Age-related alterations to working memory and to pyramidal neurons in the prefrontal cortex of rhesus monkeys begin in early middle-age and are partially ameliorated by dietary curcumin.

Layer 3 (L3) pyramidal neurons in aged rhesus monkey lateral prefrontal cortex (LPFC) exhibit significantly elevated excitability in vitro and reduced spine density compared to neurons in young subjects. The time-course of these alterations, and whether they can be ameliorated in middle age by the powerful anti-oxidant curcumin is unknown. We compared the properties of L3 pyramidal neurons from the LPFC of behaviorally characterized rhesus monkeys over the adult lifespan using whole-cell patch clamp recordings and neuronal reconstructions. Working memory (WM) impairment, neuronal hyperexcitability, and spine loss began in middle age. There was no significant relationship between neuronal properties and WM performance. Middle-aged subjects given curcumin exhibited better WM performance and less neuronal excitability compared to control subjects. These findings suggest that the appropriate time frame for intervention for age-related cognitive changes is early middle age, and points to the efficacy of curcumin in delaying WM decline. Because there was no relationship between excitability and behavior, the effects of curcumin on these measures appear to be independent.

[Acute angle closure : An ophthalmological emergency in the emergency room]. / Akuter Winkelblock : Ein ophthalmologischer Notfall in der medizinischen Notaufnahme.

BACKGROUND: The symptoms of acute angle closure, an emergency event that can lead to irreversible blindness without timely treatment, are diverse. Initially, these can be interpreted as internal or neurological diseases if headaches, pupil rigidity or nausea are in the foreground. The aim of our study was to assess the rate of harming and invasive diagnostics after primary presentation of patients with acute primary angle closure to nonophthalmologists. METHODS: Retrospective single center study of patients with acute primary angle closure. To analyze these patients, all patients who were treated by surgical iridectomy (5-133.0) or iridotomy by laser (5-136.1) in the period 2014-2018 at the Eye Center at Medical Center, University of Freiburg (Germany), were identified. Subsequently, data analysis was carried out through file inspection to check the inclusion and exclusion criteria and the course of the disease. RESULTS: In total, 91 patients with acute primary angle closure were included. Of these, 28% (n = 25) initially presented to nonophthalmological disciplines. In this patient group 56% (n = 11) received nontargeted diagnostics, with cranial imaging in 32% (n = 8) and lumbar puncture in 8% (n = 2). CONCLUSION: Acute primary angle closure is associated with a high rate of nontargeted diagnostics by nonophthalmologists. Therefore, the clinical picture of acute angle closure should be in mind across all disciplines. With unspecific symptoms such as headaches, nausea and vomiting as well as pupil rigidity, the possibility of an acute increase in intraocular pressure caused by acute angle closure must be considered and early consultation with an ophthalmologist is recommended.

Toric IOL positioning with a no-touch head-up display axis alignment.

PURPOSE: To compare a new no-touch alignment technique for toric intraocular lenses (IOL) with the conventional technique that uses a manual pendulum. METHODS: In this retrospective case-control study, patients who underwent toric IOL implantation using two different alignment techniques (digital Callisto® system vs. manual-pendulum-based marking) were compared in a vector analysis using the Alpins method and an analysis of variance regarding corrected and uncorrected visual acuity and the deviation of the achieved IOL axis from the targeted axis. RESULTS: Sixty-one eyes were included into analysis. Thirty-six of these surgeries were performed via the Callisto® system and 25 eyes via pendulum-based corneal markings. Median IOL axis misalignment was 3° in both groups. Median uncorrected distance visual acuity was 0.097 logMAR versus 0.200. Median best-corrected visual acuity was 0.000 logMAR versus 0.097. All these data were below the range of statistical significance (p > 0.05). Vector analysis showed no significant difference for TIA [median of 3.14 diopters (D) vs. 2.73 D], SIA (median of 3.82 D vs. 3.79 D), DV (1.18 D vs. 1.08 D), and CI (1.23 vs. 1.29). Median angle of error was 1.96° versus - 0.44° (p > 0.05). CONCLUSIONS: We found no significant difference in the refractive results, the IOL positioning, and the best-corrected and uncorrected distance visual acuity between the two compared methods. Nevertheless, the Callisto® IOL alignment system delivers a standardized and easy-to-use technology. In particular, less-experienced surgeons might benefit from this marking technique.

Bleb-related infections and long-term follow-up after trabeculectomy.

PURPOSE: Bleb-related infections are serious complications after trabeculectomy. They can be limited to the bleb or disseminate and lead to endophthalmitis. We herein report on all bleb-related infections that have been diagnosed at the Eye Center of the University of Freiburg, Germany, since 1999. METHODS: We reviewed a total of 1816 consecutive trabeculectomies that were performed at our hospital between the years 1999 and 2014 (353 without and 1463 with intraoperative application of mitomycin C). All bleb-related infections that were diagnosed at our clinic during the same period were included in the analysis. We fitted a Cox proportional hazards model to characterize risk factors for bleb-related infections. RESULTS: We diagnosed a total of 19 bleb-related infections in this period. Three patients with bleb-related infections that came to our clinic had their trabeculectomy performed elsewhere. The overall percentage of bleb-related infections was 0.1% after 2 years (Kaplan-Meier estimate at median follow-up). Nine eyes suffered from only localized infection of the bleb. Seven eyes developed endophthalmitis. Four infections occurred during the first postoperative month. The median age on the day of diagnosis was 71 years; the median age at surgery was 69 years. In the Cox model, intraoperative application of mitomycin C and a fornix-based conjunctival flap were identified as significant risk factors (hazard ratio: 79.02, 4.69; p < 0.01, p < 0.01). The whole group showed a reduction of visual acuity in the median from logMAR 0.12 to 0.2. Eyes that suffered from endophthalmitis showed a loss from 0.3 to 0.96, while the localized infections had a reduction from 0.04 to 0.07. CONCLUSION: Bleb-related infections are a rare complication following trabeculectomy and can be localized on the bleb or can lead to endophthalmitis, thereby threatening visual acuity. The risks and benefits of mitomycin C-augmented trabeculectomies should be taken into consideration.

The effect of additional trabecular aspiration to phacoemulsification and trabectome as a triple procedure in pseudoexfoliation glaucoma: a case-matched study.

BACKGROUND: Trabectome surgery combined with phacoemulsification is effective in lowering (intraocular pressure) IOP in pseudoexfoliation glaucoma. Trabectome surgery usually aims to remove two to four clock hours of the trabecular meshwork. When adding trabecular aspiration, the remaining meshwork can be treated as well, and therefore 360 degrees of the meshwork can be reached. This study was conducted to investigate the additional benefits and risks of adding trabecular aspiration to the combination of phacoemulsification and trabectome as a triple procedure. METHODS: Two groups of patients from two centres were compared. The first group underwent phacoemulsification and trabectome (Freiburg), and the second group underwent additional trabecular aspiration (Düsseldorf). Using a case-matched retrospective study design, 50 patients were included into each group. The clinical endpoint was the intraocular pressure at follow up. RESULTS: The mean follow up was 22 months. Mean intraocular pressure decreased in all 100 patients from 25.0 (SD 4.3) to 14.9 mmHg (SD 4.0). Comparing the two groups, the IOP reduction was from 25.0 (SD 5.0) to 14.1 (SD 4.4) mmHg in the triple procedure group compared to a reduction from 25.0 (3.6) to 15.7 (SD 3.4) mmHg in the phaco/trabectome group. The difference was statistically significant (p = 0.03). The number of medication after surgery was reduced from 2.2 (SD 0.9) to 1.7 (SD 0.9) while in the phaco/trabectome group the medication score was reduced from 2.2 (SD 1.0) to 1.1 (SD 0.1) (p < 0.001). CONCLUSIONS: This study shows that the combination of trabectome surgery and phacoemulsification leads to a clinically significant reduction of IOP over several years in patients with pseudoexfoliation glaucoma. The addition of trabecular aspiration as a triple procedure results in further lowering of IOP without causing more side effects. This finding might be biased by the higher medication score in the triple procedure group.

[Bruch's Membrane Opening in OCT in Correlation with Corneal Correction - a Pilot Study]. / Abhängigkeit der Größe der OCT-Bruch-Membran-Öffnung von Hornhautkorrekturfaktoren ­ eine Pilotstudie.

Background: The measurement by optical coherence tomography (OCT) of the Bruch membrane opening (BMO) and the thinnest retinal fiber nerve layer in relation to it (BMO-MRW) has been performed in clinical routine since 2014. To compensate for astigmatism, or increased or decreased corneal power, a correction is performed through the mean K-value of the measured eye. The aim of this study was to measure the value of this correction and its influence on the measurement results. Methods: The sectors of BMO-MRW and BMO values of the five right eyes of five healthy patients were measured five times each with Heidelberg Spectralis OCT. Corneal compensation was systematically raised with each single measurement (7.1, 7.4, 7.7, 8.0, 8.3 mm). RESULTS: The data showed almost linear dependence on the given corneal compensation values, with intraindividual variability. For the BMO-MRW, only small effects of compensation were found (0.85 up to 1.97 % per K-value difference of 0.3 mm). For BMO, the effect was greater, with a mean change of 7.71 % for every 0.3 mm change in compensation. Conclusion: For BMO-MRW, corneal compensation is of low clinical relevance. BMO is more dependent on this correction. In follow-up measurements, the compensation might not account for significant changes, although we recommend using correct corneal compensation values when obtaining single or first-time measurements.

Dendritic spine changes associated with normal aging.

Given the rapid rate of population aging and the increased incidence of cognitive decline and neurodegenerative diseases with advanced age, it is important to ascertain the determinants that result in cognitive impairment. It is also important to note that much of the aged population exhibit 'successful' cognitive aging, in which cognitive impairment is minimal. One main goal of normal aging studies is to distinguish the neural changes that occur in unsuccessful (functionally impaired) subjects from those of successful (functionally unimpaired) subjects. In this review, we present some of the structural adaptations that neurons and spines undergo throughout normal aging and discuss their likely contributions to electrophysiological properties and cognition. Structural changes of neurons and dendritic spines during aging, and the functional consequences of such changes, remain poorly understood. Elucidating the structural and functional synaptic age-related changes that lead to cognitive impairment may lead to the development of drug treatments that can restore or protect neural circuits and mediate cognition and successful aging.

Age-related increase of sI(AHP) in prefrontal pyramidal cells of monkeys: relationship to cognition.

Reduced excitability, due to an increase in the slow afterhyperpolarization (and its underlying current sI(AHP)), occurs in CA1 pyramidal cells in aged cognitively-impaired, but not cognitively-unimpaired, rodents. We sought to determine whether similar age-related changes in the sI(AHP) occur in pyramidal cells in the rhesus monkey dorsolateral prefrontal cortex (dlPFC). Whole-cell patch-clamp recordings were obtained from layer 3 and layer 5 pyramidal cells in dlPFC slices prepared from young (9.6 ± 0.7 years old) and aged (22.3 ± 0.7 years old) behaviorally characterized subjects. The amplitude of the sI(AHP) was significantly greater in layer 3 (but not layer 5) cells from aged-impaired compared with both aged-unimpaired and young monkeys, which did not differ. Aged layer 3, but not layer 5, cells exhibited significantly increased action potential firing rates, but there was no relationship between sI(AHP) and firing rate. Thus, in monkey dlPFC layer 3 cells, an increase in sI(AHP) is associated with age-related cognitive decline; however, this increase is not associated with a reduction in excitability.

Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs.

In the rTg4510 mouse model, expression of the mutant human tau variant P301L leads to development of neurofibrillary tangles (NFTs), neuronal death, and memory impairment, reminiscent of the pathology observed in human tauopathies. In the present study, we examined the effects of mutant tau expression on the electrophysiology and morphology of individual neurons using whole-cell patch-clamp recordings and biocytin filling of pyramidal cells in cortical slices prepared from rTg4510 (TG) and wild-type (WT) littermate mice. Among the TG cells, 42% contained a clear Thioflavin-S positive inclusion in the soma and were categorized as NFT positive (NFT+), while 58% had no discernable inclusion and were categorized as NFT negative (NFT-). The resting membrane potential (V(r)) was significantly depolarized (+8 mV) in TG cells, and as a consequence, evoked repetitive action potential (AP) firing rates were also significantly increased. Further, single APs were significantly shorter in duration in TG cells and the depolarizing voltage deflection or "sag" evoked by hyperpolarization was significantly greater in amplitude. In addition to these functional electrophysiological changes, TG cells exhibited significant morphological alterations, including loss or significant atrophy of the apical tuft, reduced dendritic complexity and length, and reduced spine density. Importantly, NFT- and NFT+ TG cells were indistinguishable with regard to both morphological and electrophysiological properties. Our observations show that expression of mutated tau results in significant structural and functional changes in neurons, but that these changes occur independent of mature NFT formation.

Synapses are lost during aging in the primate prefrontal cortex.

An electron microscopic analysis has been carried out on the effects of age on the numerical density of both excitatory (asymmetric) and inhibitory (symmetric) synapses in the neuropil of layers 2/3 and of layer 5 in area 46 from the frontal cortex of behaviorally tested rhesus monkeys. There is no change in the lengths of synaptic junctions with age or in the percentage distribution of synapses relative to the postsynaptic spines and dendritic shafts. However, in layers 2/3 there is an overall loss of about 30% of synapses from 5 to 30 years of age, and both asymmetric and symmetric synapses are lost at the same rate. In layer 5 the situation is different; the overall loss of synapses is only 20% and this is almost entirely due to a loss of asymmetric synapses, since there is no significant loss of symmetric synapses from this layer with age. When the synapse data are correlated with the overall cognitive impairment shown by the monkeys, it is found that there is a strong correlation between the numerical density of asymmetric synapses in layers 2/3 and cognitive impairment, with a weaker correlation between symmetric synapse loss and cognitive impairment. In layer 5 on the other hand there is no correlation between synapse loss and cognitive impairment. However synapse loss is not the only factor causing cognitive impairment, since in previous studies of area 46 we have found that age-related alteration in myelin in this frontal area also significantly contributes to cognitive decline. The synapse loss is also considered in light of earlier studies, which show that the frequency of spontaneous excitatory synaptic responses is reduced with age in layers 2/3 neurons.

Effects of aging on the electrophysiological properties of layer 5 pyramidal cells in the monkey prefrontal cortex.

A significant decline in executive system function mediated by the prefrontal cortex (PFC) often occurs with normal aging. In vitro slice studies have shown that layer 2/3 pyramidal cells in the monkey PFC exhibit increased action potential (AP) firing rates which may, in part, contribute to this decline. Given that layer 5 cells also play a role in executive system function, it is important to determine if similar age-related changes occur in these cells. Whole-cell patch-clamp recordings in in vitro slices prepared from the PFC of young and aged behaviorally characterized rhesus monkeys were employed to answer this question. Basic membrane and repetitive AP firing properties were unaltered with age. Aged cells exhibited significantly decreased single AP amplitude, duration and fall time and increased slow afterhyperpolarization (sAHP) amplitude, but these changes were not associated with cognitive performance. This study demonstrates that layer 5 pyramidal cells, unlike layer 2/3 pyramidal cells, undergo only modest electrophysiological changes with aging, and that these changes are unlikely to contribute to age-related cognitive decline.

Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies.

Serotonin 5-hydroxytryptamine type 3 receptors (5HT3R) are Ca2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT3Rs, as it was mimicked by the selective 5HT3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT3R-mediated increase in GABA release was blocked by 100 microM cadmium or by omitting Ca2+ in external solutions, indicating the Ca2+-dependence of the effect. The high voltage-activated Ca2+ channel blockers omega-conotoxin GVIA and omega-conotoxin MVIIC and 10 microM cadmium had no significant effect on the 5-HT3R-mediated enhancement of GABA release, indicating that Ca2+ influx through the 5-HT3R facilitates GABA release. Taken together, these data provide direct evidence that Ca2+ entry via presynaptic 5HT3Rs facilitates the release of GABA from hippocampal interneurons.

Normal aging results in decreased synaptic excitation and increased synaptic inhibition of layer 2/3 pyramidal cells in the monkey prefrontal cortex.

Executive system function, mediated largely by the prefrontal cortex (PFC), often declines significantly with normal aging in humans and non-human primates. The neural substrates of this decline are unknown, but age-related changes in the structural properties of PFC neurons could lead to altered synaptic signaling and ultimately to PFC dysfunction. The present study addressed this issue using whole-cell patch clamp assessment of excitatory and inhibitory postsynaptic currents (PSCs) in layer 2/3 pyramidal cells in in vitro slices of the PFC from behaviorally characterized young (< or =12 years old) and aged (> or =19 years old) rhesus monkeys. Behaviorally, aged monkeys were significantly impaired in performance on memory and executive system function tasks. Physiologically, the frequency of spontaneous glutamate receptor-mediated excitatory PSCs was significantly reduced in cells from aged monkeys, while the frequency of spontaneous GABAA receptor-mediated inhibitory PSCs was significantly increased. In contrast, there was no effect of age on the frequency, amplitude, rise time or decay time of action potential-independent miniature excitatory and inhibitory PSCs. The observed change in excitatory-inhibitory synaptic balance likely leads to significantly altered signaling properties of layer 2/3 pyramidal cells in the PFC with age.

Development and modulation of GABA(A) receptor-mediated neurotransmission in the CA1 region of prenatally protein malnourished rats.

Prenatal protein malnutrition has been demonstrated to result in alterations in the serotonergic and GABAergic neurotransmitter systems in the rat hippocampus. In the present study, whole-cell patch clamp recordings of CA1 pyramidal cells were employed in an effort to gain insight into the specific cellular locus and functional consequences of the previously reported changes. Hippocampal slices were prepared from Sprague-Dawley rats whose dams were fed either a normal (25% casein) or low (6% casein) protein diet during pregnancy. The development of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) and their modulation by the benzodiazipine agonist zolpidem were compared in cells from the two nutritional groups at postnatal days 7, 14, 21 and >90. The modulation of mIPSCs by serotonin was also examined in cells from 21 day old rats. No significant differences were observed in the characteristics of mIPSCs in cells from control vs. prenatally protein malnourished rats at any of the ages studied, although there was a trend for a higher frequency of mIPSCs in adult (>p90) prenatally protein malnourished rats. At all ages, zolpidem produced a significant increase in the mean decay time of mIPSCs that was not significantly different in cells from the two nutritional groups. Serotonin application resulted in a significant increase in the frequency of mIPSCs in CA1 pyramidal cells but there was no significant difference between cells from the two nutritional groups in the characteristics of this effect. These data demonstrate that the previously observed alterations in the serotonergic and GABAergic systems that result from prenatal protein malnutrition do not have significant functional consequences at a single cell level in the CA1 region of the rat hippocampus as measured in vitro.

Prenatal protein malnutrition results in increased frequency of miniature inhibitory synaptic currents in rat CA1 pyramidal cells.

There is growing evidence for an effect of prenatal protein malnutrition on the GABAergic neurotransmitter system in the rat hippocampus and associated structures. In the present study, we examined the functional electrophysiological consequences of observed alterations in GABA(A) and benzodiazepine receptor systems. Whole-cell patch clamp recordings of spontaneous and of miniature inhibitory postsynaptic currents (mIPSCs) generated by CA1 pyramidal cells were performed in in vitro hippocampal slices prepared from control and prenatally protein malnourished adult male rats. The characteristics of spontaneous synaptic currents were unaltered by the prenatal insult, as were the amplitudes and kinetics of GABA(A) receptor-mediated mIPSCs. The frequency of mIPSCs, however, was significantly increased in CA1 pyramidal cells in slices prepared from prenatally malnourished vs. control rats. The effect of the benzodiazepine receptor agonist chlordiazepoxide on the characteristics of mIPSCs was also examined and found to be the same in cells from both nutritional groups. The increased frequency of mIPSCs together with the lack of a change in amplitude, kinetics, or modulation by benzodiazepines of mIPSCs in response to prenatal protein malnutrition indicate a presynaptic locus of effect of this insult.

Electrophysiological properties of rat hippocampal principal cells are unaltered by prenatal protein malnutrition.

There is growing evidence that prenatal protein malnutrition alters the development of the hippocampal formation in rats (Morgane et al., 1993; Galler et al., 1996; Almeida et al., 1996, for reviews). Little is known, however, of the possible functional consequences of prenatal malnutrition on the physiology of principal cells in the hippocampus. We have addressed this issue by comparing the electrophysiological properties of hippocampal neurons (dentate granule cells and CA1 pyramidal cells) in slices prepared from control and from prenatally protein malnourished adult male and female Sprague-Dawley rats. We found no significant effect of the prenatal protein malnutrition insult upon a number of intrinsic membrane properties measured with whole-cell current clamp recordings, including: resting membrane potential, input resistance, and membrane time constant, or on action potential characteristics such as threshold, amplitude, and/or firing frequency. Additionally, we saw no effect of prenatal malnutrition upon extracellular measures of glutamatergic synaptic transmission such as the presynaptic fiber volley, excitatory postsynaptic potential or population spike amplitude at the perforant path to dentate granule cell synapse or at the Schaffer collateral to CA1 pyramidal cell synapse. In conclusion, we have demonstrated that prenatal protein malnutrition does not result in significant alterations of the cellular physiological properties of these two types of principal neurons in the adult rat hippocampus.

Morphology and electrophysiology of dentate granule cells in the rhesus monkey: comparison with the rat.

The morphologic and electrophysiologic properties of dentate granule cells in the young adult rhesus monkey (Macaca mulatta) were examined for the first time with whole-cell patch clamp recordings and intracellular biocytin filling in in vitro hippocampal slice preparations. Data from monkeys were compared with data generated in an identical manner from adult Sprague-Dawley rats. Intracellularly filled monkey and rat granule cells were identical in numerous morphologic parameters, including area of somata, total dendritic length, dendritic spread, segment number and length, and branching pattern. The single statistically significant difference in morphology was the vertical extent of the dendritic tree (distance from soma to fissure), which was 20% greater in the monkey. The passive membrane properties (resting membrane potential, input resistance, and membrane time constant) measured under current clamp conditions were virtually identical. The thresholds and amplitudes of action potentials were the same, but significant differences were seen in the kinetics of single action potentials. Monkey granule cell action potentials were significantly longer in duration (with slower rise and fall times) than action potentials in rat cells. These differences were likely due to a much smaller fast after hyperpolarization in the monkey as compared with the rat cells. Thus, with the exception of action potential properties, the principal finding of this study is that there is significant conservation of both form and function in dentate granule cells in these two species, despite the enormous phylogenetic separation. This suggests that granule cell properties may be extremely stable across diverse mammalian species.

Exocytotic Ca2+ channels in mammalian central neurons.

Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters.

Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms.

The voltage dependence of gamma-aminobutyric-acid- and norepinephrine-induced inhibition of N-type calcium current in cultured embryonic chick dorsal-root ganglion neurons was studied with whole-cell voltage-clamp recording. The inhibitory action of the neurotransmitters was comprised of at least two distinct modulatory components, which were separable on the basis of their differential voltage dependence. The first component, which we term "kinetic slowing", is associated with a slowing of the activation kinetics--an effect that subsides during a test pulse. The kinetic-slowing component is largely reversed at depolarized voltages (i.e., it is voltage-dependent). The second component, which we term "steady-state inhibition", is by definition not associated with a change in activation kinetics and is present throughout the duration of a test pulse. The steady-state inhibition is not reversed at depolarized voltages (i.e., it is voltage-independent). Although the two components can be separated on the basis of their voltage dependence, they appear to be indistinguishable in their time courses for onset and recovery as well as their rates of desensitization following multiple applications of transmitter. Furthermore, neither component requires cell dialysis, as both are observed using perforated-patch as well as whole-cell recording configurations. The co-existence in nerve terminals of both voltage-dependent and -independent mechanisms to modulate calcium channel function could offer a means of differentially controlling synaptic transmission under conditions of low- and high-frequency presynaptic discharge.