Novel methods for the assessment of safety pharmacology and toxicology parameters in anesthetized and ventilated dogs receiving inhaled drugs.

INTRODUCTION: For nonclinical drug development, it is optimal if safety pharmacology and toxicology studies are performed in a model that reasonably represents the patient the drug is intended to treat. To simulate prolonged inhalation therapy in ventilated patients, GLP inhalation toxicology methods, including safety pharmacology endpoints, in anesthetized, intubated and mechanically ventilated dogs were developed. This model required establishment of a canine intensive care unit (ICU) capable of providing prolonged anesthesia (propofol infusion and morphine titration) and partial parenteral nutrition (dextrose, amino acids and lipids) while safety parameters were monitored. METHOD: Telemetry was used to continuously monitor heart rate, ECG and blood pressure. Blood gas parameters were periodically measured while oxygen saturation and core temperature were reported continuously. Glucose was measured hourly while other standard clinical pathology (hematology, coagulation, clinical chemistry) samples were evaluated approximately every 12 h. Aerosols were administered continuously over 48 h by inhalation using a mesh nebulizer (Aerogen Solo) fed by a syringe pump into a humidified circuit of a critical care ventilator (LTV® 1000) ending in an endotracheal tube placed in the trachea. Animals were ventilated with pressure control ventilation targeting a respiratory minute volume of 2.0-3.5 l per minute (LPM). Peak inspiratory pressure (PIP) was maintained between 10 and 17 cm H2O and inspiratory time was set to 1 s with an inspiratory:expiratory (I:E) ratio of 1:2. Ventilator parameters and anesthesia were adjusted to maintain normal PaCO2 levels and adequate sedation, respectively. Novel methods were developed to determine dose and particle size in vitro as on-line measurements were not feasible during in vivo aerosol delivery. RESULTS AND DISCUSSION: Acceptable baseline measurements were established for all parameters over the 48-h evaluation period, qualifying the method as appropriate for assessment of GLP safety pharmacology and toxicology studies.

Expanded Characterization of a Hemi-Body Shielded Göttingen Minipig Model of Radiation-induced Gastrointestinal Injury Incorporating Oral Dosing Procedures.

In collaboration with the Biomedical Advanced Research and Development Authority (BARDA), the authors recently conducted a pilot study in a hemi-body shielded model of radiation-induced gastrointestinal (GI) injury in Göttingen minipigs following exposure to radiation dose levels between 8-16 Gy. Herein, the impact of oral dosing procedures is assessed, as well as the specific causes of death in animals exposed to radiation doses of 14 and 16 Gy (n = 64; 32 male, 32 female, between 6 and 8 mo of age). Oral dosing using a 2-tablet placebo system comprised of both immediate release and enteric-coated tablets starting 24 h post-irradiation resulted in inhibited gastric emptying of the enteric-coated tablets, which were found to be retained in the stomach and/or regurgitated. This finding appears to be species-specific, as similar findings have not been reported for other large animal species (e.g., non-human primates). Mortality was primarily dictated by decreased activity, body weight loss (>35%), and/or respiratory distress, despite shielding of the lung. The cause of respiratory distress in animals that were pre-terminally euthanized varied according to the timing of death, with interstitial inflammation and extensive fibrosis observed >20 days post-irradiation. Kidney damage was also identified in most animals after day 10. Changes in the GI tract were consistent with previous studies and included collagen deposition/fibrosis. Observations of inflammatory infiltrates and interstitial inflammation/fibrosis in both shielded and unshielded organs support a strong secondary inflammatory syndrome post-irradiation.

Pilot Study of Radiation-induced Gastrointestinal Injury in a Hemi-body Shielded Göttingen Minipig Model.

Development of medical countermeasures (MCMs) for gastrointestinal (GI) injury following acute radiation exposure requires well-characterized models that can assess not only survival but also secondary endpoints, including structural and functional characteristics of GI damage and recovery that ultimately contribute to long-term survival. The authors conducted a pilot study in a hemi-body shielded Göttingen minipig model of radiation-induced GI injury that enables radiation damage to the GI tract to be evaluated and reduces the potential for hemorrhage and/or damage in other more sensitive organ systems. With shielding of the head, chest, and front legs, radiation dose levels of 14 Gy were required to see significant GI-related morbidity, while dose levels of 16 Gy resulted in significant mortality by day 45 post-irradiation. Periodic scheduled necropsies showed significant reduction in and slow recovery of intestinal crypt count at 14 and 16 Gy. Intestinal proliferative activity was initially increased and then gradually decreased over the course of the study. Histological evidence of marked inflammatory infiltrates was noted in the GI tract at day 5, while collagen deposition, indicative of fibrosis, was observed as early as day 15, peaking at day 30. The radiation dose-responsive indicators of GI damage identified in this model (i.e., intestinal crypt count and proliferative activity) may serve as useful endpoints for evaluation of the efficacy of potential MCMs.

An overview of the safety pharmacology society strategic plan.

Safety Pharmacology studies are conducted to characterize the confidence by which biologically active new chemical entities (NCE) may be anticipated as safe. Non-clinical safety pharmacology studies aim to detect and characterize potentially undesirable pharmacodynamic activities using an array of in silico, in vitro and in vivo animal models. While a broad spectrum of methodological innovation and advancement of the science occurs within the Safety Pharmacology Society, the society also focuses on partnerships with health authorities and technology providers and facilitates interaction with organizations of common interest such as pharmacology, physiology, neuroscience, cardiology and toxicology. Education remains a primary emphasis for the society through content derived from regional and annual meetings, webinars and publication of its works it seeks to inform the general scientific and regulatory community. In considering the future of safety pharmacology the society has developed a strategy to successfully navigate forward and not be mired in stagnation of the discipline. Strategy can be defined in numerous ways but generally involves establishing and setting goals, determining what actions are needed to achieve those goals, and mobilizing resources within the society to accomplish the actions. The discipline remains in rapid evolution and its coverage is certain to expand to provide better guidance for more systems in the next few years. This overview from the Safety Pharmacology Society will outline the strategic plan from 2016 to 2018 and beyond and provide insight into the future of the discipline which builds upon a previous strategic plan established in 2009.

Citrulline as a Biomarker for Gastrointestinal-Acute Radiation Syndrome: Species Differences and Experimental Condition Effects.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13-17 Gy and 8-16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72-13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 µM and 63.1 ± 2.1 µM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 µM, very similar to that of humans (∼40 µM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

Rat cardiovascular telemetry: Marginal distribution applied to positive control drugs.

Cardiovascular effects are considered frequent during drug safety testing. This investigation aimed to characterize the pharmacological response of the conscious telemetered rat in vivo model to known cardiovascular active agents. These effects were analyzed using statistical analysis and cloud representation with marginal distribution curves for the contractility index and heart rate as to assess the effect relationship between cardiac variables. Arterial blood pressure, left ventricular pressure, electrocardiogram and body temperature were monitored. The application of data cloud with marginal distribution curves to heart rate and contractility index provided an interesting tactic during the interpretation of drug-induced changes particularly during selective time resolution (i.e. marginal distribution curves restricted to Tmax). Taken together, the present data suggests that marginal distribution curves can be a valuable interpretation strategy when using the rat cardiovascular telemetry model to detect drug-induced cardiovascular effects. Marginal distribution curves could also be considered during the interpretation of other inter-dependent parameters in safety pharmacology studies.

Evaluation of a novel ECG lead placement method in telemetered freely moving cynomolgus monkeys: assessment of an intravascular biopotential lead.

INTRODUCTION: ECG is considered as a critical biomarker of cardiac safety pharmacology. ECG signal quality is essential for accurate interval quantification and automated arrhythmia detection. METHODS: We evaluated ECG signal quality over a 6 month period from 15 cynomolgus monkeys with radiotelemetry transmitters using biopotential leads where the negative lead was inserted in the jugular vein and advanced to the superior vena cava (intravascular lead) and the positive lead was placed on the diaphragm at the apex of the heart (diaphragmatic lead). Signal noise and signal-to-noise ratio from this implantation methodology were compared with signals obtained from animals with subcutaneous ECG lead. Macroscopic pathology and histopathology associated with the intravascular lead were evaluated at 6 months post-implantation in six monkeys. RESULTS: The ECG morphology obtained with the intravascular/diaphragmatic lead placement was comparable to conventional subcutaneous ECG (emulating Lead II) but presented higher amplitudes (P-wave +50.0%; R-wave +30.0%). Signal noise showed a circadian cycle of changes in magnitude for subcutaneous ECG leads that was not observed with this method. The intravascular/diaphragmatic lead placement presented a higher signal-to-noise ratio than subcutaneous ECG leads. No macroscopic abnormality was observed to be associated with the intravascular lead. Mild thickening of the intima/subintima with mild intimal proliferation of the cranial vena cava surrounding the intravascular lead were noted at histopathological examination. DISCUSSION: The intravascular/diaphragmatic ECG lead placement in cynomolgus monkeys provided reduced signal noise and elevated P-QRS-T amplitudes. The intravascular lead was well tolerated and appeared suitable for chronically instrumented cardiovascular safety pharmacology studies. Further assessments would be warranted to evaluate the potential of this methodology in other species.

Innovation in safety pharmacology testing.

This issue of the Journal of Pharmacological and Toxicological Methods (JPTM) is themed. It is the eighth in a series, arising from the Annual Safety Pharmacology Society (SPS) meeting. The SPS is now in its 10th year as an independent branch of biological sciences (distinct from pharmacology and toxicology) and is the primary forum for driving advances in safety pharmacology. The theme of the meeting and this journal issue is innovation, and the focus is non-clinical safety assessment of new chemical entity (NCEs). The content is informed by regulatory guidance documents (S7A and S7B) prior to first in human (FIH) studies. The manuscripts cover a broad spectrum of safety pharmacology topics from theory to practice, with interrogation of state-of-the-art techniques, and profiling of methods that are in development for safety assessment. Philosophical and strategic issues are addressed, with consideration of the use of novel methods for population pharmacokinetic (PK) analysis, abuse liability, electrocardiogram (ECG) analysis algorithms, in vitro cardiac slice preparations, human pluripotent stem cells, and a brief discussion regarding the assessment of changes in the QRS complex of the ECG indicative of drug-induced blockade of cardiac sodium channels. Safety pharmacology methods continue to evolve.

Cardiovascular and respiratory safety pharmacology in Göttingen minipigs: Pharmacological characterization.

INTRODUCTION: Similarities between pigs and humans support the relevance of Göttingen minipigs for regulatory safety pharmacology. The minipig is the species of choice for cardiovascular safety pharmacology when pivotal repeat toxicology studies are conducted in this species. METHODS: 4 male Göttingen minipigs with cardiovascular telemetry transmitters received intravenous saline, esmolol (0.5, 1, 2, 4 and 8mg/kg), medetomidine (0.04mg/kg), remifentanil (0.5, 1, 2, 4, 8 and 16µg/kg) and dopamine (2, 8, 10, 20, 30 and 50µg/kg/min) and oral sotalol (3 and 10mg/kg). Respiratory monitoring was conducted in 3 male and 3 female Göttingen minipigs receiving intravenous saline and methacholine (0, 3.4, 13.5 and 68µg/kg). RESULTS: Heart rate (HR) corrected QT was optimal with a method based on analysis of covariance (QTca) followed by Fridericia's standard formula. Esmolol induced a decrease in HR. Medetomidine was associated with an initial hypertension with bradycardia followed by sustained hypotension, bradycadia and prolonged QTc. Remifentanil induced a dose-dependent QTc shortening with an increase in arterial pressures. Sotalol caused a decrease in HR and systolic arterial pressure with an increase in PR and QTc intervals. Dopamine induced an increase in arterial and pulse pressures. Methacholine increased tidal volume, respiratory rate and minute volume. DISCUSSION: The results suggest that the minipig is a valid alternative to other non-rodent species for cardiovascular and respiratory safety pharmacology studies when this species is justified.

Non-clinical models: validation, study design and statistical consideration in safety pharmacology.

The current issue of the Journal of Pharmacological and Toxicological Methods (JPTM) focuses exclusively on safety pharmacology methods. This is the 7th year the Journal has published on this topic. Methods and models that specifically relate to methods relating to the assessment of the safety profile of a new chemical entity (NCE) prior to first in human (FIH) studies are described. Since the Journal started publishing on this topic there has been a major effort by safety pharmacologists, toxicologists and regulatory scientists within Industry (both large and small Pharma as well as Biotechnology companies) and also from Contract Research Organizations (CRO) to publish the surgical details of the non-clinical methods utilized but also provide important details related to standard and non-standard (or integrated) study models and designs. These details from core battery and secondary (or ancillary) drug safety assessment methods used in drug development programs have been the focus of these special issues and have been an attempt to provide validation of methods. Similarly, the safety pharmacology issues of the Journal provide the most relevant forum for scientists to present novel and modified methods with direct applicability to determination of drug safety-directly to the safety pharmacology scientific community. The content of the manuscripts in this issue includes the introduction of additional important surgical methods, novel data capture and data analysis methods, improved study design and effects of positive control compounds with known activity in the model.

Patlak plot analysis CT-GFR for the determination of renal function: comparison of normal dogs with autologous kidney transplant dogs.

Glomerular filtration rate (GFR) can be determined using Patlak plot analysis with single-slice dynamic computed tomography (CT). Acute autologous graft failure has several causes, all of which induce a measurable decrease in glomerular filtration rate. This study demonstrated in an experimental model of canine autologous renal transplant that CT-derived renal plasma clearance was significantly lower (p = 0.002) in dogs having undergone transplant (0.077 +/- 0.058 ml min(-1) ml renal tissue(-1)) compared with control dogs (0.396 +/- 0.139 ml min(-1) ml renal tissue(-1)). A significant negative curvilinear relationship was seen between serum creatinine and total renal plasma clearance (R(2) = 0.84, p = 0.0001). Alterations in renal time attenuation curve shape in dogs having undergone transplant may have been related to increased renal vascular resistance related to tubular necrosis. CT-GFR may be a useful experimental tool in the evaluation of renal dysfunction in transplant models.

Combined cardiopulmonary assessments with implantable telemetry device in conscious freely moving cynomolgus monkeys.

Female cynomolgus monkeys were surgically implanted with telemetry transmitters recording ECG (DII), arterial pressure, physical activity, body temperature, and tidal volume. Respiratory rate (RR) and tidal volume (TV) were monitored simultaneously with the telemetry transmitter using impedance. Impedance-based monitoring of RR and TV by telemetry correlated with controlled TV and with pneumotachometer (>98%) in restrained animals. Control drugs with cardiovascular and respiratory effects, including saline, medetomidine (0.01, 0.02 and 0.04mg/kg) and cocaine (0.5, 1.0 and 1.5mg/kg) were administered intravenously. An averaging epoch of 5min was used for analysis of respiratory data. Medetomidine induced significant respiratory depression with decrease in RR and TV in freely moving animals while cocaine increased TV, RR and minute ventilation (MV) with concomitant increase in heart rate when compared with time matched values from saline-treated animals. The onset, duration and magnitude of cardiovascular and respiratory changes were correlated. This highlights the dependency of the cardiovascular and respiratory systems. The use of cardiopulmonary monitoring can allow continuous monitoring including during night time when variability of respiratory parameters is lower. Monitoring of cardiovascular and respiratory parameters in the same animals could also help to decrease the number of animals used in research.

Video-electroencephalography in conscious non human primate using radiotelemetry and computerized analysis: refinement of a safety pharmacology model.

INTRODUCTION: Electroencephalography (EEG) investigations are occasionally required as follow-up studies for safety pharmacology core battery (S7A). Video-EEG monitoring is a standard diagnostic tool in humans but limited data is available on its use in telemetered freely moving macaque monkeys for safety pharmacology investigations. While proconvulsant risk evaluations are routinely conducted in rodents, pharmacological or pharmacokinetic considerations lead to the use of non human primates in toxicology and safety pharmacology in some cases. METHODS: Cynomolgus monkeys were instrumented with telemetry implants. Placement of EEG electrode was based on the 10-20 system using three derivations (C3-O1, Cz-Oz and C4-O2). EEG trace analysis was carried out using NeuroScore software. After 24 h of continuous video-EEG monitoring, animals received pentylenetetrazole (PTZ, 10 mg/kg/15 min) until convulsions were noted. Convulsions were immediately treated with diazepam (1.0 mg/kg). A seizure detection protocol with a dynamic spike train threshold was used for the entire EEG monitoring period (total of 44 h) including periods when PTZ was administered. Spectral analysis was done to quantify the absolute and relative amplitude of EEG frequency bands (delta, theta, alpha, sigma and beta waves). Sleep stages were quantified and EEGs during seizures were analyzed using fast Fourier transformation (FFT) to assess dominant frequencies. RESULTS: Spike trains were detected by computerized analysis in all animals presenting PTZ-induced seizures while paroxysmal activities were systematically predictive (at least 4-min prior to generalized seizures). Beta activity increased with visual stimulation using monkey treats. Characteristics of EEG for all sleep stages (I, II, III and IV) were present in all animals. Delta activity was predominant in normal awake EEG as well as in all sleep stages. Seizure peak frequency was 3-6 Hz on FFT, corresponding to the discharge of the underlying generator. DISCUSSION: EEG-video monitoring can be useful when using non human primates to characterize neurological adverse effects with unpredictable onset. Computerized video-EEG analysis was a valuable tool for safety pharmacology investigations including proconvulsant risk assessment, spectral analysis of frequency bands and sleep stage determination.

Principles of safety pharmacology.

Safety Pharmacology is a rapidly developing discipline that uses the basic principles of pharmacology in a regulatory-driven process to generate data to inform risk/benefit assessment. The aim of Safety Pharmacology is to characterize the pharmacodynamic/pharmacokinetic (PK/PD) relationship of a drug's adverse effects using continuously evolving methodology. Unlike toxicology, Safety Pharmacology includes within its remit a regulatory requirement to predict the risk of rare lethal events. This gives Safety Pharmacology its unique character. The key issues for Safety Pharmacology are detection of an adverse effect liability, projection of the data into safety margin calculation and finally clinical safety monitoring. This article sets out to explain the drivers for Safety Pharmacology so that the wider pharmacology community is better placed to understand the discipline. It concludes with a summary of principles that may help inform future resolution of unmet needs (especially establishing model validation for accurate risk assessment). Subsequent articles in this issue of the journal address specific aspects of Safety Pharmacology to explore the issues of model choice, the burden of proof and to highlight areas of intensive activity (such as testing for drug-induced rare event liability, and the challenge of testing the safety of so-called biologics (antibodies, gene therapy and so on.).

Conscious and anesthetized non-human primate safety pharmacology models: hemodynamic sensitivity comparison.

INTRODUCTION: Drug-induced cardiovascular effects identified in conscious cynomolgus monkeys equipped with tethers and prepared for radiotelemetry were compared with results from anesthetized non-human primate (cynomolgus and rhesus) models. METHODS: Remifentanil (4.0 microg/kg, bolus), esmolol (2.0 mg/kg, bolus) and dopamine (0.05 mg/kg/min, 30 min infusion) were given intravenously to all models. RESULTS: Remifentanil decreased heart rate (HR), systolic, mean and diastolic systemic arterial pressures (SAP) in anesthetized animals while conscious monkeys presented an increase in HR, systolic, mean and diastolic SAP, as seen in humans for the respective state of consciousness (conscious and anesthetized). Esmolol decreased HR, systolic, mean and diastolic SAP in anesthetized monkeys while only HR, systolic and mean SAP achieved a statistically significant decrease in the conscious model. The amplitude of SAP reduction was greater in anesthetized models, while the amplitude of HR reduction was greater in the conscious and anesthetized cynomolgus models than in the anesthetized rhesus model. Dopamine induced a significant increase in HR, systolic, mean and diastolic SAP in anesthetized models without any statistically significant effect on HR and SAP in the conscious model. DISCUSSION: The amplitude of hemodynamic and chronotropic alterations induced by positive control drugs was generally greater in anesthetized than in conscious models and statistical significance was achieved more often with the anesthetized models. These results suggest that an anesthetized model may be valuable as part of a drug screening program for cardiovascular safety evaluations in addition to a conscious model.

Nasal response to capsaicin in patients with allergic rhinitis and in healthy volunteers: effect of colchicine.

We studied the effect of nasal administration of capsaicin in eight patients with allergic rhinitis and eight healthy subjects. We also studied the effect of colchicine, a drug known to inhibit microtubular axonal transport of peptides, on nasal response to capsaicin in these subjects. Colchicine or placebo was administered orally in a double-blind, randomized, crossover manner with a 35 day wash-out interval. Nasal challenge was performed on the last day of each period of treatment, using increasing doses of capsaicin (10(-9) to 3 x 10(-5) mmol). Capsaicin induced a dose-dependent decrease in nasal airflow conductance (active posterior rhinomanometry) (p < 0.002) that was greater in patients with allergic rhinitis (0.40 +/- 0.02 to 0.20 +/- 0.03) than in healthy subjects (0.44 +/- 0.01 to 0.35 +/- 0.02) (p = 0.0001). Capsaicin provoked a greater number of sneezes in patients with allergic rhinitis than in healthy subjects (p < 0.001), but the amount of nasal secretions was similar in these two groups of subjects. In nasal lavage fluid, capsaicin induced an increase in total, epithelial, and neutrophil cell counts in patients with allergic rhinitis (each comparison, p < 0.05), but not in healthy subjects. Capsaicin induced a slight, although not significant, increase in the concentration of elastase in nasal lavage fluid in patients with allergic rhinitis (p = 0.07), but not in healthy subjects. Albumin concentration decreased in nasal lavage fluid in both groups of subjects (p < 0.05). The tendency of capsaicin to increase neutrophil elastase in nasal lavage fluid of patients with allergic rhinitis was not observed after treatment with colchicine.(ABSTRACT TRUNCATED AT 250 WORDS)

A model of frequency tuning in the basilar papilla of the Tokay gecko, Gekko gecko.

This paper uses the quantitative details of the anatomy of the auditory papilla in the Tokay gecko Gekko gecko (as described in the companion paper) to make a quantitative model predicting the tonotopic organization of two of the three papillar areas. Assuming that hair-cell bundle stiffness is similar to that of other species, a model of resonance frequencies for the apical areas of the papilla was constructed, taking into account factors such as the number of hair cells per resonant unit, their bundle dimensions, the volume of the tectorial mass, etc. The model predicts that the apical pre- and postaxial areas, although anatomically adjacent, respond to different frequency ranges, a phenomenon not yet reported from any vertebrate. The model predicts that together, these areas respond best to frequencies between 1.1 and 5.3 kHz, close to the range found physiologically [Eatock et al. (1981) J. Comp. Physiol. 142, 203-218] (0.8 to 5 kHz) for the high-frequency range for this species. Only physiological experiments tracing responses to specific papillar nerve fibres can confirm or refute these interesting predictions of the model. The model also indicates that, compared to free-standing hair-cell bundles, the semi-isolated tectorial structures called sallets not only lower the range of characteristic frequencies but also increase the frequency selectivity of the attached hair cells.

Quantitative anatomical basis for a model of micromechanical frequency tuning in the Tokay gecko, Gekko gecko.

The basilar papilla of the Tokay gecko was studied with standard light- and scanning electron microscopy methods. Several parameters thought to be of particular importance for the mechanical response properties of the system were quantitatively measured, separately for the three different hair-cell areas that are typical for this lizard family. In the basal third, papillar structure was very uniform. The apical two-thirds are subdivided into two hair-cell areas running parallel to each other along the papilla and covered by very different types of tectorial material. Both of those areas showed prominent gradients in hair-cell bundle morphology, i.e., in the height of the stereovillar bundles and the number of stereovilli per bundle, as well as in hair cell density and the size of their respective tectorial covering. Based on the direction of the observed anatomical gradients, a 'reverse' tonotopic organization is suggested, with the highest frequencies represented at the apical end.