Recruitment of the auditory cortex in congenitally deaf cats by long-term cochlear electrostimulation.

In congenitally deaf cats, the central auditory system is deprived of acoustic input because of degeneration of the organ of Corti before the onset of hearing. Primary auditory afferents survive and can be stimulated electrically. By means of an intracochlear implant and an accompanying sound processor, congenitally deaf kittens were exposed to sounds and conditioned to respond to tones. After months of exposure to meaningful stimuli, the cortical activity in chronically implanted cats produced field potentials of higher amplitudes, expanded in area, developed long latency responses indicative of intracortical information processing, and showed more synaptic efficacy than in naïve, unstimulated deaf cats. The activity established by auditory experience resembles activity in hearing animals.

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants.

Pichia pastoris is a yeast capable of expressing large amounts of some proteins. When expression vectors are introduced into P. pastoris, individual transformants typically express widely varying amounts of protein. Because clones expressing the highest level of protein occur infrequently during the transformation process, finding them can be very labor-intensive. We developed an immunological based filter screening method that rapidly detects transformants secreting large amounts of a heterologous protein. We have applied this method to the expression of a soluble trimeric form of CD40L, a molecule that regulates B-cell responses. Using this method, we identified transformants with one to 13 copies of the CD40L expression cassette. Maximum expression was obtained with clones containing eight or more copies of the expression cassette, and a clone with eight copies was selected for further analysis. High cell density fermentation of this clone using a mixed glycerol:methanol feed yielded 255 mg CD40L per liter of supernatant.

Performance of the avian inner ear.

In spite of morphological similarities, the avian inner ear has apparently developed mechanisms of sound transduction that differ from the mammalian solution. This paper is a compilation of the present knowledge.

Monitoring of anaesthesia in neurophysiological experiments.

Cortical activity can be substantially changed by the type of anaesthetic used, and by its dose level. For easy monitoring of depth of anaesthesia we describe the changes in electroencephalogram and electrocardiogram accompanying changes in depth of anaesthesia in the cat. Anaesthesia was induced by the volatile anaesthetic isoflurane. The high-frequency components (around 30 Hz) in the electroencephalogram disappear in deep anaesthesia. The electrocardiogram also shows substantial changes in contamination due to muscle fasciculations with anaesthesia level. Fasciculations appear as noise in the electrocardiogram. The amplitude of the electrical muscle activity contaminating the ECG can be easily used for the maintainance of a constant level of anaesthesia during a neurophysiological experiment.

Artifact-free application of drugs into the avian endolymphatic space.

A push-pull system is described that allows artifact-free application of drugs into very small volumes such as, e.g., the endolymphatic space of the inner ear. The system consists of two syringes, the pistons of which are fixed together and driven by an Oriel motordrive. The syringes are connected to the pipette holders and microcapillaries by PVC tubes. One capillary is used to expel a certain volume, whilst the other one aspirates the same volume. A balanced exchange of fluids within a given space without an overall change in volume is therefore possible.

A stimulator with wireless power and signal transmission for implantation in animal experiments and other applications.

Functional electrical stimulation in alert experimental animals is often hampered, as free mobility of the animals is limited by the leads. On the other hand, portable devices carrying the power supply are not always accepted by the animal. For wireless transmission of power and stimulus code a system designed for implantation is described below. The power for the implant is supplied inductively by a rotating magnetic field. The stimulation signal is radio-transmitted by FM in the 140 MHz range and processed by the implant. Finally, a current source is driven for electrical stimulation. The present design is intended to be used for electrical cochlear stimulation. However, the circuit can also be used for other types of functional electrical stimulation.

Discharge properties of pigeon single auditory nerve fibers after recovery from severe acoustic trauma.

The time course of recovery of compound action potential (CAP) thresholds was observed in individual adult pigeons after severe acoustic trauma. Each bird had electrodes implanted on the round window of both ears. One ear was exposed to a tone of 0.7 kHz at 136-142 dB SPL for 1 hr under general anesthesia. Recovery of CAP audiograms was monitored twice a week after trauma. Single unit recordings from auditory nerve fibers were made after 3 weeks and after 4 or more months of the exposure. The CAP was abolished immediately after overstimulation in all animals. Based on the temporal patterns of functional recovery of the CAP three groups of animals were identified. The first group was characterized by fast functional recovery starting immediately after trauma followed by a return to pre-exposure values within 3 weeks. In the second group, slow functional recovery of threshold started 1-2 weeks after trauma followed by a return to pre-exposure values by 4-5 weeks. A mean residual hearing loss of 26.3 dB at 2 kHz remained. The third group consisted of animals that did not recover after trauma. Three weeks after the exposure, tuning curves of single auditory nerve fibers were very broad and sometimes irregular in shape. Their thresholds hovered around 120 dB SPL. Spontaneous firing rate and driven rate were much reduced. Four or more months after exposure, the thresholds and sharpness of tuning of many single units were almost completely recovered. Spontaneous firing rate and driven rate were comparable to those of control animals. In the slow recovery group neuronal tuning properties showed less recovery, especially at frequencies above the exposure frequency. Thresholds and sharpness of tuning were normal at frequencies below the exposure frequency, but were much poorer at frequencies above the exposure. Spontaneous firing rate was much reduced in fibers with high characteristic frequencies. In fast recovering animals, the papilla was repopulated with hair cells after 4 months. In slow recovering animals, short (abneural) hair cells were still missing over large parts of the papilla after 4 months of recovery. Residual short (abneural) hair cell loss was largest at two areas, one more basal and the other more apical to the characteristic place of the traumatizing frequency. The results show that, in adult birds, functional recovery from severe damage to both short (abneural) and tall (neural) hair cells occurs. However, the onset of recovery is delayed and the time course is slower than after destruction of short (abneural) hair cells alone. Also, recovery is incomplete, both functionally and morphologically. There is residual permanent hearing loss, and regeneration of short (abneural) hair cells is incomplete.

Selective binding and reversible release of riboflavin by polymer-bound zinc(II) azamacrocycles.

The reversible formation of a coordinative bond between a polymer-bound Lewis-acidic metal complex and a flavin imide moiety allows complete extraction of riboflavin from aqueous solution at physiological pH and its quantitative release at pH 5.

Quantitative evaluation of ototoxic side effects of furosemide, piretanide, bumetanide, azosemide and ozolinone in the cat--a new approach to the problem of ototoxicity.

A new method for the quantitative assessment of acute ototoxic side effects of drugs is described. It is suitable for screening purposes. The method is based on the determination of the toxic dose (TD50) which causes a defined hearing loss in 50% of the animals tested. The hearing loss is defined as a complete suppression of the compound action potential (CAP) of the auditory nerve, elicited by clicks 30 dB above threshold. This is approximately equivalent to a clinical hearing loss of 30 dB. The TD50 is used to estimate the therapeutic range. With this approach ototoxic side effects of furosemide, piretanide and bumetanide were compared quantitatively in cats. The TD50 values for CAP suppression were 18.37 mg/kg for furosemide; 4.29 mg/kg for piretanide and 2.21 mg/kg for bumetanide. As equipotent diuretic doses are 2.61 mg/kg for furosemide, 0.26 mg/kg for piretanide and 1.16 mg/kg for bumetanide, it appears that the relative ototoxicity is least for piretanide and highest for bumetanide. Plasma concentrations, determined initially and when recovery of CAP to 50% of control had occurred, indicate that bumetanide may be more slowly eliminated from the cochlear spaces than furosemide and piretanide. In addition azosemide and ozolinone were tested. The TD50 for azosemide was less than 10 mg/kg. With ozolinone where there are two isomers, only the diuretic (-)ozolinone was ototoxic; the TD50 was less than 100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmission in the inner ear.

The present view on cochlear neurotransmission can be summarized as follows: There are two main types of synapses on cochlear hair cells, afferent and efferent ones. Afferent synaptic structures are abundant on inner hair cells whereas similar structures on the outer hair cells are less frequent and appear to be rudimentary. Presynaptic vesicles seem to be rare in outer hair cells. For the inner hair cell--afferent terminal--the presence of a chemical transmission mechanism is generally accepted. The transmitter substance has not yet been unequivocally demonstrated. Glycine, catecholamines, GABA and 5-HT can be eliminated as candidates as these compounds do not activate afferent fibres. There are good reasons, however, to consider amino acids. Most of the experimental results support glutamate as the transmitter (e.g. effectiveness of glutamate, kainic acid, glutamate diethylester). Aspartate is less likely. It is not yet well understood, however, why glutamate has to be applied in concentrations of up to 10(-3) M intracochlearly in order to activate afferent fibres and why elevated glutamate levels could not be demonstrated in perilymph collected during acoustical stimulation, whereas this same perilymph was able to activate afferent nerve terminals when applied intracochlearly. Efferent endings use acetylcholine as a transmitter. Enzymes for synthesis and breakdown of acetylcholine are present; acetylcholine is effective at the synaptic junction, as are cholinergic compounds and specific blockers. However, there may be different types of efferent endings in both the cochlear and vestibular organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of infrasound sensitive neurones in the papilla basilaris of the pigeon: an HRP study.

Infrasound sensitive afferent fibres recorded in the pigeon cochlear ganglion were marked by intracellular injections of horseradish peroxidase (HRP). All stained fibres were found to innervate hair cells in the basilar membrane between 90 and 950 microns from its apical end. No fibres to the macula lagenae were found. Nine of the 10 completely stained fibres contacted hair cells located abneurally on the free basilar membrane, the tenth ended over the neural limbus near its abneural border. All fibres innervated between two and nine hair cells. This is in contrast to common auditory fibres in the bird that were reported to innervate only one hair cell located neurally over the neural limbus. This paper, therefore, demonstrates, for the first time, physiologically defined fibres that do not end on 'inner' hair cells.

Discharge pattern in the auditory nerve evoked by vowel stimuli: a comparison between acoustical and electrical stimulation.

Single channel cochlear implants only transmit the time structure of the electrically coded input signal. All nerve fibres show similar thresholds for monopolar round window stimulation, i.e., activation does not depend on their site of origin. To investigate the fine structure of the firing pattern elicited by stimulation with an analogue coded speech processing system (VIENNA 1-channel implant), cats were electrically stimulated with German steady-state vowels at the round window. Single fibre activity was recorded from primary auditory fibres and period histograms were calculated. The electrically evoked impulse patterns were compared with those from acoustic stimulation with the same vowels. With acoustic stimulation, the response of a fibre depends on the individual characteristic frequency (CF) with regard to the fundamental F0 and the formants F1, F2 and F3 of the vowels, the spontaneous activity of the fibre and the sound level. The evoked firing pattern was used to calculate period histograms, the frequency content of which was analysed by Fourier transformation. With electrical stimulation in the threshold range, an action potential is strongly synchronized to a cathodic peak of the current within one period of F0. With increasing current level 3-5 impulses can be locked to the same period. The timing of the short intervals is determined by the relative refractory period and current peaks (negative or positive) caused by the dominant higher formant F2 or F3. The acoustically evoked patterns are specific for the CF of the neuron and represent the spectral information of the different vowels.(ABSTRACT TRUNCATED AT 250 WORDS)

The course and morphology of efferent nerve fibres in the papilla basilaris of the pigeon (Columba livia).

This paper describes the course and morphology of efferent fibres in an avian cochlea. Horseradish peroxidase stained efferent fibres in the pigeon papilla basilaris were identified by Nomarski optics and camera lucida drawings. There are at least two types of efferent fibres: Large thick fibres take mainly a transversal course and contact short and intermediate hair cells over the free basilar membrane as well as hyaline cells. Large efferent fibres contact both hair cells and hyaline cells. Small thin fibres contact short or intermediate hair cells over the free basilar membrane or tall hair cells over the neural limbus. A physiological consequence of the findings is that efferent activity will concomitantly lead to a contraction of hyaline cells and a hyperpolarization of hair cells.

A previously unknown hair cell epithelium in the pigeon cochlea: the papilla chaotica.

A previously unknown sensory epithelium can be found on the medial wall of the apical part of the pigeon cochlear duct. It comprises about 200 hair cells. These are not arranged in any regular pattern, shape and orientation of the ciliary bundles even differ in neighbouring hair cells. We therefore propose the term papilla chaotica.

The influence of DC-polarization of the endocochlear potential on single fibre activity in the pigeon cochlear nerve.

The endocochlear potential (EP) in the pigeon ear was altered by injecting current into the scala media. Simultaneous recordings from afferent fibres in the cochlear ganglion were performed. The mean rate of spontaneous activity was little affected by current injection and the consequent shifts in EP. In contrast to this lack of effect, the preferred intervals seen in some fibres in birds were accentuated by positive current injection and reduced or in some cases suppressed by negative current injection. The threshold of the tuning curve was raised by injection of negative current but the characteristic frequency showed little change. Sharpness of tuning decreased. Analysis of the results shows that current injection in the scala media produces significant changes in the filter characteristics of the cochlea, as well as altering the driving force for the transduction process (difference between EP and hair cell membrane potential).

Synchronized responses of primary auditory fibre-populations in Caiman crocodilus (L.) to single tones and clicks.

Measurements of the responses to tones and clicks were made from single primary auditory fibres of the caiman. The distribution of the amplitude and phase of the fundamental component of the response rate modulation over the best frequencies of the fibres is comparable to that reported in the cat, despite the fact that the basilar membrane in caiman is only 4.5 mm long. However, much higher intensities are needed in the caiman (75-85 dB SPL) than reported in the cat (20 dB SPL) to obtain systematic distributions of the phase of the responses, probably due to the larger scatter of the phase responses in the caiman. The slopes of the phase distributions are very similar to those in cat. Single unit phase responses as a function of stimulus frequency at 85 dB SPL can be approximated by one, or in fibres with low best frequency, two straight lines. At lower intensities the deviation of the phase-frequency responses from a straight line increases as the group delay at the best frequency becomes larger. The shortest latencies of click responses are obtained with rarefaction clicks. Group delay estimates obtained from the responses to clicks and from the straight line approximations of the phase-frequency responses are related in a way expected for linear filter systems and accurately predict the measured distributions of the phase of the responses over the neural best frequency. The obtained group delays and click latencies in the caiman are very similar to those reported by other workers in the cat, the squirrel monkey and the treefrog, despite large morphological and probably functional differences of their inner ears. The click latencies are also very similar to those in the pigeon. The results are consistent with the existence of a mechanical travelling wave reported previously on the basilar membrane of the caiman, but at the same stimulus level the phase characteristic of the present single unit responses is steeper and the wave length estimates from the neural population phase distributions are shorter than those observed directly in the motion of the basilar membrane. Since the neural responses are an indirect estimate of the basilar membrane motion it cannot be decided whether the difference between neural and mechanical data is due to deterioration of the basilar membrane responses during the direct measurements or whether the basilar membrane response is sharpened by additional tuning mechanisms.

Responses of single cells in the cat inferior colliculus to binaural masking level difference signals.

Neurones with low best frequency (less than 2 kHz) and sensitive to interaural phase differences were recorded in the dorsal part of the central nucleus of the cat inferior colliculus. Best frequency tone (S) and noise (N) bursts were delivered binaurally via closed field sound systems either in phase (S0, N0) at both ears or inverted at one ear (SII, NII). The responses to tone + noise bursts in the stimulus configurations S0N0, SIINII and SIIN0 and noise bursts (N0 and NII) were compared. The latter two tone + noise configurations (S0NII and SIIN0) give a binaural masking level difference (BMLD) of up to 15 dB in psychophysical experiments, i.e. an increase in noise level is necessary to mask perception of the tone. Most cells responded better to in phase stimuli (here called 0 cells). A minority of cells responded better to out of phase stimuli (here called II cells). Each cell's response was correlated with the level of acoustic stimulus (tone or masker) in the preferred configuration and not with the BMLD situation: for the 0 cells, the responses were therefore maximal for S0N0 and minimal for SIINII. For II cells, the gradation was reversed: the response to SIINII was maximal and that to S0N0 minimal. For both populations, the responses to S0NII and SIIN0 were intermediate between the S0N0 and SIINII responses. Cells that responded best to S0NII or SIIN0, i.e. cells selectively coding BMLD, were not found. This was also true for the synchronized spike rates of those cells showing phase locked responses to the stimulus frequency. Some cells appeared to be strongly suppressed by the addition of an non-preferred masker (i.e. in the configuration that resulted in less response to a noise-alone burst; e.g. NII for the 0 cells). Other cells were more suppressed by the addition of a preferred masker (N0 for the 0 cells). The difference in the number of spikes evoked by the tone + noise and the noise burst was analyzed according to signal detection theory and neuronal masked threshold determined. Some 0 cells showed lower thresholds in the configuration S0NII whereas others had higher thresholds in this configuration. This correlated with the binaural suppression effects noted above: when the noise in the preferred configuration (N0) gave more suppression, the threshold was lower for S0NII; when NII gave more suppression the threshold was higher for S0NII. Over the whole population, these effects cancelled out and the neuronal threshold was not significantly affected by the BMLD configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of temperature on tuning of primary-like units in the guinea pig cochlear nucleus.

The effect of temperature changes on the response area of primary-like single units recorded extracellularly in the anteroventral cochlear nucleus of the guinea pig was studied for cochlear temperatures in the range 27.4-40.3 degrees C, while maintaining a normal rectal temperature of 38.5 degrees C. A new approach to the cochlear nucleus was developed which involved opening of the temporal bone overlying the flocculus cavity and aspirating a small portion of the paraflocculus. Irrespective of characteristic frequency CF (0.8-16 kHz), hypothermia caused a reversible elevation of CF thresholds (1.6 dB/degrees C), a small loss of the sharpness of threshold tuning and reductions of the saturation and mean spontaneous rates. The CFs were unaffected, in contrast to single units in birds and cold-blooded animals. From a comparison of the effects of temperature on different species it is proposed that the processes that define CF and sharpness of tuning are different within a cochlea, and that these processes can be differentially influenced.