Comparative neurotoxicity of dietary methylmercury and waterborne inorganic mercury in fish: Evidence of optic tectum vulnerability through morphometric and histopathological assessments.

This work investigated the effects of inorganic mercury (iHg) and methylmercury (MeHg) on the fish optic tectum morphology, viz. in relation to: (i) vulnerability of specific optic tectum layers; (ii) preferential targeting of Hg forms to neurons or glial cells; (iii) comparative toxicity of iHg and MeHg in this brain area that is in the maintenance of several fish behaviors. Two experiments exposing juvenile white seabream (Diplodus sargus) to waterborne iHg [HgCl2 (2 µg L-1)] and dietary MeHg (8.7 µg g-1) were performed, comprising both exposure (7 and 14 days; E7 and E14, respectively) and post-exposure (28 days; PE28) periods. Morphometric assessments were performed using stereological methods where the layers of the optic tectum were outlined, while its area and the number of neurons and glial cells were estimated. A histopathological assessment was also performed per section and per layer of optic tectum. iHg exposure did not trigger the loss of neurons during the exposure periods, while a decrease of glial cells was detected in a single layer of the optic tectum at E14. Differently, upon MeHg exposure, a decrease on the number of neurons and glial cells was found in several layers of optic tectum. In the post-exposure, both Hg forms triggered the loss of neurons, while only MeHg exposure led to a decrease on the number of glia cells. The histopathological assessment pointed out a higher toxicity of MeHg in the optic tectum layers, particularly in the post-exposure period, while no significant alterations were found in fish exposed to iHg. Hg forms targeted preferentially neurons. iHg and MeHg are relevant neurotoxicants to fish, with MeHg exposure leading to a higher toxicity than iHg in the optic tectum. After 28 days of post-exposure, iHg and MeHg neurotoxicity remained prominent, suggesting long-term effects of these toxicants.

Qualitative investigation of µ- and κ-opioid receptor distribution in the brains of budgerigars (Melopsittacus undulatus).

OBJECTIVE: To perform a qualitative analysis of the distribution of µ- and κ-opioid receptor mRNA in the forebrain and midbrain of budgerigars (Melopsittacus undulatus). SAMPLE: 8 brains of male budgerigars. PROCEDURES: Custom-made RNA hybridization probes (RNAscope; Advanced Cell Diagnostics Inc) were used for fluorescent in situ hybridization (FISH) assays performed on selected fresh frozen prepared sections of brain tissue to identify µ- and κ-opioid receptor mRNA. RESULTS: There was κ-opioid receptor mRNA present in the nucleus dorsomedialis posterior thalami, lateral striatum, mesopallium, tractus corticohabenularis et corticoseptalis, griseum et fibrosum, stratum griseum centrale, medial striatum, and area parahippocampalis. There was µ-opioid receptor mRNA present in the stratum griseum centrale, stratum opticum, dorsomedialis posterior thalami, area parahippocampalis, medial striatum, and nidopallium intermedium. CLINICAL RELEVANCE: Consistent with previous studies in pigeons and domestic chicks, κ-opioid receptors were more abundant than µ-opioid receptors in the samples of the present study. The results of this study may also help explain the hyperexcitability or lack of response that can occur with administration of pure µ-opioid receptor agonists, but not κ-opioid receptor agonists. This study was not quantitative, so further research should endeavor to compare the various regions of the brain using FISH technology.

Effects of Light Isoflurane Anesthesia on Organization of Direction and Orientation Selectivity in the Superficial Layer of the Mouse Superior Colliculus.

The superior colliculus (SC) is the midbrain center for integrating visual and multimodal sensory information. Neurons in the SC exhibit direction and orientation selectivity. Recent studies reported that neurons with similar preferences formed clusters in the mouse SC (Ahmadlou and Heimel, 2015; Feinberg and Meister, 2015; de Malmazet et al., 2018; Li et al., 2020). However, it remains controversial as to how these clusters are organized within the SC (Inayat et al., 2015; Chen et al., 2021). Here, we found that different brain states (i.e., awake or anesthetized with isoflurane) changed the selectivity of individual SC neurons and organizations of the neuronal population in both male and female mice. Using two-photon Ca2+ imaging, we examined both individual neuronal responses and the spatial patterns of their population responses. Under isoflurane anesthesia, orientation selectivity increased and a larger number of orientation-selective cells were observed when compared with the awake condition, whereas the proportions of direction-selective cells were similar in both conditions. Furthermore, direction- and orientation-selective cells located at closer positions showed more similar preferences, and cluster-like spatial patterns were enhanced. Inhibitory responses of direction-selective neurons were also reduced under isoflurane anesthesia. Thus, the changes in the spatial organization of response patterns were considered to be because of changes in the balance of excitation and inhibition, with excitation dominance, in the local circuits. These results provide new insights into the possibility that the functional organization of feature selectivity in the brain is affected by brain state.SIGNIFICANCE STATEMENT Recent large-scale recording studies are changing our view of visual maps in the superior colliculus (SC), including findings of cluster-like localizations of direction- and orientation-selective neurons. However, results from several laboratories are conflicting regarding the presence of cluster-like organization. Here, we demonstrated that light isoflurane anesthesia affected the direction- and orientation-tuning properties in the mouse superficial SC and that their cluster-like localization pattern was enhanced by the anesthesia. Furthermore, the effect of anesthesia on direction selectivity appeared to be different in the excitatory and inhibitory populations in the SC. Our results suggest that the functional organization of direction and orientation selectivity might be regulated by the excitation-inhibition balance that depends on the brain state.

Anatomy and function of retinorecipient arborization fields in zebrafish.

In 1994, Burrill and Easter described the retinal projections in embryonic and larval zebrafish, introducing the term "arborization fields" (AFs) for the retinorecipient areas. AFs were numbered from 1 to 10 according to their positions along the optic tract. With the exception of AF10 (neuropil of the optic tectum), annotations of AFs remained tentative. Here we offer an update on the likely identities and functions of zebrafish AFs after successfully matching classical neuroanatomy to the digital Max Planck Zebrafish Brain Atlas. In our system, individual AFs are neuropil areas associated with the following nuclei: AF1 with the suprachiasmatic nucleus; AF2 with the posterior parvocellular preoptic nucleus; AF3 and AF4 with the ventrolateral thalamic nucleus; AF4 with the anterior and intermediate thalamic nuclei; AF5 with the dorsal accessory optic nucleus; AF7 with the parvocellular superficial pretectal nucleus; AF8 with the central pretectal nucleus; and AF9d and AF9v with the dorsal and ventral periventricular pretectal nuclei. AF6 is probably part of the accessory optic system. Imaging, ablation, and activation experiments showed contributions of AF5 and potentially AF6 to optokinetic and optomotor reflexes, AF4 to phototaxis, and AF7 to prey detection. AF6, AF8 and AF9v respond to dimming, and AF4 and AF9d to brightening. While few annotations remain tentative, it is apparent that the larval zebrafish visual system is anatomically and functionally continuous with its adult successor and fits the general cyprinid pattern. This study illustrates the synergy created by merging classical neuroanatomy with a cellular-resolution digital brain atlas resource and functional imaging in larval zebrafish.

Functional, molecular and morphological heterogeneity of superficial interneurons in the larval zebrafish tectum.

The superficial interneurons, SINs, of the zebrafish tectum, have been implicated in a range of visual functions, including size discrimination, directional selectivity, and looming-evoked escape. This raises the question if SIN subpopulations, despite their morphological similarities and shared anatomical position in the retinotectal processing stream, carry out diverse, task-specific functions in visual processing, or if they have simple tuning properties in common. Here we have further characterized the SINs through functional imaging, electrophysiological recordings, and neurotransmitter typing in two transgenic lines, the widely used Gal4s1156t and the recently reported LCRRH2-RH2-2:GFP. We found that about a third of the SINs strongly responded to changes in whole-field light levels, with a strong preference for OFF over ON stimuli. Interestingly, individual SINs were selectively tuned to a diverse range of narrow luminance decrements. Overall responses to whole-field luminance steps did not vary with the position of the SIN cell body along the depth of the tectal neuropil or with the orientation of its neurites. We ruled out the possibility that intrinsic photosensitivity of Gal4s1156t+ SINs contribute to the measured visual responses. We found that, while most SINs express GABAergic markers, a substantial minority express an excitatory neuronal marker, the vesicular glutamate transporter, expanding the possible roles of SIN function in the tectal circuitry. In conclusion, SINs represent a molecularly, morphologically, and functionally heterogeneous class of interneurons, with subpopulations that detect a range of specific visual features, to which we have now added narrow luminance decrements.

Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy.

Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 µm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

Anatomy and Connectivity of the Torus Longitudinalis of the Adult Zebrafish.

This study describes the cytoarchitecture of the torus longitudinalis (TL) in adult zebrafish by using light and electron microscopy, as well as its main connections as revealed by DiI tract tracing. In addition, by using high resolution confocal imaging followed by digital tracing, we describe the morphology of tectal pyramidal cells (type I cells) that are GFP positive in the transgenic line Tg(1.4dlx5a-dlx6a:GFP)ot1. The TL consists of numerous small and medium-sized neurons located in a longitudinal eminence attached to the medial optic tectum. A small proportion of these neurons are GABAergic. The neuropil shows three types of synaptic terminals and numerous dendrites. Tracing experiments revealed that the main efference of the TL is formed of parallel-like fibers that course within the marginal layer of the optic tectum. A toral projection to the thalamic nucleus rostrolateralis is also observed. Afferents to the TL come from visual and cerebellum-related nuclei in the pretectum, namely the central, intercalated and the paracommissural pretectal nuclei, as well as from the subvalvular nucleus in the isthmus. Additional afferents to the TL may come from the cerebellum but their origins could not be confirmed. The tectal afferent projection to the TL originates from cells similar to the type X cells described in other cyprinids. Tectal pyramidal neurons show round or piriform cell bodies, with spiny apical dendritic trees in the marginal layer. This anatomical study provides a basis for future functional and developmental studies focused on this cerebellum-like circuit in zebrafish.

Effects of Locomotion on Visual Responses in the Mouse Superior Colliculus.

Visual responses are extensively shaped by internal factors. This effect is drastic in the primary visual cortex (V1), where locomotion profoundly increases visually-evoked responses. Here we investigate whether a similar effect exists in another major visual structure, the superior colliculus (SC). By performing two-photon calcium imaging of head-fixed male and female mice running on a treadmill, we find that only a minority of neurons in the most superficial lamina of the SC display significant changes during locomotion. This modulation includes both increase and decrease in response amplitude and is similar between excitatory and inhibitory neurons. The overall change in the SC is small, whereas V1 responses almost double during locomotion. Additionally, SC neurons display lower response variability and less spontaneous activity than V1 neurons. Together, these experiments indicate that locomotion-dependent modulation is not a widespread phenomenon in the early visual system and that the SC and V1 use different strategies to encode visual information.SIGNIFICANCE STATEMENT Visual information captured by the retina is processed in parallel through two major pathways, one reaching the primary visual cortex through the thalamus, and the other projecting to the superior colliculus. The two pathways then merge in the higher areas of the visual cortex. Recent studies have shown that behavioral state such as locomotion is an essential component of vision and can strongly affect visual responses in the thalamocortical pathway. Here we demonstrate that neurons in the mouse superior colliculus and primary visual cortex display striking differences in their modulation by locomotion, as well as in response variability and spontaneous activity. Our results reveal an important "division of labor" in visual processing between these two evolutionarily distinct structures.

A Descending Circuit Derived From the Superior Colliculus Modulates Vibrissal Movements.

The superior colliculus (SC) is an essential structure for the control of eye movements. In rodents, the SC is also considered to play an important role in whisking behavior, in which animals actively move their vibrissae (mechanosensors) to gather tactile information about the space around them during exploration. We investigated how the SC contributes to vibrissal movement control. We found that when the SC was unilaterally lesioned, the resting position of the vibrissae shifted backward on the side contralateral to the lesion. The unilateral SC lesion also induced an increase in the whisking amplitude on the contralateral side. To explore the anatomical basis for SC involvement in vibrissal movement control, we then quantitatively evaluated axonal projections from the SC to the brainstem using neuronal labeling with a virus vector. Neurons of the SC mainly sent axons to the contralateral side in the lower brainstem. We found that the facial nucleus received input directly from the SC, and that the descending projections from the SC also reached the intermediate reticular formation and pre-Bötzinger complex, which are both considered to contain neural oscillators generating rhythmic movements of the vibrissae. Together, these results indicate the existence of a neural circuit in which the SC modulates vibrissal movements mainly on the contralateral side, via direct connections to motoneurons, and via indirect connections including the central pattern generators.

Colocalization of Tectal Inputs With Amygdala-Projecting Neurons in the Macaque Pulvinar.

Neuropsychological and neuroimaging studies have suggested the presence of a fast, subcortical route for the processing of emotionally-salient visual information in the primate brain. This putative pathway consists of the superior colliculus (SC), pulvinar and amygdala. While the presence of such a pathway has been confirmed in sub-primate species, it has yet to be documented in the primate brain using conventional anatomical methods. We injected retrograde tracers into the amygdala and anterograde tracers into the colliculus, and examined regions of colocalization of these signals within the pulvinar of the macaque. Anterograde tracers injected into the SC labeled axonal projections within the pulvinar, primarily within the oral, lateral and medial subdivisions. These axonal projections from the colliculus colocalized with cell bodies within the pulvinar that were labeled by retrograde tracer injected into the lateral amygdala. This zone of overlap was most notable in the medial portions of the medial (PM), oral (PO) and inferior pulvinar (PI), and was often densely concentrated in the vicinity of the brachium of the SC. These data provide an anatomical basis for the previously suggested pathway mediating fast processing of emotionally salient information.

Causal Role of Neural Signals Transmitted From the Frontal Eye Field to the Superior Colliculus in Saccade Generation.

The frontal eye field (FEF) and superior colliculus (SC) are major and well-studied components of the oculomotor system. The FEF sends strong projections to the SC directly, and neurons in these brain regions transmit a variety of signals related to saccadic eye movements. Electrical microstimulation and pharmacological manipulation targeting the FEF or SC affect saccadic eye movements. These data suggest the causal contribution of each region to saccade generation. To understand how the brain generates behavior, however, it is critical not only to identify the structures and functions of individual regions, but also to elucidate how they interact with each other. In this review article, we first survey previous works that aimed at investigating whether and how the FEF and SC interact to regulate saccadic eye movements using electrophysiological and pharmacological techniques. These works have reported what signals FEF neurons transmit to the SC and what roles such signals play in regulating oculomotor behavior. We then highlight a recent attempt of our own that has applied an optogenetic approach to stimulate the neural pathway from the FEF to the SC in nonhuman primates. This study has shown that optogenetic stimulation of the FEF-SC pathway is sufficiently effective not only to modulate SC neuron activity, but also to evoke saccadic eye movements. Although the oculomotor system is a complex neural network composed of numbers of cortical and subcortical regions, the optogenetic approach will provide a powerful strategy for elucidating the role of each neural pathway constituting this network.

Luminance Changes Drive Directional Startle through a Thalamic Pathway.

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting.

Cocaine- and amphetamine-regulated transcript peptide and calcium binding proteins immunoreactivity in the superficial layers of the superior colliculus in the guinea pig: Implications for visual sensory processing.

The purpose of this study was to investigate the distribution and colocalization of cocaine- and amphetamine-regulated transcript peptide (CART) and three calcium-binding proteins (calbindin, calretinin and parvalbumin) in the superficial layers of the superior colliculus (SCs) in the guinea pig. The CART immunoreactivity was observed exclusively in the solitary fibers and neuropil, which formed various CART-ir tiers, that corresponded partially or entirely to anatomically defined layers of the SCs. The CART-ir structures exhibited a characteristic morphology with bundles of densely intermingled neuronal fibers and terminals. This pattern of CART immunoreactivity in the visually driven SCs strongly indicates that CART peptide as a putative neurotransmitter may play an important role in processing of visual information. Double-labeling immunofluorescence showed that CART did not colocalize with either calcium binding proteins (CaBPs). Immunolabeling for CaBPs revealed the presence of different neuronal populations, which were concentrated in variously pronounced tiers. Contrary to CART, the CaBPs immunoreactivity in perikarya was relatively high and CaBPs containing neurons displayed a variety of sizes and somatodendritic morphologies. Generally, CaBPs patterns in the SCs of the guinea pig differ, to some extent, from those of other rodents. These results prove the importance of studying the neurochemical cytoarchitecture of diverse mammals.

A GABAergic tecto-tegmento-tectal pathway in pigeons.

Previous studies have demonstrated that the optic tecta of the left and right brain halves reciprocally inhibit each other in birds. In mammals, the superior colliculus receives inhibitory γ-aminobutyric acid (GABA)ergic input from the basal ganglia via both the ipsilateral and the contralateral substantia nigra pars reticulata (SNr). This contralateral SNr projection is important in intertectal inhibition. Because the basal ganglia are evolutionarily conserved, the tectal projections of the SNr may show a similar pattern in birds. Therefore, the SNr could be a relay station in an indirect tecto-tectal pathway constituting the neuronal substrate for the tecto-tectal inhibition. To test this hypothesis, we performed bilateral anterograde and retrograde tectal tracing combined with GABA immunohistochemistry in pigeons. Suprisingly, the SNr has only ipsilateral projections to the optic tectum, and these are non-GABAergic. Inhibitory GABAergic input to the contralateral optic tectum arises instead from a nearby tegmental region that receives input from the ipsilateral optic tectum. Thus, a disynaptic pathway exists that possibly constitutes the anatomical substrate for the inhibitory tecto-tectal interaction. This pathway likely plays an important role in attentional switches between the laterally placed eyes of birds. J. Comp. Neurol. 524:2886-2913, 2016. © 2016 Wiley Periodicals, Inc.

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma.

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (µ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by µ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.

Subcortical connections of area V4 in the macaque.

Area V4 has numerous, topographically organized connections with multiple cortical areas, some of which are important for spatially organized visual processing, and others which seem important for spatial attention. Although the topographic organization of V4's connections with other cortical areas has been established, the detailed topography of its connections with subcortical areas is unclear. We therefore injected retrograde and anterograde tracers in different topographical regions of V4 in nine macaques to determine the organization of its subcortical connections. The injection sites included representations ranging from the fovea to far peripheral eccentricities in both the upper and lower visual fields. The topographically organized connections of V4 included bidirectional connections with four subdivisions of the pulvinar, two subdivisions of the claustrum, and the interlaminar portions of the lateral geniculate nucleus, and efferent projections to the superficial and intermediate layers of the superior colliculus, the thalamic reticular nucleus, and the caudate nucleus. All of these structures have a possible role in spatial attention. The nontopographic, or converging, connections included bidirectional connections with the lateral nucleus of the amygdala, afferent inputs from the dorsal raphe, median raphe, locus coeruleus, ventral tegmentum and nucleus basalis of Meynert, and efferent projections to the putamen. Any role of these structures in attention may be less spatially specific.

Functional imaging in the zebrafish retinotectal system using RGECO.

Genetically encoded calcium indicators (GECIs) allow repeated, non-invasive measurements of neural activity in defined populations of neurons, but until recently GECIs based on single fluorescent proteins have been limited to the green region of the color spectrum. Recent efforts in protein engineering have expanded the color palette of GECIs. One of these new GECIs, the red RGECO, is spectrally separate from the traditional GFP-based sensors such as GCaMP, and therefore opens the way for simultaneous, multicolor imaging of neural activity. While RGECO has been shown to report spontaneous calcium fluctuations in neurons, the precise relationship of RGECO signal to evoked-neural activity is not known. Measurements of neural activity using RGECO in vivo have also not been reported. Using dissociated hippocampal neurons we performed a systematic analysis of two forms of RGECO- a cytosolic form and a presynaptically localized form generated by fusion of RGECO to the presynaptic protein, synaptophysin (SyRGECO). We find that RGECO and GCaMP3 are comparable in terms of dynamic range, signal-to-noise ratios and kinetics but that RGECO is a more reliable reporter of single action potentials. In terms of performance SyGCaMP3 and SyRGECO are comparable, and both are more sensitive reporters of activity than the cytosolic form of each probe. Using the zebrafish retinotectal system we show that SyRGECO and RGECO are can report neural activity in vivo and that RGECO expression permits detailed structural analysis of neuronal arbors. We have exploited these attributes to provide a morphological and functional description of tectal cells selective for motion along the vertical axis. These results open up the possibility of using zebrafish to functionally image genetically defined pre- and postsynaptic circuit components, separable by color, which will be a powerful approach to studying neural interactions in the brain.

Projections of the superior colliculus to the pulvinar in prosimian galagos (Otolemur garnettii) and VGLUT2 staining of the visual pulvinar.

An understanding of the organization of the pulvinar complex in prosimian primates has been somewhat elusive due to the lack of clear architectonic divisions. In the current study we reveal features of the organization of the pulvinar complex in galagos by examining superior colliculus (SC) projections to this structure and comparing them with staining patterns of the vesicular glutamate transporter, VGLUT2. Cholera toxin subunit ß (CTB), Fluoro-ruby (FR), and wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were placed in topographically different locations within the SC. Our results showed multiple topographically organized patterns of projections from the SC to several divisions of the pulvinar complex. At least two topographically distributed projections were found within the lateral region of the pulvinar complex, and two less obvious topographical projection patterns were found within the caudomedial region, in zones that stain darkly for VGLUT2. The results, considered in relation to recent observations in tree shrews and squirrels, suggest that parts of the organizational scheme of the pulvinar complex in primates are present in rodents and other mammals.

Broad characterization of endogenous peptides in the tree shrew visual system.

Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.