An effective CTL peptide vaccine for Ebola Zaire Based on Survivors' CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for COVID-19 vaccine design.

The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple Class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.

Differential morphology of the superior olivary complex of Meriones unguiculatus and Monodelphis domestica revealed by calcium-binding proteins.

In mammals, the superior olivary complex (SOC) of the brainstem is composed of nuclei that integrate afferent auditory originating from both ears. Here, the expression of different calcium-binding proteins in subnuclei of the SOC was studied in distantly related mammals, the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica) to get a better understanding of the basal nuclear organization of the SOC. Combined immunofluorescence labeling of the calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k, and calretinin as well as pan-neuronal markers displayed characteristic distribution patterns highlighting details of neuronal architecture of SOC nuclei. Parvalbumin was found in almost all neurons of SOC nuclei in both species, while calbindin and calretinin were restricted to specific cell types and axonal terminal fields. In both species, calbindin displayed a ubiquitous and mostly selective distribution in neurons of the medial nucleus of trapezoid body (MNTB) including their terminal axonal fields in different SOC targets. In Meriones, calretinin and calbindin showed non-overlapping expression patterns in neuron somata and terminal fields throughout the SOC. In Monodelphis, co-expression of calbindin and calretinin was observed in the MNTB, and hence both CaBPs were also co-localized in terminal fields within the adjacent SOC nuclei. The distribution patterns of CaBPs in both species are discussed with respect to the intrinsic neuronal SOC circuits as part of the auditory brainstem system that underlie the binaural integrative processing of acoustic signals as the basis for localization and discrimination of auditory objects.

Eliciting cytotoxic T-lymphocyte responses from synthetic vectors containing one or two epitopes in a C57BL/6 mouse model using peptide-containing biodegradable microspheres and adjuvants.

We describe a vaccine delivery mechanism consisting of a synthetic, non-living vector of large d,l poly(lactic-co-glycolic) acid (PLGA) microspheres that carry specific cytotoxic T lymphocyte (CTL) epitopes. We demonstrate in mice that it can be used to elicit substantial interferon gamma ELISPOT responses to more than one specific epitope in the same individual. Our data suggest that a superior adjuvant configuration for the formulation is to place a TLR-9 agonist CpG inside the microsphere and a TLR-4 agonist MPLA in the injectate solution. This finding contrasts with the observations of others. Our approach provides a means to elicit immune responses efficiently to select epitopes, which may be important for an effective vaccine against HIV.

Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling.

Precise regulation of the chloride homeostasis crucially determines the action of inhibitory transmitters GABA and glycine and thereby endows neurons or even discrete neuronal compartments with distinct physiological responses to the same transmitters. In mammals, the signaling mediated by GABAA/glycine receptors shifts during early postnatal life from depolarization to hyperpolarization, due to delayed maturation of the chloride homeostasis system. While the activity of the secondary active, K(+)-Cl(-)-extruding cotransporter KCC2, renders GABA/glycine hyperpolarizing in auditory brainstem nuclei of altricial rodents, the mechanisms contributing to the initially depolarizing transmembrane gradient for Cl(-) in respective neurons remained unknown. Here we used gramicidin-perforated patch recordings, non-invasive Cl(-) and Ca(2+) imaging, and immunohistochemistry to identify the Cl(-)-loading transporter that renders depolarizing effects of GABA/glycine in early postnatal life of spherical bushy cells in the cochlear nucleus of gerbil. Our data identify the 1Na(+):1K(+):2Cl(-) cotransporter 1 (NKCC1) as the major Cl(-)-loader responsible for depolarizing action of GABA/glycine at postnatal days 3-5 (P3-5). Extracellular GABA/muscimol elicited calcium signaling through R-, L-, and T-type channels, which was dependent on bumetanide- and [Na(+)]e-sensitive Cl(-) accumulation. The "adult like", low intracellular Cl(-) concentration is established during the second postnatal week, through a mechanism engaging the NKCC1-down regulation between P5 and P15 and ongoing KCC2-mediated Cl(-)-extrusion.

Development of auditory localization accuracy and auditory spatial discrimination in children and adolescents.

The present study investigated the development of two parameters of spatial acoustic perception in children and adolescents with normal hearing, aged 6-18 years. Auditory localization accuracy was quantified by means of a sound source identification task and auditory spatial discrimination acuity by measuring minimum audible angles (MAA). Both low- and high-frequency noise bursts were employed in the tests, thereby separately addressing auditory processing based on interaural time and intensity differences. Setup consisted of 47 loudspeakers mounted in the frontal azimuthal hemifield, ranging from 90° left to 90° right (-90°, +90°). Target signals were presented from 8 loudspeaker positions in the left and right hemifields (±4°, ±30°, ±60° and ±90°). Localization accuracy and spatial discrimination acuity showed different developmental courses. Localization accuracy remained stable from the age of 6 onwards. In contrast, MAA thresholds and interindividual variability of spatial discrimination decreased significantly with increasing age. Across all age groups, localization was most accurate and MAA thresholds were lower for frontal than for lateral sound sources, and for low-frequency compared to high-frequency noise bursts. The study also shows better performance in spatial hearing based on interaural time differences rather than on intensity differences throughout development. These findings confirm that specific aspects of central auditory processing show continuous development during childhood up to adolescence.

Unique features of extracellular matrix in the mouse medial nucleus of trapezoid body--implications for physiological functions.

The medial nucleus of the trapezoid body (MNTB) is a vital structure of sound localization circuits in the auditory brainstem. Each principal cell of MNTB is contacted by a very large presynaptic glutamatergic terminal, the calyx of Held. The MNTB principal cells themselves are surrounded by extracellular matrix components forming prominent perineuronal nets (PNs). Throughout the CNS, PNs, which form lattice-like structures around the somata and proximal dendrites, are associated with distinct types of neurons. PNs are highly enriched in hyaluronan and chondroitin sulfate proteoglycans therefore providing a charged surface structure surrounding the cell body and proximal neurites of these neurons. The localization and composition of PNs have lead investigators to a number of hypotheses about their functions including: creating a specific extracellular ionic milieu around these neurons, stabilizing synapses, and influencing the outgrowth of axons. However, presently the precise functions of PNs are still quite unclear primarily due to the lack of an ideal experimental model system that is highly enriched in PNs and in which the synaptic transmission properties can be precisely measured. The MNTB principal cells could offer such a model, since they have been extensively characterized electrophysiologically. However, extracellular matrix (ECM) in these neurons has not yet been precisely detailed. The present study gives a detailed examination of the ECM organization and structural differences in PNs of the mouse MNTB. The different PN components and their distribution pattern are scrutinized throughout the MNTB. The data are complemented by electron microscopic investigations of the unique ultrastructural localization of PN-components and their interrelation with distinct pre- and postsynaptic MNTB cell structures. Therefore, we believe this work identifies the MNTB as an ideal system for studying PN function.

Development of the chloride homeostasis in the auditory brainstem.

Inhibitory neurotransmission plays a substantial role in encoding of auditory cues relevant for sound localization in vertebrates. While the anatomical organization of the respective afferent auditory brainstem circuits shows remarkable similarities between mammals and birds, the properties of inhibitory neurotransmission in these neural circuits are strikingly different. In mammals, inhibition is predominantly glycinergic and endowed with fast kinetics. In birds, inhibition is mediated by gamma-Aminobutiric acid (GABA) and too slow to convey temporal information. A further prominent difference lies in the mechanism of inhibition in the respective systems. In auditory brainstem neurons of mammals, [Cl(-)](i) undergoes a developmental shift causing the actions of GABA and glycine to gradually change from depolarization to the 'classic' hyperpolarizing-inhibition before hearing onset. Contrary to this, in the mature avian auditory brainstem Cl(-) homeostasis mechanisms accurately adjust the Cl(-) gradient to enable depolarizing, but still very efficient, shunting inhibition. The present review considers the mechanisms underlying development of the Cl(-) homeostasis in the auditory system of mammals and birds and discusses some open issues that require closer attention in future studies.

Multilinear models of single cell responses in the medial nucleus of the trapezoid body.

The representation of acoustic stimuli in the brainstem forms the basis for higher auditory processing. While some characteristics of this representation (e.g. tuning curve) are widely accepted, it remains a challenge to predict the firing rate at high temporal resolution in response to complex stimuli. In this study we explore models for in vivo, single cell responses in the medial nucleus of the trapezoid body (MNTB) under complex sound stimulation. We estimate a family of models, the multilinear models, encompassing the classical spectrotemporal receptive field and allowing arbitrary input-nonlinearities and certain multiplicative interactions between sound energy and its short-term auditory context. We compare these to models of more traditional type, and also evaluate their performance under various stimulus representations. Using the context model, 75% of the explainable variance could be predicted based on a cochlear-like, gamma-tone stimulus representation. The presence of multiplicative contextual interactions strongly reduces certain inhibitory/suppressive regions of the linear kernels, suggesting an underlying nonlinear mechanism, e.g. cochlear or synaptic suppression, as the source of the suppression in MNTB neuronal responses. In conclusion, the context model provides a rich and still interpretable extension over many previous phenomenological models for modeling responses in the auditory brainstem at submillisecond resolution.

A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3.

Despite the apparent robustness of language learning in humans, a large number of children still fail to develop appropriate language skills despite adequate means and opportunity. Most cases of language impairment have a complex etiology, with genetic and environmental influences. In contrast, we describe a three-generation German family who present with an apparently simple segregation of language impairment. Investigations of the family indicate auditory processing difficulties as a core deficit. Affected members performed poorly on a nonword repetition task and present with communication impairments. The brain activation pattern for syllable duration as measured by event-related brain potentials showed clear differences between affected family members and controls, with only affected members displaying a late discrimination negativity. In conjunction with psychoacoustic data showing deficiencies in auditory duration discrimination, the present results indicate increased processing demands in discriminating syllables of different duration. This, we argue, forms the cognitive basis of the observed language impairment in this family. Genome-wide linkage analysis showed a haplotype in the central region of chromosome 12 which reaches the maximum possible logarithm of odds ratio (LOD) score and fully co-segregates with the language impairment, consistent with an autosomal dominant, fully penetrant mode of inheritance. Whole genome analysis yielded no novel inherited copy number variants strengthening the case for a simple inheritance pattern. Several genes in this region of chromosome 12 which are potentially implicated in language impairment did not contain polymorphisms likely to be the causative mutation, which is as yet unknown.

Glycine-mediated changes of onset reliability at a mammalian central synapse.

Glycine is an inhibitory neurotransmitter activating a chloride conductance in the mammalian CNS. In vitro studies from brain slices revealed a novel presynaptic site of glycine action in the medial nucleus of the trapezoid body (MNTB) which increases the release of the excitatory transmitter glutamate from the calyx of Held. Here, we investigate the action of glycine on action potential firing of single MNTB neurons from the gerbil under acoustic stimulation in vivo. Iontophoretic application of the glycine receptor antagonist strychnine caused a significant decrease in spontaneous and sound-evoked firing rates throughout the neurons' excitatory response areas, with the largest changes at the respective characteristic frequency (CF). The decreased firing rate was accompanied by longer and more variable onset latencies of sound-evoked responses. Outside the neurons' excitatory response areas, firing rates increased during the application of strychnine due to a reduction of inhibitory sidebands, causing a broadening of frequency tuning. These results indicate that glycine enhances the efficacy for on-CF stimuli, while simultaneously suppressing synaptic transmission for off-CF stimuli. These in vivo results provide evidence of multiple excitatory and inhibitory glycine effects on the same neuronal population in the mature mammalian CNS.

The medial nucleus of the trapezoid body: comparative physiology.

Principal cells of the medial nucleus of the trapezoid body (MNTB) receive their excitatory input through large somatic terminals, the calyces of Held, which arise from axons of globular bushy cells located in the contralateral ventral cochlear nucleus. Discharges of MNTB neurons are characterized by high stimulus evoked firing rates, temporally precise onset responses, and a high degree of phase-locking to either pure tones or stimulus envelopes. Since the calyx of Held synapse is accessible to in vitro and to in vivo recordings, it serves as one of the most elaborate models for studying synaptic transmission in the mammalian brain. Although in such studies, the major emphasis is on synaptic physiology, the interpretation of the data will benefit from an understanding of the MNTB's contribution to auditory signal processing, including possible functional differences in different species. This implies the consideration of possible functional differences in different species. Here, we compare single unit recordings from MNTB principal cells in vivo in three different rodent species: gerbil, mouse and rat. Because of their good low-frequency hearing gerbils are often used in in vivo preparations, while mice and rats are predominantly used in slice preparations. We show that MNTB units in all three species exhibit high firing rates and precise onset-timing. Still there are species-specific specializations that might suggest the preferential use of one species over the others, depending on the scope of the respective investigation.

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling.

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

Pharmacokinetics and pharmacodynamics of inhaled versus intravenous morphine in healthy volunteers.

BACKGROUND: A new pulmonary drug delivery system produces aerosols from disposable packets of medication. This study compared the pharmacokinetics and pharmacodynamics of morphine delivered by an AERx prototype with intravenous morphine. METHODS: Fifteen healthy volunteers were enrolled. Two subjects were administered four inhalations of 2.2 mg morphine each at 1-min intervals or 4.4 mg over 3 min by intravenous infusion. Thirteen subjects were given twice the above doses, i.e., eight inhalations or 8.8 mg intravenously over 7 min. Arterial blood sampling was performed every minute during administration and at 2, 5, 7, 10, 15, 20, 45, 60, 90, 120, 150, 180, and 240 min after administration. The effect of morphine was assessed by measuring pupil diameter and ventilatory response to a hypercapnic challenge. Pharmacokinetic and pharmacodynamic analyses were performed simultaneously using mixed-effect models. RESULTS: The pharmacokinetic data after intravenous administration were described by a three-exponent decay model preceded by a lag time. The pharmacokinetic model for administration by inhalation consisted of the three-exponent intravenous pharmacokinetic model preceded by a two-exponent absorption model. The authors found that, with administration by inhalation, the total bioavailability was 59%, of which 43% was absorbed almost instantaneously and 57% was absorbed with a half-life of 18 min. The median times to the half-maximal miotic effects of morphine were 10 and 5.5 min after inhalation and intravenous administration, respectively (P < 0.01). The pharmacodynamic parameter ke0 was approximately 0.003 min-1. CONCLUSIONS: The onset and duration of the effects of morphine are similar after intravenous administration or inhalation via this new pulmonary drug delivery system. Morphine bioavailability after such administration is 59% of the dose loaded into the dosage form.

Comparison of in vitro and in vivo efficiencies of a novel unit-dose liquid aerosol generator and a pressurized metered dose inhaler.

Gamma scintigraphic imaging was employed in 10 healthy volunteers to compare the total and regional lung deposition of aerosols generated by two delivery platforms that permitted microprocessor-controlled actuation at an optimal point during inhalation. An aqueous solution containing 99mTc-DTPA was used to assess the deposition of aerosols delivered by inhalation from two successive unit-dosage forms (44 microl volume) using a prototype of a novel liquid aerosol system (AERx Pulmonary Delivery System). This was compared with aerosol deposition after inhalation of two 50 microl puffs of a 99mTc-HMPAO-labeled solution formulation from a pressurized metered dose inhaler (MDI). The in vitro size characteristics of the radiolabeled aerosols were determined by cascade impaction. For the AERx system, the predicted lung delivery efficiency based on the product of emitted dose (60.8%, coefficient of variation (CV)=12%) and fine particle fraction (% by mass of aerosol particles <5.7 microm in diameter) was 53.3% (CV=13%). For the solution MDI, the emitted dose was 62.9% (CV=13%) and the predicted lung dose was 44. 9% (CV=15%). The AERx system demonstrated efficient and reproducible dosing characteristics in vivo. Of the dose loaded into the device, the mean percent reaching the lungs was 53.3% (CV=10%), with only 6. 9% located in the oropharynx/stomach. In contrast, the lung deposition from the solution MDI was significantly less (21.7%) and more variable (CV=31%), with 42.0% of the radiolabel detected in the oropharynx/stomach. Analysis of the regional deposition of the radioaerosol indicated a homogeneous pattern of deposition after delivery from the AERx system. A predominantly central pattern of distribution occurred after MDI delivery, where the pattern of deposition was biased towards a central zone depicting the conducting airways. The AERx system, in contrast to MDIs, seems highly suited to the delivery of systemically active agents via pulmonary administration.

Pulmonary insulin administration using the AERx system: physiological and physicochemical factors influencing insulin effectiveness in healthy fasting subjects.

BACKGROUND: Orally inhaled insulin may provide a convenient and effective therapy for prandial glucose control in patients with diabetes. This study evaluated the influence of formulation pH and concentration and different respiratory maneuvers on pharmacokinetic and pharmacodynamic properties of inhaled insulin. METHODS: Three, open-label crossover studies in a total of 23 healthy subjects were conducted in which the safety, pharmacokinetics, and pharmacodynamics of insulin inhalation were compared to subcutaneous (SC) injection into the abdomen of commercially available regular insulin. A novel, aerosol generating system (AERx Diabetes Management System, Aradigm Corporation, Hayward, CA) was used to deliver aqueous insulin bolus aerosols to the lower respiratory tract from formulations at pH 3.5 or 7.4 and concentrations of U250 (250 U/mL) or U500 (500 U/mL). RESULTS: Time to maximum insulin concentration in serum (Tmax) after SC dosing occurred approximately 50-60 minutes with the time to minimum plasma glucose concentration (i.e., maximum hypoglycemic effect), (TGmin), occurring later, at around 100-120 minutes. In contrast, pulmonary delivery led to a significantly earlier Tmax (7-20 minutes) and TGmin (60-70 minutes), parameters that were shown to be largely unaffected by changing the pH or concentration of the insulin. However, investigation of changes in inhaled volume (achieved by different programming of the AERx system) for administration of the same sized aerosol bolus revealed significant effects. Significantly slower absorption and time to peak hypoglycemic activity occurred when aerosol delivery of insulin occurred during a shallow (approximately 40% vital capacity) as opposed to a deep (approximately 80% vital capacity) inspiration. In addition, it was shown that serum concentration of insulin increased immediately after a series of forced expiraratory maneuvers 30 minutes after inhaled delivery. CONCLUSIONS: Pulmonary delivery of aqueous bolus aerosols of insulin in healthy subjects resulted in rapid absorption with an associated hypoglycemic effect quicker than is achieved after subcutaneous dosing of regular insulin. Inhaled insulin pharmacokinetics and pharmacodynamics were independent of formulation variables (pH, concentration) but affected by certain respiratory maneuvers.

Inhalation delivery systems with compliance and disease management capabilities.

Non-compliance with prescribed medication is a major reason for poor therapeutic outcomes, leading to unnecessary contributions to healthcare costs. Poor technique in self-administration of inhalation therapy is a special type of non-compliance associated with this route of administration. However, pulmonary drug delivery has fundamental advantages for therapy of diseases of the respiratory tract because it is site-directed. The lung is also a promising portal for drug delivery into the systemic circulation. Incorporation of microprocessors into pulmonary drug delivery systems facilitates sophisticated compliance management of chronic diseases such as asthma and diabetes. Microprocessor-assisted systems afford control of patients' administration technique during the therapeutic inhalation event, thus leading to efficient and reproducible regional deposition of the inhaled drug or diagnostic agent. SmartMist is a hand-held asthma disease management device that aids patients to use optimally metered dose inhalers. It also measures pulmonary lung function and provides a long term downloadable electronic record of the therapeutic and diagnostic events. The AERx pulmonary delivery system utilizes similar microprocessor capabilities; however, it employs a novel means of generating aqueous aerosols from unit dose packages, thus providing a broad inhalation technology base for delivery of a wide variety of therapeutic and diagnostic agents into the respiratory tract, and via the lung into the systemic circulation.

Pulmonary administration of aerosolised fentanyl: pharmacokinetic analysis of systemic delivery.

AIMS: Pulmonary drug delivery is a promising noninvasive method of systemic administration. Our aim was to determine whether a novel breath-actuated, microprocessor-controlled metered dose oral inhaler (SmartMist, Aradigm Corporation) could deliver fentanyl in a way suitable for control of severe pain. METHODS: Aersolised pulmonary fentanyl base 100-300 microg was administered to healthy volunteers using SmartMist and the resultant plasma concentration-time data were compared with those from the same doses administered by intravenous (i.v.) injection in the same subjects. RESULTS: Plasma concentrations from SmartMist were similar to those from i.v. injection. Time-averaged bioavailability based upon nominal doses averaged approximately 100%, and was > 50% within 5 min of delivery. Fentanyl systemic pharmacokinetics were similar to those previously reported with no trends to dose-dependence from either route. Side-effects (e.g. sedation, lightheadedness) were the same from both routes. CONCLUSIONS: Fentanyl delivery using SmartMist can provide analgetically relevant plasma drug concentrations. This, combined with its ease of noninvasive use and transportability, suggests a strong potential for field and domicilliary use, and for patient controlled analgesia without the need for i.v. cannulae.

Development of single- and two-tone responses of anteroventral cochlear nucleus neurons in gerbil.

Responses of anteroventral cochlear nucleus (AVCN) neurons in developing gerbils were obtained to single-tone stimuli, and two-tone stimuli elicited by best frequency probes presented over a range of intensities. Neurons displayed Type I, Type I/III, and Type III receptive field patterns. Best frequencies ranged from 1.5 to 10.0 kHz. Two-tone suppression (2TS) was first observed in 5 of 16 neurons examined at 14 dab. and in all neurons examined in gerbils aged 15 to 60 dab. Suppression areas grew larger, and discharge rate reductions became greater with age. Features of the two-tone responses that were highly correlated with single-tone responses across age groups include maximum rate reductions and suppression area thresholds. The intensity level of the CF probe-tone also influenced these features of 2TS. Maximum rate reductions to below spontaneous rate levels of activity were common across age groups. Results suggest that the cochlear amplifier is present and fundamentally adult-like by 15 dab for the regions of the cochlea coding the mid frequencies in gerbil. Over the subsequent week, contributions to the developing two-tone responses by the cochlear amplifier increase slightly. Two-tone responses are influenced by central inhibitory mechanisms as early as 14 dab.

Cortical perineuronal nets in the gray short-tailed opossum (Monodelphis domestica): a distribution pattern contrasting with that shown in placental mammals.

Extracellular matrix proteoglycans accumulated in perineuronal nets and in certain neuropil zones have been shown to influence the immediate neuronal microenvironment, and to contribute to the chemoarchitectonic characteristics of neuronal networks. Studies in different placental mammals, including the human, have suggested that the major principles of extracellular matrix distribution remained constant during phylogenesis of the different mammalian strains. However, the comparison of matrix distribution between various species also indicates that striking deviations from the basic pattern may occur, although their functional significance appears unknown as yet. This study examines the extracellular matrix in the forebrain of a basic American marsupial, which has evolved independently of placental mammals for more than 100 million years. Brain sections obtained from adult gray short-tailed opossums (Monodelphis domestica) were stained for extracellular matrix components using the N-acetylgalactosamine-binding lectin Wisteria floribunda agglutinin (WFA), a polyclonal antibody against chondroitin sulfate proteoglycans (CSPG), and biotinylated hyaluronectin for the detection of hyaluronan. In subcortical regions, the distribution patterns of WFA-stained and CSPG-immunoreactive perineuronal nets were similar to those reported previously in placental mammals. In contrast, a unique distribution was found in the neocortex. This distribution was characterized by the presence of perineuronal nets around pyramidal cells and matrix components within the adjacent neuropil that together form a continuously labeled zone in layer V. Weakly stained nets ensheathed less numerous pyramidal cells in the upper layers II/III and a few multipolar cortical neurons. Dual staining experiments showed that cortical net-associated neurons were rarely immunopositive for parvalbumin. This fact, in addition to the predominant association of extracellular matrix components with layer V pyramidal cells, differentiates the neocortex in Monodelphis from that of all placental mammals studied to date. Regarding the basic phylogenetic position of this marsupial species it remains to be shown if these distribution characteristics of extracellular matrix may represent also a basic feature of cortical organization.