Morphology and Dendrite-Specific Synaptic Properties of Midbrain Neurons Shape Multimodal Integration.

Multimodal integration facilitates object recognition and response to sensory cues. This depends on spatiotemporal coincidence of sensory information, recruitment of NMDA-type glutamate receptors and inhibitory feedback. Shepherd's crook neurons (SCNs) in the avian optic tectum (TeO) are an ideal model for studying cellular mechanism of multimodal integration. They receive different sensory modalities through spatially segregated dendrites, are important for stimulus selection and have an axon-carrying dendrite (AcD). We performed whole-cell patch-clamp experiments in chicken midbrain slices of both sexes. We emulated visual and auditory input in vitro by stimulating presynaptic afferents electrically. Simultaneous stimulation enhanced responses inversely depending on stimulation amplitude demonstrating the principle of inverse effectiveness. Contribution of NMDA-type glutamate receptors prolonged postsynaptic events for visual inputs only, causing a strong modality-specific difference in synaptic efficacy. We designed a multicompartment model to study the effect of morphological and physiological parameters on multimodal integration by varying the distance between soma and axonal origin and the amount of NMDA receptor (NMDAR) contribution. These parameters changed the preference of the model for one input channel and adjusted the range of input rates at which multimodal enhancement occurred on naturalistic stimulation. Thus, the unique morphology and synaptic features of SCNs shape the integration of input at different dendrites and generates an enhanced multimodal response.SIGNIFICANCE STATEMENT Multimodal integration improves perception and responses to objects. The underlying cellular mechanism depends on a balance between excitation and inhibition, and NMDA-type glutamate receptors that are involved in the multiplicative nature of enhancement following the principle of inverse effectiveness. Based on a detailed analysis of an identified multimodal cell type in the vertebrate midbrain, we studied the influence of cellular morphology and unimodal synaptic properties on multimodal integration. We can show that the combination of cellular morphology and modality-specific synaptic properties including NMDA receptor (NMDAR) contribution is optimal for nonlinear, multimodal enhancement and determines the dynamic response range of the integrating neuron. Our findings mechanistically explain how synaptic properties and cellular morphology of a midbrain neuron contribute to multimodal enhancement.

Profiling of the human monocytic cell secretome by quantitative label-free mass spectrometry identifies stimulus-specific cytokines and proinflammatory proteins.

The immune response to pathogens or injury relies on the concerted release of cytokines and proteins with biological activity important for host protection, host defense, and wound healing. Consequently, the secretome of immune cells provides a promising resource for discovery of specific molecular markers and targets for pharmacological intervention. Here, we employ label-free MS for unbiased, quantitative profiling of the human monocytic cell secretome under different proinflammatory stimuli. The quantitative secretome profiles reveal the highly stimulus-dependent cellular response and differential, specific secretion of more than 200 proteins, including important proinflammatory proteins and cytokines.

Altered hypothalamus-pituitary-adrenal axis function: A relevant factor in the comorbidity of atopic eczema and attention deficit/hyperactivity disorder?

Epidemiological data show a significant association between childhood atopic eczema (AE) and an increased risk to develop attention deficit/hyperactivity disorder (ADHD). However, the underlying mechanisms of the comorbidity of AE and ADHD are mostly unknown. We investigated whether alterations of hypothalamus-pituitary-adrenal (HPA) axis function represent a shared feature of AE and ADHD potentiating AE-ADHD comorbidity. Children aged 6-12 years with AE, ADHD, or comorbid AE + ADHD and healthy control (HC) children were examined cross-sectionally (N = 145). To evaluate HPA axis function, salivary cortisol in response to psychosocial stress (Trier Social Stress Test for Children, TSST-C), after awakening (cortisol awakening response, CAR), and throughout the day (short diurnal profile) and hair cortisol capturing long-term HPA axis activity were assessed. Quantile regression analyses showed an attenuated cortisol response (% maximum change) to the TSST-C in children with ADHD compared to HC. A diminished cortisol response to acute stress was also observed in the comorbid AE + ADHD group, in which the reduction was numerically even more pronounced. Contrary to our previous findings, no alteration of the cortisol response to the TSST-C was observed in children with AE. However, in children with AE, increased ADHD-like behavior (i.e., inattention, impulsivity, and overall ADHD symptom severity) was associated with a reduced HPA axis response to acute stress. No such associations were observed in children without AE. Groups did not differ in CAR, short diurnal profile, and hair cortisol. These findings underscore the potential relevance of HPA axis function in the pathophysiology of AE and ADHD with emphasis on stress reactivity. Additional studies are required to further explore the separate and joint role of the HPA axis in the pathophysiology of AE and ADHD.

A candidate pathway for a visual instructional signal to the barn owl's auditory system.

Many organisms use multimodal maps to generate coherent neuronal representations that allow adequate responses to stimuli that excite several sensory modalities. During ontogeny of these maps, one modality typically acts as the dominant system the other modalities are aligned to. A well studied model for the alignment of sensory maps is the calibration of the auditory space map by the visual system in the optic tectum of the barn owl. However, a projection from the optic tectum to the site of plasticity in the auditory pathway that could deliver an instructive signal has not been found so far. We have analyzed the development of the connectivity between the bimodal (visual and auditory) map of space in the barn owl's optic tectum and the auditory space map in the inferior colliculus with tracing methods and intracellular fills. Neurons in the tectal stratum griseum centrale were found to be suited to deliver an alignment signal from the visual midbrain to the auditory pathway. These neurons are presumably part of the efferent tectal projection pathway that mediates head saccades. The implications of a sensory alignment signal possibly being delivered by a (pre)motor command pathway are discussed.

Chattering and differential signal processing in identified motion-sensitive neurons of parallel visual pathways in the chick tectum.

At least three identified cell types in the stratum griseum centrale (SGC) of the chick optic tectum mediate separate pathways from the retina to different subdivisions of the thalamic nucleus rotundus. Two of these, SGC type I and type II, constitute the major direct inputs to rotundal subdivisions that process various aspects of visual information, e.g., motion and luminance changes. Here, we examined the responses of these cell types to somatic current injection and synaptic input. We used a brain slice preparation of the chick tectum and applied whole-cell patch recordings, restricted electrical stimulation of dendritic endings, and subsequent labeling with biocytin. Type I neurons responded with regular sequences of bursts ("chattering") to depolarizing current injection. Electrical stimulation of retinal afferents evoked a sharp-onset EPSP/burst response that was blocked with CNQX. The sharp-onset EPSP/burst response to synaptic stimulation persisted when the soma was hyperpolarized, thus suggesting the presence of dendritic spike generation. In contrast, the type II neurons responded to depolarizing current injection solely with an irregular sequence of individual spikes. Electrical stimulation of retinal afferents led to slow and long-lasting EPSPs that gave rise to one or several action potentials. In conclusion, the morphological distinct SGC type I and II neurons also have different response properties to retinal inputs. This difference is likely to have functional significance for the differential processing of visual information in the separate pathways from the retina to different subdivisions of the thalamic nucleus rotundus.

Bottlebrush dendritic endings and large dendritic fields: motion-detecting neurons in the mammalian tectum.

The widefield vertical neurons of the lower stratum griseum superficiale (SGS3) and upper stratum opticum (SO) of the superior colliculus provide an extrageniculate route for visual information to reach the pulvinar. Previous physiological studies indicate that SGS3/SO neurons have large receptive fields and respond to small moving stimuli. We sought to better characterize the dendritic morphology of SGS3/SO neurons with intracellular filling in slice preparations of the ground squirrel superior colliculus. We found that dendrites of widefield vertical cells end in monostratified arrays of spiny terminal specializations called "bottlebrush" dendritic endings. Two major subtypes of neurons are described. Type I neurons have somata restricted to the SGS3 and bottlebrush endings in the most superficial sublayer of the SGS. Type II neurons are found at the base of the SGS and in the upper SO, and have bottlebrush endings arrayed within the middle sublayers of the SGS. Bottlebrush endings may sample and integrate laminated afferents to the superior colliculus, and cellular subtypes may underlie multiple information streams within the tectopulvinar pathway. A similar dendritic morphology and projection pattern can be found in cells of the avian optic tectum that project upon the nucleus rotundus, a thalamic nucleus homologous to the mammalian caudal/inferior pulvinar. Because motion processing is a dominant feature of the avian tectorotundal pathway, the current results suggest that both dendritic morphology and motion processing are conserved features of widefield vertical cells in the tectopulvinar pathway of vertebrates.

Bottlebrush dendritic endings and large dendritic fields: motion-detecting neurons in the tectofugal pathway.

In avian and mammalian brains, visual information from the retina is conveyed to the telencephalon via two separate pathways: the thalamofugal and the tectofugal pathways. Recently, Karten et al. ([1997] J. Comp. Neurol. 387:449-465) examined a portion of the tectofugal pathway, the projection from the optic tectum to the nucleus rotundus thalami, in pigeons. They defined two distinct subpopulations of tectal neurons projecting from the stratum griseum centrale (SGC; tectal layer 13) to specific divisions of the rotundus. The goal of this study in chick was to verify the existence of the type I and type II SGC neurons, as defined by Karten et al., and then examine in greater detail the connectivity and morphology of these SGC neurons. Furthermore, our results suggest how the unique morphological characteristics of SGC neurons contribute to the large receptive fields (20-50 degrees) found in physiological recordings and the SGC neuronal response to extremely small (ca. 0.05 degree), fast-moving (100 degrees/second) stimuli. By injecting retrograde tracer into various divisions of the chick rotundus, we verified that, indeed, the chick did possess type I and type II SGC neurons, as well as a "new" type of SGC neuron, type III, that is not found in the pigeon. We then used intracellular cell-filling techniques to define further these three types of SGC neurons. Our examination revealed the following: Type I SGC neurons had large, circular dendritic fields (average diameter, 1,725 microns) composed of smooth dendrites and ending in spine-rich, bottlebrush endings located in retinorecipient tectal layer 5b; type II SGC neurons had elliptical dendritic fields (average 1,447 microns) and dendritic endings located never more superficially than tectal layer 8; and type III SGC neurons had large dendritic fields (average 1,800 microns) of unknown shape and bottlebrush dendritic endings located in retinorecipient tectal layer 4. We suggest that the neuronal features of the SGC neurons (i.e., bottlebrush dendritic endings and large dendritic fields) are key morphological characteristics for the detection of motion within the tectofugal pathway. Furthermore, because neurons with similar morphology have also been found in the tecta of both mammals and reptiles, we suggest that these neuronal features are fundamental components of a phylogenetically conserved system used for the "extrastriate" detection of motion in vertebrates.

Selective synaptic cadherin expression by traced neurons of the chicken visual system.

The stable and specific locking-in of pre- and postsynaptic membranes in synaptogenesis may be mediated by integral membrane proteins, such as members of the cadherin family. Cadherins are ideal candidate molecules for mediating synaptic specificity because they are differentially expressed in functionally connected brain structures. We studied the expression of four classic cadherins (R-cadherin, N-cadherin, cadherin-6B and cadherin-7) at the synaptic level on the somata and the proximal neurites of identified neuron populations that were traced selectively in the developing chicken visual system. Three major findings were observed. (1) Synapses on somata of shepherd's crook cells of the optic tectum are associated preferentially with one cadherin subtype. (2) In an isthmic nucleus that contains a mixed population of cells expressing different cadherins, somatic synapses tend to express the same cadherin subtype as the rest of the cell. (3) In the oculomotor complex, two cadherin subtypes are expressed only by synapses on the axon hillock. However, another neuron type that projects from the tectum to the isthmic nucleus does not show such selective synaptic cadherin staining. Our findings support the idea that a cadherin-based adhesive mechanism can mediate synaptic specificity.

The use of in vitro preparations of the isolated amphibian central nervous system in neuroanatomy and electrophysiology.

In the present study an isolated preparation of the complete anuran central nervous system (CNS) is described which can be kept alive for several days and allows tracing, immunohistochemical and electrophysiological studies. A simple perfusion chamber is being used in which the isolated CNS preparation is superfused with oxygenated Ringer. The use of an isolated CNS has many advantages including: (1) virtually all areas are easily accessible at the same time without having the problem of blood vessels that hinder access; (2) large lesions and massive tracer applications are possible without survival problems of the animal, and tracers will not be translocated by blood circulation; (3) since pulsations caused by the pressure changes of blood circulation do not occur, intracellular recordings are comparatively easy and stable; and (4) this approach offers the possibility of working on the same brain for several days by storing the preparation in a refrigerator overnight at low temperatures, thus allowing extensive utilization of a single preparation and reduction in the number of experimental animals required. Some applications to the anuran auditory system illustrate that the isolated anuran CNS is well-suited for a variety of neuroanatomical and physiological techniques.

Development of retino-recipient projection neurons in the optic tectum of the chicken.

The dendritic development of a well-characterized retino-recipient neuronal type in the chicken optic tectum has been traced with intracellular labeling. Normal dendritic development can be divided into three phases: extension, differentiation and pruning. During the first phase, cells extend their dendrites, generate large dendritic fields and position their distal endings in a certain retino-recipient tectal layer. In the second phase, these dendritic endings arborize into characteristic bottlebrush-like structures, while the overall morphology of the neurons remains unaltered. After hatching, the number and width of the bottlebrush endings are reduced. The findings are discussed with respect to the innervation of the optic tectum by retinal afferents and possible guidance mechanisms for synapse formation in this system.

Pretecto-tectal interactions: effects of lesioning and stimulating the pretectum on field potentials in the optic tectum of salamanders in vitro.

Interactions between pretectum and optic tectum of salamanders were analyzed by recording evoked potentials (EPs) in the optic tectum in vitro in response to stimulation of the contralateral optic nerve. Neither lesioning of the pretectum nor ablation of the medulla oblongata including the nucleus isthmi altered the shape of tectal EPs, suggesting that the tectal EP in this preparation reflects activation of tectal circuitry by retinal afferents without a major contribution from non-retinal afferents. To analyze the effect of stimulation of the pretectum on the tectal EP, we stimulated the pretectal area pharmacologically. Amplitudes of tectal EPs decreased rapidly after stimulation of the pretectum and recovered within minutes (glutamate) or hours (kainic acid). The pretectal influence on the tectal EP might act presynaptically on retinal afferents or by modulating the response of inhibitory interneurons.

Morphology and axonal projection patterns of auditory neurons in the midbrain of the painted frog, Discoglossus pictus.

Acoustic signals are extensively used for guiding various behaviors in frogs such as vocalization and phonotaxis. While numerous studies have investigated the anatomy and physiology of the auditory system, our knowledge of intrinsic properties and connectivity of individual auditory neurons remains poor. Moreover, the neural basis of audiomotor integration still has to be elucidated. We determined basic response patterns, dendritic arborization and axonal projection patterns of auditory midbrain units with intracellular recording and staining techniques in an isolated brain preparation. The subnuclei of the torus semicircularis subserve different tasks. The principal nucleus, the main target of the ascending auditory input, has mostly intrinsic neurons, i.e., their dendrites and axons are restricted to the torus itself. In contrast, neurons of the magnocellular and the laminar nucleus project to various auditory and non-auditory processing centers. The projection targets include thalamus, tegmentum, periaqueductal gray, medulla oblongata, and in the case of laminar neurons--the spinal cord. Additionally, tegmental cells receive direct auditory input and project to various targets, including the spinal cord. Our data imply that both auditory and premotor functions are implemented in individual toral and tegmental neurons. Their axons constitute parallel descending pathways to several effector systems and might be part of the neural substrate for differential audiomotor integration.

Audio-motor interface in anurans.

Like males of many anuran species, fire-bellied toads (Bombina orientalis) call antiphonally, which demonstrates an auditory input into the call-generating network. Males produce their calls by an inspiratory airstream, which is generated exclusively by contraction of the muscles of the buccal cavity. The painted frog (Discoglossus pictus) possesses a combined inspiratory and expiratory call mechanism, and also uses only buccal muscles. These muscles are controlled by branchial motoneurons, which receive vocal premotor input mainly from the pretrigeminal nucleus. The interconnections between the auditory pathway and the vocal pathway were examined by neuroanatomical tracing and intracellular recording. Mesencephalic auditory nuclei, laminar and magnocellular nucleus of the torus semicircularis, and tegmental nuclei constitute strong descending efferents, which, in turn, form collaterals that terminate in vocal premotor nuclei. These findings imply fast audio-vocal interfacing, which is a prerequisite for the control of antiphonal calling.

Active sensing--closing multiple loops.

In this contribution, it is argued that the prevailing view of the sensory system as an intricate, but passive processor of external information does not capture the full complexity of brain performance. Instead, we try to reinforce a notion that sees the brain as a system embedded within the environment and actively exploring it. We will attempt to emphasize the bidirectional interaction between brain and environment at all levels of processing and at different scales of the system description. This modified approach to the understanding of the brain has profound consequences for experimental investigations, starting with the experimental design and extending into data analysis and interpretation.

Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.

BACKGROUND: Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience. NEW METHOD: We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer. RESULTS: In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution. COMPARISON WITH EXISTING METHODS: Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals. CONCLUSIONS: With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.

Development of the delay lines in the nucleus laminaris of the chicken embryo revealed by optical imaging.

One strategy in localizing a sound source in the azimuthal plane is the comparison of arrival times of sound stimuli at the two ears. The processing of interaural time differences (ITDs) in the auditory brainstem was suggested by the Jeffress model in 1948. In chicks, binaural neurons in the nucleus laminaris (NL) receive input from both ipsilateral and contralateral nucleus magnocellularis (NM) neurons, with the axons of the latter acting as delay lines. A given neuron in the NL responds maximally to coinciding input from both NM neurons. To achieve maximum resolution of sound localization in the NL, the conduction velocity along these delay lines must be precisely tuned. Here, we examined the development of this velocity between embryonic days (E)12 and E18. Our optical imaging approach visualizes the contralateral delay lines along almost the complete NL of the chicken embryo. Optical imaging with the voltage-sensitive dye RH 795 showed no significant differences in the velocity between E12 and E15, but a significant increase from E15 to E18, at both 21 degrees C and 35 degrees C. Surprisingly, at 21 degrees C the conduction velocity in the dorso-lateral part of the NL was significantly higher compared to the situation in the ventro-medial part. The observed development in contralateral conduction velocity may be due to a developmental increase in myelination of the NM axons. Indeed, antibody staining against myelin-associated glycoprotein (alpha-MAG) showed no myelination of the NM axon branches within the NL at E12 and E15. On the other hand, a clear alpha-MAG immunoreactivity occurred at E18. Our results therefore describe the developmental physiological properties of the delay line in the chicken embryo.

Chemoarchitecture of the anuran auditory midbrain.

The anuran torus semicircularis consists of several subnuclei that are part of the ascending auditory pathway as well as audiomotor interface structures. Additionally, recent anatomical studies suggest that the midbrain tegmentum is an integral part of the audiomotor network. To describe the chemoarchitecture of these nuclei, taking into account the toral subdivisions, we investigated the distribution of serotonin, leucine-enkephalin, substance P, tyrosine-hydroxylase, dopamine D2-receptor, parvalbumin, aspartate, GABA, and estrogen-binding protein-immunoreactivity in the midbrain of Bombina orientalis, Discoglossus pictus and Xenopus laevis. In the torus semicircularis, the highest density of immunoreactive fibers and terminals for all transmitters was found in the laminar nucleus. Parvalbumin-like immunoreactivity was highest in the principal nucleus, and D2-receptor-like immunoreactivity was uniformly distributed throughout the torus. In the tegmentum, axons and/or dendrites were stained with all antibodies except estrogen-binding protein. Additionally, heavily stained enkephalin and substance P-immunopositive fiber plexus were found in the lateral and dorsal tegmentum. The immunostainings revealed no qualitative differences between the three species. Immunopositive cell bodies were labeled in several brain areas, the connectivity of which with torus and tegmentum is discussed on the background of functional questions. The putative neuromodulatory innervation of both the laminar nucleus of the torus semicircularis and the tegmentum may be the anatomical basis for the influence of the animal's endogenous state on the behavioral reaction to sensory stimuli. These data corroborate earlier anatomical and physiological findings that the neurons of these nuclei are key elements in the audio-motor interface.

Connectivity of the salamander pretectum: an in-vitro (whole-brain) intracellular tracing study.

The amphibian optic tectum and pretectum have been analyzed in detail anatomically and physiologically, and a specific model for tecto-pretectal interaction in the context of the visual guidance of behavior has been proposed. However, anatomical evidence for this model, particularly the precise pattern of pretectotectal connectivity, is lacking. Therefore, we stained pretectal neurons intracellularly in an in-vitro preparation of the salamanders Plethodon jordani and Hydromantes genei. Our results demonstrate that the projections of neurons of the nucleus praetectalis profundus are divergent and widespread. Individual neurons may project divergently to telencephalic (ipsilateral amygdala and striatum), diencephalic (ipsi-and contralateral thalamus, contralateral pretectum), and mesencephalic (ipsi- and contralateral tectum and tegmentum) centers, and to the ipsi- and contralateral medulla oblongata and rostral spinal cord. The projection of pretectal cells to the optic tectum is bilateral; axonal structures do not show discernible patterns and are present in all layers of the superficial white matter. A classification of pretectal neurons on the basis of axonal termination pattern or dendritic arborization has not been possible. Our results do not support the hypothesis that a distinct class of pretectal neurons projects to a particular subset of tectal cells. Rather, the pretectum appears to influence the tectum indirectly, acting either on retinal afferents or modulating inhibitory interneurons.