Forward masking in the superior paraolivary nucleus of the rat.

In natural acoustic environments, perception of acoustic stimuli depends on the recent contextual history. Forward masking describes a phenomenon whereby the detection threshold of a probe stimulus is markedly increased when it is preceded by a masking stimulus. The aim of this study was to characterize the offset response of single units in the superior paraolivary nucleus (SPON) to a forward masking paradigm. We observed two distinct response types to forward-masked stimuli, namely inhibited and facilitated responses. In the presence of a default masking stimulus, inhibited responses to probe stimuli were characterized by elevated thresholds and/or diminished spike counts, whereas facilitated responses were characterized by reduced thresholds and increased spike counts. In units with inhibited responses to the probe stimuli, probe thresholds increased and spike counts decreased as masker intensity was raised or the masker-to-probe delay was shortened. Conversely, in units with facilitated responses to the probe stimuli, probe thresholds decreased and spike counts increased as masker intensity was raised or the masker-to-probe delay was shortened. Neither inhibited nor facilitated responses to the forward masking paradigm were significantly dependent on masker frequency. These findings suggest that SPON responses are not themselves consistently subject to the same forward masking properties observed in other nuclei along the ascending auditory pathway. The potential neural mechanisms of the forward masking responses observed in the SPON are discussed.

Organization of the Zone of Transition between the Pretectum and the Thalamus, with Emphasis on the Pretectothalamic Lamina.

The zone of transition between the pretectum, derived from prosomere 1, and the thalamus, derived from prosomere 2, is structurally complex and its understanding has been hampered by cytoarchitectural and terminological confusion. Herein, using a battery of complementary morphological approaches, including cytoarchitecture, myeloarchitecture and the expression of molecular markers, we pinpoint the features or combination of features that best characterize each nucleus of the pretectothalamic transitional zone of the rat. Our results reveal useful morphological criteria to identify and delineate, with unprecedented precision, several [mostly auditory] nuclei of the posterior group of the thalamus, namely the pretectothalamic lamina (PTL; formerly known as the posterior limitans nucleus), the medial division of the medial geniculate body (MGBm), the suprageniculate nucleus (SG), and the ethmoid, posterior triangular and posterior nuclei of the thalamus. The PTL is a sparsely-celled and fiber rich flattened nucleus apposed to the lateral surface of the anterior pretectal nucleus (APT) that marks the border between the pretectum and the thalamus; this structure stains selectively with the Wisteria floribunda agglutinin (WFA), and is essentially immunonegative for the calcium binding protein parvalbumin (PV). The MGBm, located medial to the ventral division of the MGB (MGBv), can be unequivocally identified by the large size of many of its neurons, its dark immunostaining for PV, and its rather selective staining for WFA. The SG, which extends for a considerable caudorostral distance and deviates progressively from the MGB, is characterized by its peculiar cytoarchitecture, the paucity of myelinated fibers, and the conspicuous absence of staining for calretinin (CR); indeed, in many CR-stained sections, the SG stands out as a blank spot. Because most of these nuclei are small and show unique anatomical relationships, the information provided in this article will facilitate the interpretation of the results of experimental manipulations aimed at the auditory thalamus and improve the design of future investigations. Moreover, the previously neglected proximity between the MGBm and the caudal region of the scarcely known PTL raises the possibility that certain features or roles traditionally attributed to the MGBm may actually belong to the PTL.

Forward masking in the medial nucleus of the trapezoid body of the rat.

Perception of acoustic stimuli is modulated by the temporal and spectral relationship between sound components. Forward masking experiments show that the perception threshold for a probe tone is significantly impaired by a preceding masker stimulus. Forward masking has been systematically studied at the level of the auditory nerve, cochlear nucleus, inferior colliculus and auditory cortex, but not yet in the superior olivary complex. The medial nucleus of the trapezoid body (MNTB), a principal cell group of the superior olive, plays an essential role in sound localization. The MNTB receives excitatory input from the contralateral cochlear nucleus via the calyces of Held and innervates the ipsilateral lateral and medial superior olives, as well as the superior paraolivary nucleus. Here, we performed single-unit extracellular recordings in the MNTB of rats. Using a forward masking paradigm previously employed in studies of the inferior colliculus and auditory nerve, we determined response thresholds for a 20-ms characteristic frequency pure tone (the probe), and then presented it in conjunction with another tone (the masker) that was varied in intensity, duration, and frequency; we also systematically varied the masker-to-probe delay. Probe response thresholds increased and response magnitudes decreased when a masker was presented. The forward suppression effects were greater when masker level and masker duration were increased, when the masker frequency approached the MNTB unit's characteristic frequency, and as the masker-to-probe delay was shortened. Probe threshold shifts showed an exponential decay as the masker-to-probe delay increased.

The superior paraolivary nucleus shapes temporal response properties of neurons in the inferior colliculus.

The mammalian superior paraolivary nucleus (SPON) is a major source of GABAergic inhibition to neurons in the inferior colliculus (IC), a well-studied midbrain nucleus that is the site of convergence and integration for the majority ascending auditory pathways en route to the cortex. Neurons in the SPON and IC exhibit highly precise responses to temporal sound features, which are important perceptual cues for naturally occurring sounds. To determine how inhibitory input from the SPON contributes to the encoding of temporal information in the IC, a reversible inactivation procedure was conducted to silence SPON neurons, while recording responses to amplitude-modulated tones and silent gaps between tones in the IC. The results show that SPON-derived inhibition shapes responses of onset and sustained units in the IC via different mechanisms. Onset neurons appear to be driven primarily by excitatory inputs and their responses are shaped indirectly by SPON-derived inhibition, whereas sustained neurons are heavily influenced directly by transient offset inhibition from the SPON. The findings also demonstrate that a more complete dissection of temporal processing pathways is critical for understanding how biologically important sounds are encoded by the brain.

Development of on-off spiking in superior paraolivary nucleus neurons of the mouse.

The superior paraolivary nucleus (SPON) is a prominent cell group in the auditory brain stem that has been increasingly implicated in representing temporal sound structure. Although SPON neurons selectively respond to acoustic signals important for sound periodicity, the underlying physiological specializations enabling these responses are poorly understood. We used in vitro and in vivo recordings to investigate how SPON neurons develop intrinsic cellular properties that make them well suited for encoding temporal sound features. In addition to their hallmark rebound spiking at the stimulus offset, SPON neurons were characterized by spiking patterns termed onset, adapting, and burst in response to depolarizing stimuli in vitro. Cells with burst spiking had some morphological differences compared with other SPON neurons and were localized to the dorsolateral region of the nucleus. Both membrane and spiking properties underwent strong developmental regulation, becoming more temporally precise with age for both onset and offset spiking. Single-unit recordings obtained in young mice demonstrated that SPON neurons respond with temporally precise onset spiking upon tone stimulation in vivo, in addition to the typical offset spiking. Taken together, the results of the present study demonstrate that SPON neurons develop sharp on-off spiking, which may confer sensitivity to sound amplitude modulations or abrupt sound transients. These findings are consistent with the proposed involvement of the SPON in the processing of temporal sound structure, relevant for encoding communication cues.

Sound rhythms are encoded by postinhibitory rebound spiking in the superior paraolivary nucleus.

The superior paraolivary nucleus (SPON) is a prominent structure in the auditory brainstem. In contrast to the principal superior olivary nuclei with identified roles in processing binaural sound localization cues, the role of the SPON in hearing is not well understood. A combined in vitro and in vivo approach was used to investigate the cellular properties of SPON neurons in the mouse. Patch-clamp recordings in brain slices revealed that brief and well timed postinhibitory rebound spiking, generated by the interaction of two subthreshold-activated ion currents, is a hallmark of SPON neurons. The I(h) current determines the timing of the rebound, whereas the T-type Ca(2+) current boosts the rebound to spike threshold. This precisely timed rebound spiking provides a physiological explanation for the sensitivity of SPON neurons to sinusoidally amplitude-modulated (SAM) tones in vivo, where peaks in the sound envelope drive inhibitory inputs and SPON neurons fire action potentials during the waveform troughs. Consistent with this notion, SPON neurons display intrinsic tuning to frequency-modulated sinusoidal currents (1-15Hz) in vitro and discharge with strong synchrony to SAMs with modulation frequencies between 1 and 20 Hz in vivo. The results of this study suggest that the SPON is particularly well suited to encode rhythmic sound patterns. Such temporal periodicity information is likely important for detection of communication cues, such as the acoustic envelopes of animal vocalizations and speech signals.

Connections of the Superior Paraolivary Nucleus of the Rat: II. Reciprocal Connections with the Tectal Longitudinal Column.

The superior paraolivary nucleus (SPON), a prominent GABAergic center of the mammalian auditory brainstem, projects to the ipsilateral inferior colliculus (IC) and sends axons through the commissure of the IC (CoIC). Herein we demonstrate that the SPON is reciprocally connected with the recently discovered tectal longitudinal column (TLC). The TLC is a long and narrow structure that spans nearly the entire midbrain tectum longitudinally, immediately above the periaqueductal gray matter (PAG) and very close to the midline. Unilateral injections of biotinylated dextran into the SPON of the rat label abundant terminal fibers in the TLC of both sides, with an ipsilateral predominance. The SPON provides a dense innervation of the entire rostrocaudal extent of the ipsilateral TLC, and a relatively sparser innervation of the caudal and rostral portions of the contralateral TLC. SPON fibers reach the TLC by two routes: as collaterals of axons of the CoIC, and as axons that circumvent the ipsilateral IC before traveling in the deep layers of the superior colliculus (SC). The density of these projections identifies SPON as a significant source of input to the TLC. Other targets of the SPON discovered in this study include the deep layers of the SC and the PAG. The same experiments reveal numerous labeled cell bodies in the TLC, interspersed among the labeled SPON fibers. This observation suggests that the SPON is a significant target of TLC projections. The discovery of novel reciprocal connections between the SPON and the TLC opens unexpected avenues for investigation of sound processing in mammalian brainstem circuits.

Molecular guidance cues necessary for axon pathfinding from the ventral cochlear nucleus.

During development, multiple guidance cues direct the formation of appropriate synaptic connections. Factors that guide developing axons are known for various pathways throughout the mammalian brain; however, signals necessary to establish auditory connections are largely unknown. In the auditory brainstem the neurons whose axons traverse the midline in the ventral acoustic stria (VAS) are primarily located in the ventral cochlear nucleus (VCN) and project bilaterally to the superior olivary complex (SOC). The circumferential trajectory taken by developing VCN axons is similar to that of growing axons of spinal commissural neurons. Therefore, we reasoned that netrin-DCC and slit-robo signaling systems function in the guidance of VCN axons. VCN neurons express the transcription factor, mafB, as early as embryonic day (E) 13.5, thereby identifying the embryonic VCN for these studies. VCN axons extend toward the midline as early as E13, with many axons crossing by E14.5. During this time, netrin-1 and slit-1 RNAs are expressed at the brainstem midline. Additionally, neurons within the VCN express RNA for DCC, robo-1, and robo-2, and axons in the VAS are immunoreactive for DCC. VCN axons do not reach the midline of the brainstem in mice mutant for either the netrin-1 or DCC gene. VCN axons extend in pups lacking netrin-1, but most DCC-mutant samples lack VCN axonal outgrowth. Stereological cell estimates indicate only a modest reduction of VCN neurons in DCC-mutant mice. Taken together, these data show that a functional netrin-DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem.

Distinct roles for glycine and GABA in shaping the response properties of neurons in the superior paraolivary nucleus of the rat.

The superior paraolivary nucleus (SPON) is a prominent periolivary cell group of the superior olivary complex. SPON neurons use gamma-aminobutyric acid (GABA) as their neurotransmitter and are contacted by large numbers of glycinergic and GABAergic punctate profiles, representing a dense inhibitory innervation from the medial nucleus of the trapezoid body (MNTB) and from collaterals of SPON axons, respectively. SPON neurons have low rates of spontaneous activity, respond preferentially to the offset of pure tones, and phase-lock to amplitude-modulated tones. To determine the roles of glycine and GABA in shaping SPON responses, we recorded from single units in the SPON of anesthetized rats before, during, and after application of the glycine receptor antagonist strychnine, the GABA(A) receptor antagonist bicuculline, or both drugs applied simultaneously. Strychnine caused a major increase in spike counts during the stimulus presentation, followed by the disappearance of offset spikes. In half of the recorded units, bicuculline caused moderately increased firing during the stimulus. However, in 86% of units bicuculline also caused a large increase in the magnitude of the offset response. Application of the drug cocktail caused increased spontaneous activity, dramatically increased spike counts during the stimulus presentation, and eliminated the offset response in most units. We conclude that glycinergic inhibition from the MNTB suppresses SPON spiking during sound stimulation and is essential in generating offset responses. GABAergic inhibition, presumably from intrinsic SPON collaterals, plays a subtler role, contributing in some cells to suppression of firing during the stimulus and in most cells to restrict firing after stimulus offset.

Apoptosome impairment during development results in activation of an autophagy program in cerebral cortex.

The deficiency of upstream regulators of the mitochondrial death pathway has been recently shown to trigger in vitro a cellular process of self-clearance with features of autophagy. We show here that, when Apaf1 (responsible for apoptosome formation) is downregulated in vivo in cortical precursors, cells express markers of neuronal differentiation, accumulate in ectopic cortical masses and show hallmarks of the beclin-1-dependent pathway of autophagy, probably activated by a depletion in growth factors in the cells' microenvironment. To visualize this process in a cell culture model system, we also used a neural precursor cell line to mimic growth factor starvation in the absence of the apoptosome and tracked autophagolysosome formation. Our findings demonstrate the existence of an interplay between the autophagy and apoptosis pathways in vivo in brain development, and possibly link the absence of apoptosis to the occurrence of pathological conditions associated with peculiar cellular morphotypes.

Distinct subtypes of somatostatin-containing neocortical interneurons revealed in transgenic mice.

GABA-releasing inhibitory interneurons in the cerebral cortex can be classified by their neurochemical content, firing patterns, or axonal targets, to name the most common criteria, but whether classifications using different criteria converge on the same neuronal subtypes, and how many such subtypes exist, is a matter of much current interest and considerable debate. To address these issues, we generated transgenic mice expressing green fluorescent protein (GFP) under control of the GAD67 promoter. In two of these lines, named X94 and X98, GFP expression in the barrel cortex was restricted to subsets of somatostatin-containing (SOM+) GABAergic interneurons, similar to the previously reported "GIN" line (Oliva et al., 2000), but the laminar distributions of GFP-expressing (GFP+) cell bodies in the X94, X98, and GIN lines were distinct and nearly complementary. We compared neurochemical content and axonal distribution patterns of GFP+ neurons among the three lines and analyzed in detail electrophysiological properties in a dataset of 150 neurons recorded in whole-cell, current-clamp mode. By all criteria, there was nearly perfect segregation of X94 and X98 GFP+ neurons, whereas GIN GFP+ neurons exhibited intermediate properties. In the X98 line, GFP expression was found in infragranular, calbindin-containing, layer 1-targeting ("Martinotti") cells that had a propensity to fire low-threshold calcium spikes, whereas X94 GFP+ cells were stuttering interneurons with quasi fast-spiking properties, residing in and targeting the thalamo-recipient neocortical layers. We conclude that much of the variability previously attributed to neocortical SOM+ interneurons can be accounted for by their natural grouping into distinct subtypes.

Low-frequency hearing loss in prenatally stressed rats.

We investigated the possibility that hearing thresholds are altered in prenatally stressed rats raised in a normal auditory environment. Pregnant dams were assigned randomly to prenatally stressed and control groups. Half of the dams were subjected to the mild stressors of handling, exposure to a novel cage and saline injection at random times during lights-on daily. The hearing thresholds of young adult male offspring were assessed by recording auditory-evoked brainstem responses to 0.5, 1, 2, 4, 8, 16, 32 and 64 kHz pure tones. The resultant audiograms showed that prenatally stressed offspring had significantly higher hearing thresholds than control animals at 1, 2 and 4 kHz (t-tests, P<0.05). The threshold shifts caused by prenatal stress averaged 7.7 dB across frequencies. We conclude that prenatal stress causes low-frequency hearing loss, possibly due to increased vulnerability to noise-induced hearing loss, accelerated cochlear degeneration and/or disrupted cochlear development.

Neurons in the medial nucleus of the trapezoid body and superior paraolivary nucleus of the rat may play a role in sound duration coding.

We describe neurons in two nuclei of the superior olivary complex that display differential sensitivities to sound duration. Single units in the medial nucleus of the trapezoid body (MNTB) and superior paraolivary nucleus (SPON) of anesthetized rats were studied. MNTB neurons produced primary-like responses to pure tones and displayed a period of suppressed spontaneous activity after stimulus offset. In contrast, neurons of the SPON, which receive a strong glycinergic input from MNTB, showed very little or no spontaneous activity and responded with short bursts of action potentials after the stimulus offset. Because SPON spikes were restricted to the same time window during which suppressed spontaneous activity occurs in the MNTB, we presume that SPON offset activity represents a form of postinhibitory rebound. Using characteristic frequency tones of 2- to 1,000-ms duration presented 20 dB above threshold, we show that the profundity and duration of the suppression of spontaneous activity in MNTB as well as the magnitude and first spike latency of the SPON offset response depend on stimulus duration as well as on stimulus intensity, showing a tradeoff between intensity and duration. Pairwise comparisons of the responses to stimuli of various durations revealed that the duration sensitivity in both nuclei is sharpest for stimuli <50 ms.

Three-dimensional reconstruction of immunolabeled neuromuscular junctions in the human thyroarytenoid muscle.

OBJECTIVES/HYPOTHESIS: The objective was to reveal the location of the neuromuscular junctions in a three-dimensional reconstruction of the human thyroarytenoid muscle within the true vocal fold. STUDY DESIGN: Immunohistochemical analysis of serially sectioned human true vocal folds was performed, followed by reconstruction in three dimensions using computer imaging software. METHODS: Six fresh human larynges from autopsy were harvested, fixed in formalin, and embedded in paraffin. Eight vocal cords were studied from these six larynges. Five-micron serial sections were collected throughout the entire vocal cord in an axial plane at 500-microm intervals. Immunohistochemical analysis was performed with anti-synaptophysin antibody. A computer-controlled imaging and reconstruction system was used to create a three-dimensional reconstruction from the serial sections and to represent the location of the clustered band of neuromuscular junctions within each true vocal fold. The vocal cord was divided into equal thirds from anterior to posterior for statistical analysis. RESULTS: The most neuromuscular junctions (74%) were located in the middle third, and the least (7%) were found in the anterior third. The difference in anterior-to-posterior distribution was statistically significant in all eight specimens by chi2 analysis (P <.001). CONCLUSION: The distribution of neuromuscular junctions is not random within the human thyroarytenoid muscle. Because neuromuscular junctions are most highly concentrated in a band within the mid belly of the muscle, botulinum toxin type A (Botox) injection in patients with spasmodic dysphonia should be targeted to this region.

Physiological response properties of neurons in the superior paraolivary nucleus of the rat.

The superior paraolivary nucleus (SPON) is a prominent nucleus of the superior olivary complex. In rats, this nucleus is composed of a morphologically homogeneous population of GABAergic neurons that receive excitatory input from the contralateral cochlear nucleus and inhibitory input from the ipsilateral medial nucleus of the trapezoid body. SPON neurons provide a dense projection to the ipsilateral inferior colliculus and are thereby capable of exerting profound modulatory influence on collicular neurons. Despite recent interest in the structural and connectional features of SPON, little is presently known concerning the physiological response properties of this cell group or its functional role in auditory processing. We utilized extracellular, in vivo recording methods to study responses of SPON neurons to broad band noise, pure tone, and amplitude-modulated pure tone stimuli. Localization of recording sites within the SPON provides evidence for a medial (high frequency) to lateral (low frequency) tonotopic representation of frequencies within the nucleus. Best frequencies of SPON neurons spanned the audible range of the rat and receptive fields were narrow with V-shaped regions near threshold. Nearly all SPON neurons responded at the offset of broad band noise and pure tone stimuli. The vast majority of SPON neurons displayed very low rates of spontaneous activity and only responded to stimuli presented to the contralateral ear, although a small population showed binaural facilitation. Most SPON neurons also generated spike activity that was synchronized to sinusoidally amplitude-modulated tones. Taken together, these data suggest that SPON neurons may serve to encode temporal features of complex sounds, such as those contained in species-specific vocalizations.

Protein expression levels of the Src activating protein AFAP are developmentally regulated in brain.

The Src family of nonreceptor tyrosine kinases plays an important role in modulating signals that affect growth cone extension, neuronal differentiation, and brain development. Recent reports indicate that the Src SH2/SH3 binding partner AFAP-110 has the capacity to modulate actin filament integrity as a cSrc activating protein and as an actin filament bundling protein. Both AFAP-110 and a brain specific isoform called AFAP-120 (collectively referred to as AFAP) exist at high levels in chick embryo brain. We sought to identify the localization of AFAP in mouse brain in order to identify its expression pattern and potential role as a cellular modulator of Src family kinase activity and actin filament integrity in the brain. In E16 mouse embryos, AFAP expression levels were very high and concentrated in the olfactory bulb, cortex, forebrain, cerebellum, and various peripheral sensory structures. In P3 mouse pups, overall expression was reduced compared to E16 embryos, and AFAP was found primarily in olfactory bulb, cortex, and cerebellum. AFAP expression levels were significantly reduced in adult mice, with high expression levels only detected in the olfactory bulb. Western blot analysis indicated that concentrated expression of AFAP correlates well with the AFAP-120 isoform, which appears to be a splice variant of AFAP-110. As the expression pattern of AFAP overlaps with the reported expression patterns of cSrc and Fyn, we hypothesize that AFAP is positioned to modulate signal transduction cascades that direct activation of these nonreceptor tyrosine kinases and concomitant cellular changes that occur in actin filaments during brain development.

Unbiased stereological estimates of neuron number in subcortical auditory nuclei of the rat.

The mammalian auditory system consists of a large number of cell groups, each containing its own complement of neuronal cell types. In recent years, much effort has been devoted to the quantitation of auditory neurons with common morphological, connectional, pharmacological or functional features. However, it is difficult to place these data into the proper quantitative perspective due to our lack of knowledge of the number of neurons contained within each auditory nucleus. To this end, we have employed unbiased stereological methods to estimate neuron number in the cochlear nuclei, superior olivary complex, lateral lemniscus, inferior colliculus and medial geniculate body. Additionally, we generated a three-dimensional model of the superior olivary complex. The utility of unbiased stereological estimates of auditory nuclei is discussed in the context of various experimental paradigms.

Histologic and immunologic observations of viral-induced rhinosinusitis in the mouse.

Viral upper respiratory infection is one of the most common diagnoses made in primary care offices. Although symptoms resolve within 1 week for many patients, a percentage develops rhinosinusitis, and many of these patients are treated with antibiotics. We have developed a model of viral rhinosinusitis using intranasal inoculation of reovirus into mice that were then killed on postinoculation days 2, 4, 7, 10, 14, or 21 and heads were embedded in paraffin for histological and immunohistochemical analyses. Reovirus-like immunoreactivity was noted in the septa and paranasal sinus mucosa in mice as early as day 2, with peak intensity seen on day 4, and scant staining seen on day 7. Complete absence of viral staining was seen by day 10, which corresponded with increased intracellular adhesion molecule 1 immunostaining in the nose. By day 10, a large mucosal influx of B cells was observed, with a moderate influx of macrophages and smaller influx of T cells. By day 14, there was a peak in the number of B cells with a corresponding, but less pronounced peak in T cells, while macrophages began to decline at this point. By day 21, the panel of immune markers returned to near normal levels. The results of this study suggest that the immune system continues to produce a response as long as 2 weeks after clearance of viral antigens. One proposed mechanism for this phenomenon is that local factors such as cytokines are released continually after infection, even in the absence of persistent viruses or bacteria.

Fos-Like Immunoreactivity Induced by Seizure in Mice Is Specifically Associated With Euchromatin in Neurons.

Administration of convulsant drugs causes the rapid induction of c-fos in identified neurons within the mouse central nervous system (Morgan et al., 1987). In particular, Fos-like immunoreactivity is evident in nuclei of granule cells of the hippocampal dentate gyrus within 30 minutes of the onset of seizure. By immunoelectron microscopy, Fos antibody binding was exclusively localized to dispersed chromatin (euchromatin) of several types of projection neurons and local circuit neurons in various brain regions and especially in the dentate gyrus, 210 minutes after a single injection of Metrazol. Fos-like immunoreactivity was not detectable in the nucleolus, nor in the characteristic peripheral and nucleolus-associated heterochromatin of hippocampal granule cells. No immunostaining was observed in nuclei of glial, ependymal or endothelial cells, and no cytoplasmic reactivity was seen in any cell type. These findings support a role for Fos in stimulus-response coupling at the level of transcriptional regulation in neurons.