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Single-cell analysis without immune-specific labelling is essential across research fields, but conventional flow cytometers (FCMs) struggle with label-free analysis. We introduce a novel microfluidic scanning flow cytometer (μSFC) designed for label-free analysis within a simple microfluidic chip. Our system outperforms traditional FCMs for label-free analysis but its signal-to-noise ratio (SNR) limits the minimum detectable size. We present three modifications to enhance SNR and improve the smallest detectable particle size: additional neutral optical density filtering, a lower noise-equivalent-power photoreceiver, and laser spot size reduction. These improvements enable reliable characterization of particles as small as 3 μm. Experimental results validate the correlation between angular profile oscillations and particle size. While reliable detection down to 1 μm is achieved, further refinement is needed. The simplicity and low setup of the μSFC make it promising for integration into multi-parametric single-cell analysis systems, facilitating comprehensive cellular characterization for diagnostic and point-of-care applications.

Abstract Background Italian technical normative in the field of cultural heritage is often considered insufficient or not suitable in practise, therefore efforts are necessary to design new and/or improve already existing ones. Results In this paper an alternative analytical procedure for the determination of water absorption (by full immersion) by stone material, described in the NORMAL 07-81 document, is proposed. Improvements concern methods accuracy and reduction of sample size; further also density data is obtained. Conclusions The new procedure was applied on three different marble samples and outcomes are encouraging, but further testing is running to better understand to what extent sample size can be reduced without worsening accuracy of results, taking into account that stone is a very heterogeneous material.

Introduction: The evaluation of biological degradation of waterlogged archeological wood is crucial to choose the conservative and protective treatments to be applied to the wooden material. The waterlogged environmental conditions are characterized by oxygen scarcity, only allowing the growth of adapted microbes capable to degrade the organic wooden material, mainly erosion bacteria and soft-rot fungi. In this work, we characterized and evaluated the biodegradation state and the microbial communities of wooden fragments preserved in storage tanks. These were preserved by waterlogging within the Neolithic village "La Marmotta," currently found under the Bracciano Lake (Lazio, Italy)., Methods: The waterlogged wood samples were first identified taxonomically with an optical microscope, also allowing an evaluation of their preservation state. The microbial community was then evaluated through the sequencing of Internal Transcribed Spacer sequences for fungi and 16S for bacteria with the Oxford Nanopore Technologies (ONT) MinION platform., Results: The identified microbial community appears to be consistent with the waterlogged samples, as many bacteria attributable to the erosion of wood and ligninolytic fungi have been sequenced., Discussion: The reported results highlight the first use of targeted metabarcoding by ONT applied to study the biodeterioration of waterlogged archeological wood., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beccaccioli, Moricca, Faino, Reale, Mineo and Reverberi.)

In cultural heritage, unaesthetic stains on carbonate stones due to their close contacts with metals are of concern for the preservation of sculptures, monumental facades and archeological finds of various origin and antiquities. Rust stains made up of various oxidized iron compounds are the most frequent forms of alteration. The presence of ferric iron on rust-stained marble surfaces was confirmed in previous studies and oriented the choice of the best cleaning method (based on complexing agents specific for ferric ions). However, the composition of rust stains may vary along their extension. As the corrosion of the metallic iron proceeds, if the oxygen levels in the surroundings are low and there are no conditions to favor the oxidation, ferrous ions can also diffuse within the carbonate structure and form a variety of intermediate compounds. In this study, the iron stains on archeological marbles were compared with those artificially produced on Carrara marbles and Travertine samples. The use of integrated techniques (optical and scanning electron microscopy as well as Mössbauer and XPS spectroscopy) with complementary analytical depths, has provided the overall information. Rust formation and diffusion mechanisms in carbonates were revealed together with the evolution of iron speciation and identification of phases such as ferrihydrite, goethite, maghemite, nanomagnetite, and hematite.

Objective: Deformability is an essential feature of red blood cells (RBCs), enabling them to undergo significant shape change in response to external forces. Impaired erythrocyte deformability is associated with several pathologic conditions, and quantitative measurement of RBC deformability is critical to understanding and diagnosing RBC related diseases. Whereas traditional approaches to cell mechanical characterization generally have limited throughput, emerging microscale technologies are opening new opportunities for high-throughput deformability cytometry at the single-cell level., Methods: In this work, we propose an innovative microfluidic system based on (i) a hyperbolic microchannel to induce erythrocyte deformation by extensional flow, and (ii) an electrical sensing zone with coplanar electrodes to evaluate the deformed cell shape., Results: RBC deformation under extensional flow is achieved, and the deformed cell shape is quantified by means of an electrical anisotropy index, at a throughput of 300 cell/s. Measurements of healthy and chemically stiffened RBCs demonstrate that the anisotropy index can be used to characterize RBC deformability, as an alternative to deformation indices based on high-speed image processing., Conclusion: A contactless and optics-free approach for RBC deformability analysis has been presented., Significance: Due to its simplicity and potential for integration, the proposed approach holds promises for fast and low-cost erythrocyte deformability assays, especially in point-of-care and resource-limited settings.

Background: Despite the well-established worldwide phenomenon of "the feminisation of medicine," in Italy, Urology remains a male-dominated field., Objective: The aims of our work are to assess data on medical students' choice of surgical specialty in Italy to investigate if a gender-biased trend exists and to find the key points that influence the decision-making process when choosing a specialty, with a focus on Urology., Design: Data about access to residency programs in 2017-2020 were analysed through descriptive statistics. Investigations concerning the decision-making process were carried through distribution of an online anonymous survey to Italian medical students., Results: Urology was among the specialties with the lowest proportion of female residents in Italy in the last 4 years: 37 (29.4%) in 2017, 27 (21.4%) in 2018, 40 (26.7%) in 2019, and 57 (25.2%) in 2020. The total number of participants of the survey was 1409, of which only 341 declared being keen to pursue a career path in surgery. Out of the 942 students not interested in surgery, 46.2% females and 22.5% males indicated a "sexist environment" as one of the reasons. Overall, the main reason for medical students not choosing Urology is the lack of interest in the specialty. Furthermore, there is a different perception of Urology as a sexist environment between female (23.4%) and male (3.2%, p  < 0.001) medical students, which may influence their decision-making process., Conclusions: In Italy, the prevalence of female medical graduates does not mirror the proportion of female doctors choosing a career in some surgical specialties, including Urology. Our survey results clearly identified that a large proportion of medical graduates are not choosing urology because of the perception of a sexist environment. While the reasons for this phenomenon remain unclear, the presence of a gender-biased perception of a sexist environment represents a possible explanation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Sofia, Luca, Simona, Simone, Serena, Stefano, Alessandra and Roberto.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Objective: In aerobiological monitoring and agriculture there is a pressing need for accurate, label-free and automated analysis of pollen grains, in order to reduce the cost, workload and possible errors associated to traditional approaches., Methods: We propose a new multimodal approach that combines electrical sensing and optical imaging to classify pollen grains flowing in a microfluidic chip at a throughput of 150 grains per second. Electrical signals and synchronized optical images are processed by two independent machine learning-based classifiers, whose predictions are then combined to provide the final classification outcome., Results: The applicability of the method is demonstrated in a proof-of-concept classification experiment involving eight pollen classes from different taxa. The average balanced accuracy is 78.7% for the electrical classifier, 76.7% for the optical classifier and 84.2% for the multimodal classifier. The accuracy is 82.8% for the electrical classifier, 84.1% for the optical classifier and 88.3% for the multimodal classifier., Conclusion: The multimodal approach provides better classification results with respect to the analysis based on electrical or optical features alone., Significance: The proposed methodology paves the way for automated multimodal palynology. Moreover, it can be extended to other fields, such as diagnostics and cell therapy, where it could be used for label-free identification of cell populations in heterogeneous samples.

Studies have shown that nitrate (NO 3 - )-rich beetroot juice (BJ) supplementation improves endurance and high-intensity intermittent exercise. The dose-response effects on taekwondo following BJ supplementation are yet to be determined. This study aimed to investigate two acute doses of 400 mg of NO 3 - (BJ-400) and 800 mg of NO 3 - (BJ-800) on taekwondo-specific performance and cognitive function tests compared with a placebo (PL) and control (CON) conditions. Eight trained male taekwondo athletes (age: 20 ± 4 years, height: 180 ± 2 cm, body mass: 64.8 ± 4.0 kg) completed four experimental trials using a randomized, double-blind placebo-controlled design: BJ-400, BJ-800, PL, and CON. Participants consumed two doses of BJ-400 and BJ-800 or nitrate-depleted PL at 2.5 h prior to performing the Multiple Frequency Speed of Kick Test (FSKT). Countermovement jump (CMJ) was performed before the (FSKT) and PSTT, whereas cognitive function was assessed (via the Stroop test) before and after supplementation and 10 min following PSTT. Blood lactate was collected before the CMJ tests immediately and 3 min after the FSKT and PSST; rating of perceived exertion (RPE) was recorded during and after both specific taekwondo tests. No significant differences ( p > 0.05), with moderate and large effect sizes, between conditions were observed for PSTT and FSKT performances. In addition, blood lactate, RPE, heart rate, and CMJ height were not significantly different among conditions ( p > 0.05). However, after the PSTT test, cognitive function was higher in BJ-400 compared to other treatments ( p < 0.05). It was concluded that acute intake of 400 and 800 mg of NO 3 - rich BJ reported a moderate to large effect size in anaerobic and aerobic; however, no statistical differences were found in taekwondo-specific performance.

Background: Carbohydrate (CHO) ingestion enhances exercise performance; however, the efficacy of CHO intake on repeated bouts of exercise simulating a taekwondo tournament is unknown. Therefore, the purpose was to compare the effects of two different doses of CHO on a sports-specific kicking test during a simulated taekwondo tournament compared to placebo (PLA)., Methods: In a double-blind, randomized-placebo controlled, cross-over trial, eleven junior male professional taekwondo athletes (age: 16 ± 0.8 years, body mass: 55.3 ± 7.3 kg) ingested one of three solutions: (i) high dose (C45): 45 g of CHO (60 g∙h - 1 ), (ii) low dose (C22.5): 22.5 g of CHO (30 g∙h - 1 ; both solutions containing 2:1 glucose:fructose), or a PLA immediately following each kicking test. The kicking test was repeated 5 times, separated by 45 mins of rest, simulating a typical taekwondo competition day. Ratings of perceived exertion (RPE) and gastrointestinal discomfort (GI) scores were collected immediately after, and blood glucose before each test., Results: The results revealed that C45 and C22.5 did not improve total, successful, or percentage of successful kicks compared to PLA (p > 0.05). Blood glucose was significantly higher following both CHO conditions compared with PLA across all five tests (p < 0.05). There were no differences between treatments or across tests for RPE (p > 0.05)., Conclusion: CHO intake, independent of the dose, did not alter taekwondo kick performance during a simulated taekwondo tournament.

Objective: Cell counting and characterization is fundamental for medicine, science and technology. Coulter-type microfluidic devices are effective and automated systems for cell/particle analysis, based on the electrical sensing zone principle. However, their throughput and accuracy are limited by coincidences (i.e., two or more particles passing through the sensing zone nearly simultaneously), which reduce the observed number of particles and may lead to errors in the measured particle properties. In this work, a novel approach for coincidence resolution in microfluidic impedance cytometry is proposed., Methods: The approach relies on: (i) a microchannel comprising two electrical sensing zones and (ii) a model of the signals generated by coinciding particles. Maximum a posteriori probability (MAP) estimation is used to identify the model parameters and therefore characterize individual particle properties., Results: Quantitative performance assessment on synthetic data streams shows a counting sensitivity of 97% and a positive predictive value of 99% at concentrations of 2×10 6 particles/ml. An application to red blood cell analysis shows accurate particle characterization up to a throughput of about 2500 particles/s. An original formula providing the expected number of coinciding particles is derived, and good agreement is found between experimental results and theoretical predictions., Conclusion: The proposed cytometer enables the decomposition of signals generated by coinciding particles into individual particle contributions, by using a Bayesian approach., Significance: This system can be profitably used in applications where accurate counting and characterization of cell/particle suspensions over a broad range of concentrations is required.

Microfluidic applications such as active particle sorting or selective enrichment require particle classification techniques that are capable of working in real time. In this paper, we explore the use of neural networks for fast label-free particle characterization during microfluidic impedance cytometry. A recurrent neural network is designed to process data from a novel impedance chip layout for enabling real-time multiparametric analysis of the measured impedance data streams. As demonstrated with both synthetic and experimental datasets, the trained network is able to characterize with good accuracy size, velocity, and cross-sectional position of beads, red blood cells, and yeasts, with a unitary prediction time of 0.4 ms. The proposed approach can be extended to other device designs and cell types for electrical parameter extraction. This combination of microfluidic impedance cytometry and machine learning can serve as a stepping stone to real-time single-cell analysis and sorting. Graphical Abstract.

The study and the characterization of cell death mechanisms are fundamental in cell biology research. Traditional death/viability assays usually involve laborious sample preparation and expensive equipment or reagents. In this work, we use electrical impedance spectroscopy as a label-free methodology to characterize viable, necrotic and apoptotic human lymphoma U937 cells. A simple three-electrode coplanar layout is used in a differential measurement scheme and thousands of cells are measured at high-throughput (≈200 cell/s). Tailored signal processing enables accurate and robust cell characterization without the need for cell focusing systems. The results suggest that, at low frequency (0.5 MHz), signal magnitude enables the discrimination between viable/necrotic cells and cell fragments, whereas phase information allows discriminating between viable cells and necrotic cells. At higher frequency (10 MHz) two subpopulations of cell fragments are distinguished. This work substantiates the prominent role of electrical impedance spectroscopy for the development of next-generation cell viability assays., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microfluidic impedance cytometry is a label-free approach for high-throughput analysis of particles and cells. It is based on the characterization of the dielectric properties of single particles as they flow through a microchannel with integrated electrodes. However, the measured signal depends not only on the intrinsic particle properties, but also on the particle trajectory through the measuring region, thus challenging the resolution and accuracy of the technique. In this work we show via simulation that this issue can be overcome without resorting to particle focusing, by means of a straightforward modification of the wiring scheme for the most typical and widely used microfluidic impedance chip., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Introduction: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood., Methods: In a close-contact co-culture model of human airway epithelial and endothelial cells, cellular crosstalk was analyzed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy, and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system, the temporal release of mediators was analyzed in the co-culture model., Results: Within 4 h of challenge, double-stranded RNA induced the release of TNF-α by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX 3 CL1 (fractalkine) by 8 h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-α., Conclusion: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signaling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation.

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.

The connection between auditory fields of the temporal lobe and prefrontal cortex has been well characterized in nonhuman primates. Little is known of temporofrontal connectivity in humans, however, due largely to the fact that invasive experimental approaches used so successfully to trace anatomical pathways in laboratory animals cannot be used in humans. Instead, we used a functional tract-tracing method in 12 neurosurgical patients with multicontact electrode arrays chronically implanted over the left (n = 7) or right (n = 5) perisylvian temporal auditory cortex (area PLST) and the ventrolateral prefrontal cortex (VLPFC) of the inferior frontal gyrus (IFG) for diagnosis and treatment of medically intractable epilepsy. Area PLST was identified by the distribution of average auditory-evoked potentials obtained in response to simple and complex sounds. The same sounds evoked little if there is any activity in VLPFC. A single bipolar electrical pulse (0.2 ms, charge-balanced) applied between contacts within physiologically identified PLST resulted in polyphasic evoked potentials clustered in VLPFC, with greatest activation being in pars triangularis of the IFG. The average peak latency of the earliest negative deflection of the evoked potential on VLPFC was 13.48 ms (range: 9.0-18.5 ms), providing evidence for a rapidly conducting pathway between area PLST and VLPFC.

Although it is well known that any material degrades faster when exposed to an aggressive environment as well as that "aggressive" cannot be univocally defined as depending also on the chemical-physical characteristics of material, few researches on the identification of the most significant parameters influencing the corrosion of metallic object are available.A series of ancient coins, coming from the archaeological excavation of Palazzo Valentini (Rome) were collected together with soils, both near and far from them, and then analysed using different analytical techniques looking for a correlation between the corrosion products covering the coins and the chemical-physical soil characteristics. The content of soluble salts in the water-bearing stratum and surfacing in the archaeological site, was also measured.The obtained results stress the influence of alkaline soils on formation of patina. Cerussite, probably due to the circulation of water in layers rich in marble and plaster fragments, was the main corrosion product identified by X-ray Diffraction (XRD). Copper, lead and vanadium were found in soil surrounding coins. By measuring conductivity, pH and soluble salts content of the washing solutions from both coins and soils, we could easily separate coins coming from different stratigraphic units of the site.Data were treated by cluster and multivariate analysis, revealing a correlation between part of the coins and the nearby soil samples.

Numerous studies have investigated the spatial sensitivity of cat auditory cortical neurons, but possible dynamic properties of the spatial receptive fields have been largely ignored. Given the considerable amount of evidence that implicates the primary auditory field in the neural pathways responsible for the perception of sound source location, a logical extension to earlier observations of spectrotemporal receptive fields, which characterize the dynamics of frequency tuning, is a description that uses sound source direction, rather than sound frequency, to examine the evolution of spatial tuning over time. The object of this study was to describe auditory space-time receptive field dynamics using a new method based on cross-correlational techniques and white-noise analysis in spherical auditory space. This resulted in a characterization of auditory receptive fields in two spherical dimensions of space (azimuth and elevation) plus a third dimension of time. Further analysis has revealed that spatial receptive fields of neurons in auditory cortex, like those in the visual system, are not static but can exhibit marked temporal dynamics. This might result, for example, in a neuron becoming selective for the direction and speed of moving auditory sound sources. Our results show that approximately 14% of AI neurons evidence significant space-time interaction (inseparability).

Two transient sounds, considered as a conditioner followed by a probe, were delivered successively from the same or different direction in virtual acoustic space (VAS) while recording from single neurons in primary auditory cortex (AI) of cats under general anesthesia. Typically, the response to the probe sound was progressively suppressed as the interval between the two sounds (ISI) was systematically reduced from 400 to 50 ms, and the sound-source directions were within the cell's virtual space receptive field (VSRF). Suppression of the cell's discharge could be accompanied by an increase in response latency. In some neurons, the joint response to two sounds delivered successively was summative or facilitative at ISIs below about 20 ms. These relationships held throughout the VSRF, including those directions on or near the cell's acoustic axis where sounds often elicit the strongest response. The strength of suppression varied systematically with the direction of the probe sound when the ISI was fixed and the conditioning sound arrived from the cell's acoustic axis. Consequently a VSRF defined by the response to the lagging probe sound was progressively reduced in size when ISIs were shortened from 400 to 50 ms. Although the presence of a previous sound reduced the size of the VSRF, for many of these VSRFs a systematic gradient of response latency was maintained. The maintenance of such a gradient may provide a mechanism by which directional acuity remains intact in an acoustic environment containing competing acoustic transients.

A spherical approximation technique is presented that affords a mathematical characterization of a virtual space receptive field (VSRF) based on first-spike latency in the auditory cortex of cat. Parameterizing directional sensitivity in this fashion is much akin to the use of difference-of-Gaussian (DOG) functions for modeling neural responses in visual cortex. Artificial neural networks and approximation techniques typically have been applied to problems conforming to a multidimensional Cartesian input space. The problem with using classical planar Gaussians is that radial symmetry and consistency on the plane actually translate into directionally dependent distortion on spherical surfaces. An alternative set of spherical basis functions, the von Mises basis function (VMBF), is used to eliminate spherical approximation distortion. Unlike the Fourier transform or spherical harmonic expansions, the VMBFs are nonorthogonal, and hence require some form of gradient-descent search for optimal estimation of parameters in the modeling of the VSRF. The optimization equations required to solve this problem are presented. Three descriptive classes of VSRF (contralateral, frontal, and ipsilateral) approximations are investigated, together with an examination of the residual error after parameter optimization. The use of the analytic receptive field model in computational models of population coding of sound direction is discussed, together with the importance of quantifying receptive field gradients. Because spatial hearing is by its very nature three dimensional or, more precisely, two dimensional (directional) on the sphere, we find that spatial receptive field models are best developed on the sphere.

Spatial receptive fields of primary auditory (AI) neurons were studied by delivering, binaurally, synthesized virtual-space signals via earphones to cats under barbiturate anesthesia. Signals were broadband or narrowband transients presented in quiet anechoic space or in acoustic space filled with uncorrelated continuous broadband noise. In the absence of background noise, AI virtual space receptive fields (VSRFs) are typically large, representing a quadrant or more of acoustic space. Within the receptive field, onset latency and firing strength form functional gradients. We hypothesized earlier that functional gradients in the receptive field provide information about sound-source direction. Previous studies indicated that spatial gradients could remain relatively constant across changes in signal intensity. In the current experiments we tested the hypothesis that directional sensitivity to a transient signal, as reflected in the gradient structure of VSRFs of AI neurons, is also retained in the presence of a continuous background noise. When background noise was introduced three major affects on VSRFs were observed. 1) The size of the VSRF was reduced, accompanied by a reduction of firing strength and lengthening of response latency for signals at an acoustic axis and on-lines of constant azimuth and elevation passing through the acoustic axis. These effects were monotonically related to the intensity of the background noise over a noise intensity range of approximately 30 dB. 2) The noise intensity-dependent changes in VSRFs were mirrored by the changes that occurred when the signal intensity was changed in signal-alone conditions. Thus adding background noise was equivalent to a shift in the threshold of a directional signal, and this shift was seen across the spatial receptive field. 3) The spatial gradients of response strength and latency remained evident over the range of background noise intensity that reduced spike count and lengthened onset latency. Those gradients along the azimuth that spanned the frontal midline tended to remain constant in slope and position in the face of increasing intensity of background noise. These findings are consistent with our hypothesis that, under background noise conditions, information that underlies directional acuity and accuracy is retained within the spatial receptive fields of an ensemble of AI neurons.

Transient broad-band stimuli that mimic in their spectrum and time waveform sounds arriving from a speaker in free space were delivered to the tympanic membranes of barbiturized cats via sealed and calibrated earphones. The full array of such signals constitutes a virtual acoustic space (VAS). The extra-cellular response to a single stimulus at each VAS direction, consisting of one or a few precisely time-locked spikes, was recorded from neurons in primary auditory cortex. Effective sound directions form a virtual space receptive field (VSRF). Near threshold, most VSRFs were confined to one quadrant of acoustic space and were located on or near the acoustic axis. Generally, VSRFs expanded monotonically with increases in stimulus intensity, with some occupying essentially all of the acoustic space. The VSRF was not homogeneous with respect to spike timing or firing strength. Typically, onset latency varied by as much as 4-5 msec across the VSRF. A substantial proportion of recorded cells exhibited a gradient of first-spike latency within the VSRF. Shortest latencies occupied a core of the VSRF, on or near the acoustic axis, with longer latency being represented progressively at directions more distant from the core. Remaining cells had VSRFs that exhibited no such gradient. The distribution of firing probability was mapped in those experiments in which multiple trials were carried out at each direction. For some cells there was a positive correlation between latency and firing probability.

Responses to two harmonically related tones, approximating the lowest formants of nine American English vowels, were recorded from single auditory-nerve fibers. Data were compiled as period histograms for tones presented singly and in combination using the fundamental frequency of the two-tone complex as the time base. The amplitudes of the primary frequency components present in a histogram were estimated by least-squares fitting a half-wave rectified sum of the stimulating sinusoids plus a constant. Nonlinear interactions resulted for most two-tone stimuli: one tone dominated the response. When one tone was equal to best frequency, that tone always controlled discharge timing, usually suppressing the response to the second tone. Complicated interactions took place when the stimulating frequencies bracketed best frequency. The tone nearest best frequency was most effective near threshold, while higher stimulus levels usually favored the low-frequency tone. Nevertheless, the suppression mechanisms appear to provide an effective spatial separation in the cochlea for the response components to each vowel approximation. Fourier analysis of the period histograms yielded qualitatively similar results.