of Ca(2+) through NMDA-R channels was used to calibrate the Ca(2+) influx signals. The measured values of fractional Ca(2+) currents, which agree with the predictions of the Goldman-Hodgkin-Katz equations, are also compatible with a two-barrier model for ion permeation, in which the differences between the energy barriers for Ca(2+) and monovalent ions are similar on the external and internal membrane sides.

Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones.

1. The Ca2+ permeability of non-NMDA and NMDA receptor channels was studied using a fluorometric flux measurement approach in somata and dendrites of CA1 pyramidal neurones in rat hippocampal slices. For this purpose, the Ca2+ fraction of the total cation current (named 'fractional Ca2+ current') was measured directly from the change in the Ca(2+)-sensitive fura-2 fluorescence at 380 nm excitation wavelength. 2. The fractional Ca2+ current through the somatic NMDA receptor channels was 10.69 +/- 2.13% (mean +/- S.D.) and that through dendritic receptor channels was 10.70 +/- 1.96%. The fractional Ca2+ current was not dependent on the extracellular Mg2+ concentration and its voltage dependence was in agreement with the Goldman-Hodgkin-Katz current equation. 3. AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) or kainate applications produced small but significant Ca2+ entry. Fractional Ca2+ currents of 0.58 +/- 0.34% were measured for somatic AMPA applications, 0.68 +/- 0.20% for somatic kainate applications, 0.66 +/- 0.25% for dendritic AMPA applications and 0.61 +/- 0.16% for dendritic kainate applications. 4. The expression pattern of glutamate receptor subunits encoding messenger ribonucleic acids (mRNAs) was analysed with the single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) approach applied to CA1 pyramidal neurones. The AMPA receptor subunits GluR-A, GluR-B and GluR-C, and the NMDA receptor subunits NR2A and NR2B were found to be abundantly expressed in all CA1 pyramidal neurones tested. 5. This study establishes the fractional Ca2+ current through somatic and dendritic NMDA and non-NMDA receptor channels in CA1 pyramidal neurones. The dendritic, presumably synaptic, NMDA receptor channels are highly Ca2+ permeable and have a fractional Ca2+ current closely resembling that of somatic extrasynaptic NMDA receptor channels. Both somatic and dendritic non-NMDA receptor channels are of the 'low Ca2+ permeable' type and have a fractional Ca2+ current that is about twenty times smaller than that of NMDA receptor channels.

Fractional calcium current through neuronal AMPA-receptor channels with a low calcium permeability.

The Ca(2+)-permeation properties of AMPA-receptor (AMPA-R) channels in Purkinje neurons in rat cerebellar slices were studied using a combination of whole-cell patch-clamp recordings, Fura-2 fluorometry, and single-cell reverse-transcription (RT)-PCR. Several lines of evidence indicate that Purkinje neurons, at both early and late stages of postnatal development, express exclusively AMPA-R channels with a low Ca2+ permeability. First, no Ca2+ signal was detected during application of either AMPA or kainate to Purkinje neurons loaded with the Ca2+ indicator Fura-2 AM. In contrast, kainate application induced large Ca2+ transients in Bergmann glia cells. Second, in ion substitution experiments, when Ca2+ is the only extracellular permeant cation, the reversal potential corresponds to that expected for AMPA-R channels with a low permeability for Ca2+. Third, using a fluorometric flux-measurement approach (Schneggenburger et al., 1993a), we found that the Ca2+ fraction of the total cation current through AMPA-R channels is approximately 0.6%. This value is approximately sixfold lower than that found for recombinant AMPA-R lacking the AMPA-R subunit GluR2. Furthermore, single-cell RT-PCR experiments revealed the presence of the AMPA-R subunits GluR1, GluR2, and GluR3 in Purkinje neurons in cerebellar slices at developmental stages corresponding to those studied electrophysiologically. The expression of GluR2 in all cells tested (n = 14) is consistent with the subunit composition predicted from studies of recombinant AMPA-R channels with a low permeability for Ca2+ (Burnashev et al., 1992b). In conclusion, this study establishes that cerebellar Purkinje neurons at all postnatal developmental stages possess AMPA-R channels with a low permeability for Ca2+.

Depolarization-induced calcium signals in the somata of cerebellar Purkinje neurons.

Cerebellar Purkinje neurons express voltage-gated Ca2+ channels that are located on their somata and dendrites. Previous reports, based on microelectrode recordings and fura-2 Ca2+ imaging, suggested that depolarization-mediated intracellular Ca2+ signaling is confined almost completely to the dendrites. We investigated the spatial distribution of depolarization-induced Ca2+ signals in Purkinje neurons by applying whole-cell patch-clamp recordings combined with fluorometric Ca2+ imaging to cerebellar slices. Under our recording conditions, depolarizing pulses produced the dendritic but also large somatic Ca2+ signals. By selective perfusion of the slice with a Ca(2+)-free EGTA-containing solution, we could isolate experimentally Ca2+ signals in somata and dendrites, respectively. Moreover, experiments performed on cerebellar slices from young rats (up to postnatal day 6), in which Purkinje neurons are almost completely devoid of dendrites, showed that Ca2+ currents produced by the activation of somatic Ca2+ channels are associated with Ca2+ transients similar to those seen in the somata of adult Purkinje neurons. Our results strongly indicate that the depolarization-induced somatic Ca2+ signals are caused by Ca2+ entry through voltage-gated channels located on the somatic membrane of Purkinje neurons.

Glutamate- and AMPA-mediated calcium influx through glutamate receptor channels in medial septal neurons.

The Ca(2+)-fraction of the ion current flowing through glutamate receptor channels activated either by glutamate or by AMPA was determined in forebrain neurons of the rat medial septum. By combining whole-cell patch-clamp and fura-2 fluorometric measurements we found that, at negative membrane potentials and at an extracellular free Ca(2+)-concentration of 1.6 mM, the Ca(2+)-fraction of the current activated by glutamate is 5.7%. A pharmacological analysis of responses produced by ionophoretically-released glutamate demonstrated a large contribution of NMDA-receptors but a small contribution of AMPA/kainate receptors to these responses. Interestingly, also AMPA-mediated currents were associated with significant changes in Ca(2+)-sensitive fluorescence. The fractional Ca2+ current of AMPA-induced responses was 1.2 +/- 0.4% (n = 5).

Fractional contribution of calcium to the cation current through glutamate receptor channels.

The Ca2+ fraction of the ion current flowing through glutamatergic NMDA and AMPA/kainate receptor channels was determined in forebrain neurons of the medial septum. The neurons were overloaded with the Ca2+ indicator dye fura-2 (1 mM) via the recording patch pipettes. This approach allowed the direct determination of the Ca2+ influx from changes in the Ca(2+)-sensitive fura-2 fluorescence. We found that, at negative membrane potentials and at an extracellular free Ca2+ concentration of 1.6 mM, the Ca2+ fraction of the current through the NMDA receptor channels is only 6.8%, about 2-fold lower than previously estimated from reversal potential measurements. Interestingly, a quite high fractional Ca2+ current of 1.4% was determined for the linearly conducting AMPA/kainate receptor channels found in these neurons.

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

Patch-clamp analysis of voltage-gated currents in intermediate lobe cells from rat pituitary thin slices.

Ionic currents of hypophyseal intermediate lobe cells were studied using a thin-slice preparation of the rat pituitary in conjunction with conventional and perforated whole-cell patch-clamp recording techniques. A majority (89%) of the cells studied generated Na+, Ca2+ and K+ currents upon depolarizing voltage steps and responded to bath application of gamma-aminobutyric acid (GABA; 20-50 microM) with inward currents (in symmetrical chloride, holding potential -80 mV). A small percentage of cells (11%) did not display inward membrane currents upon depolarization and was unresponsive to GABA. In the first type of cells, Ca2+ and K+ currents were further studied in isolation. Ca2+ tail currents showed a biphasic time course upon repolarization, with time constants and amplitudes of 2.07 +/- 0.29 ms, 123 +/- 22 pA (for the slowly deactivating component) and 0.14 +/- 0.06 ms, 437 +/- 33 pA (for the fast-deactivating component; means +/- SD of n = 4 cells). Slowly and fast-deactivating conductances were half-maximally activated at around -10 mV and +10 mV respectively. Depolarizing voltage steps elicited two types of K+ current, which were separated using a prepulse protocol. A fast-activating, transient component showed half-maximal steady-state inactivation between -65 mV and -45 mV depending on the divalent cation composition of the external solution. Its decay was fitted by single-exponential functions with time constants of 36 +/- 11 ms and 3.9 +/- 0.9 ms at -20 mV and +40 mV respectively (mean +/- SD; n = 4 cells). Whereas the peak current amplitudes of the transient K+ current component remained stable, the amplitude of the second, delayed component increased progressively throughout the course of whole-cell experiments. In cells recorded with the perforated whole-cell technique, bath application of dopamine (10 nM-1 microM) induced large hyperpolarizations from a spontaneous membrane potential of -40 mV, but did not consistently affect the amplitude of the voltage-gated K+ conductances. These data are compared to previous studies using other preparations of the intermediate lobe, and differences are discussed, thus helping to extend our knowledge of electrical excitability of hypophyseal cells.

Excitatory and inhibitory synaptic currents and receptors in rat medial septal neurones.

1. A thin-slice preparation was used to study the postsynaptic potentials and the underlying currents of visually identified rat medial septal (MS) neurones under tight-seal voltage- and current-clamp conditions. 2. Upon stimulation of the afferent fibres, all MS neurones exhibited a sequence of excitatory-inhibitory postsynaptic potentials (EPSP-IPSP). Under voltage clamp, with potassium glutamate as internal solution and at negative holding potentials (Vh), this synaptic pattern appeared as an initial inward current followed by a longer lasting outward current. 3. The inward postsynaptic current was completely abolished by 5 microM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas the outward current disappeared in the presence of 10 microM-bicuculline. Thus the major excitatory and inhibitory synaptic inputs were identified as being due to activation of quisqualate/kainate glutamatergic and gamma-aminobutyric acid (GABAA) receptors, respectively. 4. At positive Vh a CNQX-resistant component of the excitatory postsynaptic current (EPSC) was revealed. This component was slower than the one mediated by the quisqualate receptor and was abolished by 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), indicating that N-methyl-D-aspartate (NMDA) receptors are involved in excitatory synaptic transmission in MS cells. The existence of the two main subtypes (NMDA and non-NMDA) of glutamatergic receptors in MS neurones was also confirmed by the responses of the neurones to bath application of the different agonists (glutamate, quisqualate, kainate and NMDA). 5. The CNQX-sensitive EPSC had a reversal potential near 0 mV. The fast rise time (approximately 0.7 ms) indicates a somatic location of the excitatory synapses. The relaxation kinetics of the fast EPSC were fitted by a single exponential function with a time constant of 1.13 +/- 0.1 ms. This parameter was independent of Vh. Fast EPSCs were blocked by CNQX in a dose-dependent manner (dissociation constant, KD = 0.2 microM). 6. Inhibitory postsynaptic currents (IPSCs) were studied in symmetrical chloride solutions after blockade of the excitatory receptors. The current-voltage relation was linear and reversed at 0 mV. The IPSCs had a fast rise time and their decay was best fitted by the sum of two exponentials with time constant of approximately 20 and 50 ms (Vh = -60 mV). The IPSCs were abolished by bicuculline (KD = 1 microM), a selective antagonist of GABAA receptors. As expected, bath application of GABA produced large whole-cell currents. 7. In many cells, in addition to the usual EPSP-IPSP sequence, failures of either the EPSP or the IPSP were frequently observed during the experimental protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

Gas-component analysis of laser fusion targets.

A gas-chromatographic method for analyzing the fuel content of laser fusion targets has been developed. It provides information on isotope ratios in the fuel gas, percent of molecular species, and total pressure of fuel gas in individual targets to a limit of 0.2 ng DT. The method can also be used to quantify other gaseous components not active in the thermonuclear process (e.g., H2, He, etc.).