Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Effects of Metformin on Spontaneous Ca2+ Signals in Cultured Microglia Cells under Normoxic and Hypoxic Conditions.

Microglial functioning depends on Ca2+ signaling. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5-7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10-15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10-15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.

Different effects of metformin and phenformin on hypoxia-induced Ca2+ fluxes in cultured primary neurons.

Recent evidence suggests that metformin and phenformin may exert beneficial effects against neuronal injury in the ischemic brain, however, the difference of action between these two drugs and the molecular mechanism of such protection is not clear. In this study, we investigated whether mild hypoxia-affected neurons exhibit changes in cytosolic calcium handling and whether metformin and phenformin exert any effect on calcium homeostasis in hypoxia-affected neurons. Cultured primary rat cortical cells were stained with calcium sensitive dye Oregon Green 488 BAPTA-1 AM and spontaneous calcium dependent changes of fluorescence were recorded. Using obtained fluorescence traces we estimated changes in relative amplitude of recorded spontaneous signals, changes in frequency of spontaneous activity, and changes in decay of fluorescence traces. We found that hypoxia caused reduction of the relative signal amplitude, increased the spontaneous activity, and slowed the decay of calcium concentration. After pre-treatment of cells with 0.1-0.5 mM metformin, the relative signal amplitude increased and the frequency of spontaneous signals decreased in hypoxia-affected neurons. However, pre-treatment with 1-25 µM phenformin neither increased the relative signal amplitude nor reduced the frequency of spontaneous signals. The decay of fluorescence traces became faster after application of metformin or phenformin comparing to neurons under hypoxic conditions. These results suggest different action of metformin and phenformin in improvement of Ca2+ homeostasis in hypoxia-affected neurons, which may have different effects on neuronal survival and functions after hypoxia/ischemia.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Spatial synchronization of visual stimulus-evoked gamma frequency oscillations in the rat superior colliculus.

In the superior colliculus, visual stimuli can induce gamma frequency oscillations of neuronal activity. It has been shown that in cats, these oscillations are synchronized over distances of greater than 300 µm that may contribute toward visual information processing. We investigated the spatial properties of such oscillations in a rodent because the availability of molecular tools could enable future studies on the role of these oscillations in visual information processing. Using extracellular electrode array recordings in anesthetized rats, we found that visual stimuli-induced gamma and eta frequency (30-115 Hz) oscillations of the local field potential that were synchronized over distances of ∼ 600 µm. Multiple-unit events were phase locked to the local field potential signal and showed prominent oscillations during OFF responses. The rate of lower than 5 ms cross-electrode coincidences was in line with the response-corrected predictions for each electrode. These data suggest that the synchronized superior colliculus neuronal activity is largely network driven, whereas common synaptic inputs play a minor role.

Visual Stimuli Evoked Action Potentials Trigger Rapidly Propagating Dendritic Calcium Transients in the Frog Optic Tectum Layer 6 Neurons.

The superior colliculus in mammals or the optic tectum in amphibians is a major visual information processing center responsible for generation of orientating responses such as saccades in monkeys or prey catching avoidance behavior in frogs. The conserved structure function of the superior colliculus the optic tectum across distant species such as frogs, birds monkeys permits to draw rather general conclusions after studying a single species. We chose the frog optic tectum because we are able to perform whole-cell voltage-clamp recordings fluorescence imaging of tectal neurons while they respond to a visual stimulus. In the optic tectum of amphibians most visual information is processed by pear-shaped neurons possessing long dendritic branches, which receive the majority of synapses originating from the retinal ganglion cells. Since the first step of the retinal input integration is performed on these dendrites, it is important to know whether this integration is enhanced by active dendritic properties. We demonstrate that rapid calcium transients coinciding with the visual stimulus evoked action potentials in the somatic recordings can be readily detected up to the fine branches of these dendrites. These transients were blocked by calcium channel blockers nifedipine CdCl2 indicating that calcium entered dendrites via voltage-activated L-type calcium channels. The high speed of calcium transient propagation, >300 µm in <10 ms, is consistent with the notion that action potentials, actively propagating along dendrites, open voltage-gated L-type calcium channels causing rapid calcium concentration transients in the dendrites. We conclude that such activation by somatic action potentials of the dendritic voltage gated calcium channels in the close vicinity to the synapses formed by axons of the retinal ganglion cells may facilitate visual information processing in the principal neurons of the frog optic tectum.

20Hz membrane potential oscillations are driven by synaptic inputs in collision-detecting neurons in the frog optic tectum.

Although the firing patterns of collision-detecting neurons have been described in detail in several species, the mechanisms generating responses in these neurons to visual objects on a collision course remain largely unknown. This is partly due to the limited number of intracellular recordings from such neurons, particularly in vertebrate species. By employing patch recordings in a novel integrated frog eye-tectum preparation we tested the hypothesis that OFF retinal ganglion cells were driving the responses to visual objects on a collision course in the frog optic tectum neurons. We found that the majority (22/26) of neurons in layer 6 responding to visual stimuli fitted the definition of η class collision-detectors: they readily responded to a looming stimulus imitating collision but not a receding stimulus (spike count difference ∼10 times) and the spike firing rate peaked after the stimulus visual angle reached a threshold value of ∼20-45°. In the majority of these neurons (15/22) a slow frequency oscillation (f=∼20Hz) of the neuronal membrane potential could be detected in the responses to a simulated collision stimulus, as well as to turning off the lights. Since OFF retinal ganglion cells could produce such oscillations, our observations are in agreement with the hypothesis that 'collision' responses in the frog optic tectum neurons are driven by synaptic inputs from OFF retinal ganglion cells.

Persistent sodium current decreases transient gain in turtle motoneurons.

Voltage dependent ion channels can influence signal integration in neurons dramatically. In addition to the classical fast-inactivating Na(+) current that mediates action potentials, many neurons also express persistent sodium current (I(NaP)). Activating at membrane potentials below the threshold for action potentials, this current may amplify excitatory postsynaptic potentials and shape the firing patterns. To determine the qualitative contribution of I(NaP) to the intrinsic firing properties of motoneurons, we eliminated this current by dynamic clamp. As expected, we found that elimination of I(NaP) shifted the rheobase to more positive currents. More interestingly, elimination of I(NaP) increased the steepness of initial frequency-to-current (fI) relation. This suggests that I(NaP) decreases the transient gain and broadens the integration window for short synaptic inputs in spinal motoneurons.

Asymmetric excitatory synaptic dynamics underlie interaural time difference processing in the auditory system.

Low-frequency sound localization depends on the neural computation of interaural time differences (ITD) and relies on neurons in the auditory brain stem that integrate synaptic inputs delivered by the ipsi- and contralateral auditory pathways that start at the two ears. The first auditory neurons that respond selectively to ITD are found in the medial superior olivary nucleus (MSO). We identified a new mechanism for ITD coding using a brain slice preparation that preserves the binaural inputs to the MSO. There was an internal latency difference for the two excitatory pathways that would, if left uncompensated, position the ITD response function too far outside the physiological range to be useful for estimating ITD. We demonstrate, and support using a biophysically based computational model, that a bilateral asymmetry in excitatory post-synaptic potential (EPSP) slopes provides a robust compensatory delay mechanism due to differential activation of low threshold potassium conductance on these inputs and permits MSO neurons to encode physiological ITDs. We suggest, more generally, that the dependence of spike probability on rate of depolarization, as in these auditory neurons, provides a mechanism for temporal order discrimination between EPSPs.

The scientific heritage of Professor Aron Gutman (Commemorating the 10th anniversary of Aron Gutman's death).

Aron Gutman started his scientific research when he was a student of the Department of Physics and Mathematics, Vilnius University. At that time, he developed the theory of nonhomogenous vector relations between magnetic moments of electrons in an atom and applied it for explanation of energy spectrum of real atoms. Since 1960, he worked in Kaunas Medical Institute, and his main field of scientific interests was theoretical biophysics and electrophysiology of living tissues and cells. The earlier biophysical works of A. Gutman dealt with problems of the bioelectrical fields that underlie electroencephalogram, electrocorticogram, and electrocardiogram. The most important achievement was a theory of individual potential or postsynaptic field potential of synapses from individual axon (EEG quantum) and its role in shaping of electroencephalogram. In the later works (from 1971), he looked into properties and function of the individual nerve cells. He had created and developed the theory of nonlinear (bistable) dendrites and analyzed functional implications of such dendrites. In the last works, A. Gutman tried to relate the functioning of the nervous system at the cellular and system levels. He made efforts to find connection between the properties of individual neurones and principles (laws) of functioning of the nervous system. He had managed to relate dendritic bistability of neurones and Gelfand-Tsetlin principle of the functioning of the central nervous system (also known as the principle of minimal afferentiation). He explained some regularities in motor control by the dendritic bistability of motoneurones.

An eye-tectum preparation allowing routine whole-cell recordings of neuronal responses to visual stimuli in frog.

We propose an in vitro eye-tectum preparation enabling whole-cell recordings of tectal neurons combined with visual stimulation. The recordings were made from the tectum, which was cut in order to facilitate access to the cell bodies located in the inner tectal layers. The preparation remains viable for up to 5h while routine prolonged whole-cell recordings could be obtained from tectal neurons. Cutting of the tectum did not disrupt endogenous synaptic circuits and sensory inputs allowing examination of functional neuronal responses evoked with visual stimuli. Recordings from layer 6 tectal neurons indicated that neuronal responses were shaped by a mixture of excitation and inhibition generated by sensory input and local neuronal network. Visually evoked synaptic responses could also activate fast dendritic currents. Thus, the preparation brings about the benefits of in vivo recordings without the effects of anesthetics that could influence processing of sensory inputs. Using the proposed preparation, the network circuit function, which operates during central processing of a visual input, can be studied as well as the role of intrinsic properties of neurons in detection and processing of visual information.

Spikelet currents in frog tectal neurons with different firing patterns in vitro.

Neuronal potential-dependent membrane currents are important in shaping the integration of synaptic inputs. Our recordings in voltage-clamp mode indicate that the small fast inward currents (spikelet currents), which were several times smaller than action potential (AP) currents, are a distinguished feature of 33% of neurons from 8 to 6 layers of the frog tectum. Out of all neuronal types described previously, only phasic cells and neurons with 'sag' in response to hyperpolarizing step current injection did not show spikelet currents. These small fast inward currents were sensitive to the intracellular administration of the sodium channel blocker QX-314, but not to the extracellular application of a glutamate receptor antagonist kynurenic acid. This suggests that spikelet currents are mediated by fast voltage-dependent Na(+) channels. Since spikelet currents could also be elicited with synaptic stimulation it is possible that spikelets are generated in dendrites and, thus, are important for fast integration of visual signals in tectal neurons.

Well-timed, brief inhibition can promote spiking: postinhibitory facilitation.

Brief synaptic inhibition can overwhelm a nearly coincident suprathreshold excitatory input to preclude spike generation. Surprisingly, a brief inhibitory event that occurs in a favorable time window preceding an otherwise subthreshold excitation can facilitate spiking. Such postinhibitory facilitation (PIF) requires that the inhibition has a short (decay) time constant tauinh. The timescale ranges of tauinh and of the window (width and timing) for PIF depend on the rates of neuronal subthreshold dynamics. The mechanism for PIF is general, involving reduction by hyperpolarization of some excitability-suppressing factor that is partially recruited at rest. Here we illustrate and analyze PIF, experimentally and theoretically, using brain stem auditory neurons and a conductance-based five-variable model. In this auditory case, PIF timescales are in the sub- to few millisecond range and the primary mechanistic factor is a low-threshold potassium conductance gKLT. Competing dynamic influences create the favorable time window: hyperpolarization that moves V away from threshold and hyperexcitability resulting from reduced gKLT. A two-variable reduced model that retains the dynamics only of V and gKLT displays a similar time window. We analyze this model in the phase plane; its geometry has generic features. Further generalizing, we show that PIF behavior may occur even in a very reduced model with linear subthreshold dynamics, by using an integrate-and-fire model with an accommodating voltage-dependent threshold. Our analyses of PIF provide insights for fast inhibition's facilitatory effects in longer trains. Periodic subthreshold excitatory inputs can lead to firing, even one for one, if brief inhibitory inputs are interleaved within a range of favorable phase lags. The temporal specificity of inhibition's facilitating effect could play a role in temporal processing, in sensitivity to inhibitory and excitatory temporal patterning, in the auditory and other neural systems.

Cellular signalling properties in microcircuits.

Molecules and cells are the signalling elements in microcircuits. Recent studies have uncovered bewildering diversity in postsynaptic signalling properties in all areas of the vertebrate nervous system. Major effort is now being invested in establishing the specialized signalling properties at the cellular and molecular levels in microcircuits in specific brain regions. This review is part of the TINS Microcircuits Special Feature.

Sodium along with low-threshold potassium currents enhance coincidence detection of subthreshold noisy signals in MSO neurons.

Voltage-dependent membrane conductances support specific neurophysiological properties. To investigate the mechanisms of coincidence detection, we activated gerbil medial superior olivary (MSO) neurons with dynamic current-clamp stimuli in vitro. Spike-triggered reverse-correlation analysis for injected current was used to evaluate the integration of subthreshold noisy signals. Consistent with previous reports, the partial blockade of low-threshold potassium channels (I(KLT)) reduced coincidence detection by slowing the rise of current needed on average to evoke a spike. However, two factors point toward the involvement of a second mechanism. First, the reverse correlation currents revealed that spike generation was associated with a preceding hyperpolarization. Second, rebound action potentials are 45% larger compared to depolarization-evoked spikes in the presence of an I(KLT) antagonist. These observations suggest that the sodium current (I(Na)) was substantially inactivated at rest. To test this idea, I(Na) was enhanced by increasing extracellular sodium concentration. This manipulation reduced coincidence detection, as reflected by slower spike-triggering current, and diminished the hyperpolarization phase in the reverse-correlation currents. As expected, a small outward bias current decreased the pre-spike hyperpolarization phase, and TTX blockade of I(Na) nearly eliminated the hyperpolarization phase in the reverse correlation current. A computer model including Hodgkin-Huxley type conductances for spike generation and for I(KLT) showed reduction in coincidence detection when I(KLT) was reduced or when I(Na) was increased. We hypothesize that desirable synaptic signals first remove some inactivation of I(Na) and reduce activation of I(KLT) to create a brief temporal window for coincidence detection of subthreshold noisy signals.

Subthreshold outward currents enhance temporal integration in auditory neurons.

Many auditory neurons possess low-threshold potassium currents ( I(KLT)) that enhance their responsiveness to rapid and coincident inputs. We present recordings from gerbil medial superior olivary (MSO) neurons in vitro and modeling results that illustrate how I(KLT) improves the detection of brief signals, of weak signals in noise, and of the coincidence of signals (as needed for sound localization). We quantify the enhancing effect of I(KLT) on temporal processing with several measures: signal-to-noise ratio (SNR), reverse correlation or spike-triggered averaging of input currents, and interaural time difference (ITD) tuning curves. To characterize how I(KLT), which activates below spike threshold, influences a neuron's voltage rise toward threshold, i.e., how it filters the inputs, we focus first on the response to weak and noisy signals. Cells and models were stimulated with a computer-generated steady barrage of random inputs, mimicking weak synaptic conductance transients (the "noise"), together with a larger but still subthreshold postsynaptic conductance, EPSG (the "signal"). Reduction of I(KLT) decreased the SNR, mainly due to an increase in spontaneous firing (more "false positive"). The spike-triggered reverse correlation indicated that I(KLT) shortened the integration time for spike generation. I(KLT) also heightened the model's timing selectivity for coincidence detection of simulated binaural inputs. Further, ITD tuning is shifted in favor of a slope code rather than a place code by precise and rapid inhibition onto MSO cells (Brand et al. 2002). In several ways, low-threshold outward currents are seen to shape integration of weak and strong signals in auditory neurons.

Influence of membrane properties on spike synchronization in neurons: theory and experiments.

In the brain spike synchronization in neurons is involved in information transfer and certain forms of dysfunction. The theory of random point processes was used to relate the statistical properties of input point processes to synchronization between firing in neurons viewed as threshold devices. Derived analytical relations describe normalized synchronization in the case of shared input with balanced excitation and inhibition. For neuronal models with unbalanced shared input and spike generating Hodgkin-Huxley type conductances, the theory satisfactorily describes the temporal dependence of spike synchronization on the delay between spikes. Computer generated stochastic stimulus current was used to stimulate motoneurons in turtle spinal cord slices. Theory was able to approximate the temporal dependence of spike synchronization on the delay between spikes when the membrane time constant and the relative spike threshold measured were used in calculations. In agreement with the theoretical prediction, normalized spike synchrony was reduced when the threshold for spike generation was lowered by injection of steady depolarizing bias current. In spinal motoneurons the relative spike threshold can be lowered by a persistent inward current facilitated by activation of certain metabotropic transmitter receptors. After induction of this inward current spike synchronization was reduced several times. It is suggested that downregulation of the persistent inward current in motoneurons by disruption of brainstem modulatory systems, as in Parkinson disease, can facilitate tremor due to the increased synchrony between motoneurons.

Firing properties of frog tectal neurons in vitro.

Whole-cell recordings from frog tectal slices revealed different types of neuronal firing patterns in response to prolonged current injection. The patterns included regular spiking without adaptation, accelerating firing, adapting spiking, repetitive bursting and phasic response with only one spike. The observed firing patterns are similar to those found in the mammalian superior colliculus. The frog tectum could be a useful preparation in elucidating the relationship between neuronal function and membrane properties.

Influence of subthreshold nonlinearities on signal-to-noise ratio and timing precision for small signals in neurons: minimal model analysis.

Subthreshold voltage- and time-dependent conductances can subserve different roles in signal integration and action potential generation. Here, we use minimal models to demonstrate how a non-inactivating low-threshold outward current (I(KLT)) can enhance the precision of small-signal integration. Our integrate-and-fire models have only a few biophysical parameters, enabling a parametric study of I(KLT) effects. I(KLT) increases the signal-to-noise ratio (SNR) for firing when a subthreshold 'signal' EPSP is delivered in the presence of weak random input. The increased SNR is due to the suppression of spontaneous firings to random input. In accordance, SNR grows as the EPSP amplitude increases. SNR also grows as the unitary synaptic current's time constant increases, leading to more effective suppression of spontaneous activity. Spike-triggered reverse correlation of the injected current indicates that, to reach spike threshold, a cell with I(KLT) requires a briefer time course of injected current. Consistent with this narrowed integration time window, I(KI.T) enhances phase-locking. measured as vector strength, to a weak noisy and periodically modulated stimulus. Thus subthreshold negative feedback mediated by I(KLT) enhances temporal processing. An alternative suppression mechanism is voltage- and time-dependent inactivation of a low-threshold inward current. This feature in an integrate-and-fire model also shows SNR enhancement, in comparison with a case when the inward current is non-inactivating. Small-signal detection can be significantly improved in noisy neuronal systems by subthreshold negative feedback, serving to suppress false positives.

Enhancement of signal-to-noise ratio and phase locking for small inputs by a low-threshold outward current in auditory neurons.

Neurons possess multiple voltage-dependent conductances specific for their function. To investigate how low-threshold outward currents improve the detection of small signals in a noisy background, we recorded from gerbil medial superior olivary (MSO) neurons in vitro. MSO neurons responded phasically, with a single spike to a step current injection. When bathed in dendrotoxin (DTX), most cells switched to tonic firing, suggesting that low-threshold potassium currents (I(KLT)) participated in shaping these phasic responses. Neurons were stimulated with a computer-generated steady barrage of random inputs, mimicking weak synaptic conductance transients (the "noise"), together with a larger but still subthreshold postsynaptic conductance, EPSG (the "signal"). DTX reduced the signal-to-noise ratio (SNR), defined as the ratio of probability to fire in response to the EPSG and the probability to fire spontaneously in response to noise. The reduction was mainly attributable to the increase of spontaneous firing in DTX. The spike-triggered reverse correlation indicated that, for spike generation, the neuron with I(KLT) required faster inward current transients. This narrow temporal integration window contributed to superior phase locking of firing to periodic stimuli before application of DTX. A computer model including Hodgkin-Huxley type conductances for spike generation and for I(KLT) (Rathouz and Trussell, 1998) showed similar response statistics. The dynamic low-threshold outward current increased SNR and the temporal precision of integration of weak subthreshold signals in auditory neurons by suppressing false positives.