Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum.

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.

A specialized reciprocal connectivity suggests a link between the mechanisms by which the superior colliculus and parabigeminal nucleus produce defensive behaviors in rodents.

The parabigeminal nucleus (PBG) is the mammalian homologue to the isthmic complex of other vertebrates. Optogenetic stimulation of the PBG induces freezing and escape in mice, a result thought to be caused by a PBG projection to the central nucleus of the amygdala. However, the isthmic complex, including the PBG, has been classically considered satellite nuclei of the Superior Colliculus (SC), which upon stimulation of its medial part also triggers fear and avoidance reactions. As the PBG-SC connectivity is not well characterized, we investigated whether the topology of the PBG projection to the SC could be related to the behavioral consequences of PBG stimulation. To that end, we performed immunohistochemistry, in situ hybridization and neural tracer injections in the SC and PBG in a diurnal rodent, the Octodon degus. We found that all PBG neurons expressed both glutamatergic and cholinergic markers and were distributed in clearly defined anterior (aPBG) and posterior (pPBG) subdivisions. The pPBG is connected reciprocally and topographically to the ipsilateral SC, whereas the aPBG receives afferent axons from the ipsilateral SC and projected exclusively to the contralateral SC. This contralateral projection forms a dense field of terminals that is restricted to the medial SC, in correspondence with the SC representation of the aerial binocular field which, we also found, in O. degus prompted escape reactions upon looming stimulation. Therefore, this specialized topography allows binocular interactions in the SC region controlling responses to aerial predators, suggesting a link between the mechanisms by which the SC and PBG produce defensive behaviors.

Anatomical Specializations Related to Foraging in the Visual System of a Nocturnal Insectivorous Bird, the Band-Winged Nightjar (Aves: Caprimulgiformes).

Nocturnal animals that rely on their visual system for foraging, mating, and navigation usually exhibit specific traits associated with living in scotopic conditions. Most nocturnal birds have several visual specializations, such as enlarged eyes and an increased orbital convergence. However, the actual role of binocular vision in nocturnal foraging is still debated. Nightjars (Aves: Caprimulgidae) are predators that actively pursue and capture flying insects in crepuscular and nocturnal environments, mainly using a conspicuous "sit-and-wait" tactic on which pursuit begins with an insect flying over the bird that sits on the ground. In this study, we describe the visual system of the band-winged nightjar (Systellura longirostris), with emphasis on anatomical features previously described as relevant for nocturnal birds. Orbit convergence, determined by 3D scanning of the skull, was 73.28°. The visual field, determined by ophthalmoscopic reflex, exhibits an area of maximum binocular overlap of 42°, and it is dorsally oriented. The eyes showed a nocturnal-like normalized corneal aperture/axial length index. Retinal ganglion cells (RGCs) were relatively scant, and distributed in an unusual oblique-band pattern, with higher concentrations in the ventrotemporal quadrant. Together, these results indicate that the band-winged nightjar exhibits a retinal specialization associated with the binocular area of their dorsal visual field, a relevant area for pursuit triggering and prey attacks. The RGC distribution observed is unusual among birds, but similar to that of some visually dependent insectivorous bats, suggesting that those features might be convergent in relation to feeding strategies.

"Shepherd's crook" neurons drive and synchronize the enhancing and suppressive mechanisms of the midbrain stimulus selection network.

The optic tectum (TeO), or superior colliculus, is a multisensory midbrain center that organizes spatially orienting responses to relevant stimuli. To define the stimulus with the highest priority at each moment, a network of reciprocal connections between the TeO and the isthmi promotes competition between concurrent tectal inputs. In the avian midbrain, the neurons mediating enhancement and suppression of tectal inputs are located in separate isthmic nuclei, facilitating the analysis of the neural processes that mediate competition. A specific subset of radial neurons in the intermediate tectal layers relay retinal inputs to the isthmi, but at present it is unclear whether separate neurons innervate individual nuclei or a single neural type sends a common input to several of them. In this study, we used in vitro neural tracing and cell-filling experiments in chickens to show that single neurons innervate, via axon collaterals, the three nuclei that comprise the isthmotectal network. This demonstrates that the input signals representing the strength of the incoming stimuli are simultaneously relayed to the mechanisms promoting both enhancement and suppression of the input signals. By performing in vivo recordings in anesthetized chicks, we also show that this common input generates synchrony between both antagonistic mechanisms, demonstrating that activity enhancement and suppression are closely coordinated. From a computational point of view, these results suggest that these tectal neurons constitute integrative nodes that combine inputs from different sources to drive in parallel several concurrent neural processes, each performing complementary functions within the network through different firing patterns and connectivity.

The Medial Ventrothalamic Circuitry: Cells Implicated in a Bimodal Network.

Previous avian thalamic studies have shown that the medial ventral thalamus is composed of several nuclei located close to the lateral wall of the third ventricle. Although the general connectivity is known, detailed morphology and connectivity pattern in some regions are still elusive. Here, using the intracellular filling technique in the chicken, we focused on two neural structures, namely, the retinorecipient neuropil of the n. geniculatus lateralis pars ventralis (GLv), and the adjacent n. intercalatus thalami (ICT). We found that the GLv-ne cells showed two different neuronal types: projection cells and horizontal interneurons. The projection cells showed variable morphologies and dendritic arborizations with axons that targeted the n. lentiformis mesencephali (LM), griseum tectale (GT), ICT, n. principalis precommissuralis (PPC), and optic tectum (TeO). The horizontal cells showed a widespread mediolateral neural process throughout the retinorecipient GLv-ne. The ICT cells, on the other hand, had multipolar somata with wide dendritic fields that extended toward the lamina interna of the GLv, and a projection pattern that targeted the n. laminaris precommissuralis (LPC). Together, these results elucidate the rich complexity of the connectivity pattern so far described between the GLv, ICT, pretectum, and tectum. Interestingly, the implication of some of these neural structures in visuomotor and somatosensory roles strongly suggests that the GLv and ICT are part of a bimodal circuit that may be involved in the generation/modulation of saccades, gaze control, and space perception.

The centrifugal visual system of a palaeognathous bird, the Chilean Tinamou (Nothoprocta perdicaria).

The avian centrifugal visual system, which projects from the brain to the retina, has been intensively studied in several Neognathous birds that have a distinct isthmo-optic nucleus (ION). However, birds of the order Palaeognathae seem to lack a proper ION in histologically stained brain sections. We had previously reported in the palaeognathous Chilean Tinamou (Nothoprocta perdicaria) that intraocular injections of Cholera Toxin B subunit retrogradely label a considerable number of neurons, which form a diffuse isthmo-optic complex (IOC). In order to better understand how this IOC-based centrifugal visual system is organized, we have studied its major components by means of in vivo and in vitro tracing experiments. Our results show that the IOC, though structurally less organized than an ION, possesses a dense core region consisting of multipolar neurons. It receives afferents from neurons in L10a of the optic tectum, which are distributed with a wider interneuronal spacing than in Neognathae. The tecto-IOC terminals are delicate and divergent, unlike the prominent convergent tecto-ION terminals in Neognathae. The centrifugal IOC terminals in the retina are exclusively divergent, resembling the terminals from "ectopic" centrifugal neurons in Neognathae. We conclude that the Tinamou's IOC participates in a comparable general IOC-retina-TeO-IOC circuitry as the neognathous ION. However, the connections between the components are structurally different and their divergent character suggests a lower spatial resolution. Our findings call for further comparative studies in a broad range of species for advancing our understanding of the evolution, plasticity and functional roles of the avian centrifugal visual system.

Congruent representation of visual and acoustic space in the superior colliculus of the echolocating bat Phyllostomus discolor.

The midbrain superior colliculus (SC) commonly features a retinotopic representation of visual space in its superficial layers, which is congruent with maps formed by multisensory neurons and motor neurons in its deep layers. Information flow between layers is suggested to enable the SC to mediate goal-directed orienting movements. While most mammals strongly rely on vision for orienting, some species such as echolocating bats have developed alternative strategies, which raises the question how sensory maps are organized in these animals. We probed the visual system of the echolocating bat Phyllostomus discolor and found that binocular high acuity vision is frontally oriented and thus aligned with the biosonar system, whereas monocular visual fields cover a large area of peripheral space. For the first time in echolocating bats, we could show that in contrast with other mammals, visual processing is restricted to the superficial layers of the SC. The topographic representation of visual space, however, followed the general mammalian pattern. In addition, we found a clear topographic representation of sound azimuth in the deeper collicular layers, which was congruent with the superficial visual space map and with a previously documented map of orienting movements. Especially for bats navigating at high speed in densely structured environments, it is vitally important to transfer and coordinate spatial information between sensors and motor systems. Here, we demonstrate first evidence for the existence of congruent maps of sensory space in the bat SC that might serve to generate a unified representation of the environment to guide motor actions.

Microconnectomics of the pretectum and ventral thalamus in the chicken (Gallus gallus).

The avian pretectal and ventrothalamic nuclei, encompassing the griseum tectale (GT), n. lentiformis mesencephali (LM), and n. geniculatus lateralis pars ventralis (GLv), are prominent retinorecipient structures related to optic flow operations and visuomotor control. Hence, a close coordination of these neural circuits is to be expected. Yet the connectivity among these nuclei is poorly known. Here, using intracellular labeling and in situ hybridization, we investigated the detailed morphology, connectivity, and neurochemical identity of neurons in these nuclei. Two different cell types exist in the GT: one that generates an axonal projection to the optic tectum (TeO), LM, GLv, and n. intercalatus thalami (ICT), and a second population that only projects to the LM and GLv. In situ hybridization revealed that most neurons in the GT express the vesicular glutamate transporter (VGluT2) mRNA, indicating a glutamatergic identity. In the LM, three morphological cell types were defined, two of which project axons towards dorsal targets. The LM neurons showed strong VGluT2 expression. Finally, the cells located in the GLv project to the TeO, LM, GT, n. principalis precommisuralis (PPC), and ICT. All neurons in the GLv showed strong expression of the vesicular inhibitory amino acid transporter (VIAAT) mRNA, suggesting a GABAergic identity. Our results show that the pretectal and ventrothalamic nuclei are highly interconnected, especially by glutamatergic and GABAergic neurons from the GT and GLv, respectively. This complex morphology and connectivity might be required to organize orienting visuomotor behaviors and coordinate the specific optic flow patterns that they induce. J. Comp. Neurol. 524:2208-2229, 2016. © 2015 Wiley Periodicals, Inc.

The visual system of a palaeognathous bird: visual field, retinal topography and retino-central connections in the Chilean tinamou (Nothoprocta perdicaria).

Most systematic studies of the avian visual system have focused on Neognathous species, leaving virtually unexplored the Palaeognathae, comprised of the flightless ratites and the South American tinamous. We investigated the visual field, the retinal topography, and the pattern of retinal and centrifugal projections in the Chilean tinamou, a small Palaeognath of the family Tinamidae. The tinamou has a panoramic visual field with a small frontal binocular overlap of 20°. The retina possesses three distinct topographic specializations: a horizontal visual streak, a dorsotemporal area, and an area centralis with a shallow fovea. The maximum ganglion cell density is 61,900/ mm(2) , comparable to Falconiformes. This would provide a maximal visual acuity of 14.0 cycles/degree, in spite of relatively small eyes. The central retinal projections generally conform to the characteristic arrangement observed in Neognathae, with well-differentiated contralateral targets and very few ipsilateral fibers. The centrifugal visual system is composed of a considerable number of multipolar centrifugal neurons, resembling the "ectopic" neurons described in Neognathae. They form a diffuse nuclear structure, which may correspond to the ancestral condition shared with other sauropsids. A notable feature is the presence of terminals in deep tectal layers 11-13. These fibers may represent either a novel retinotectal pathway or collateral branches from centrifugal neurons projecting to the retina. Both types of connections have been described in chicken embryos. Our results widen the basis for comparative studies of the vertebrate visual system, stressing the conserved character of the visual projections' pattern within the avian clade.

Morphology, projection pattern, and neurochemical identity of Cajal's "centrifugal neurons": the cells of origin of the tectoventrogeniculate pathway in pigeon (Columba livia) and chicken (Gallus gallus).

The nucleus geniculatus lateralis pars ventralis (GLv) is a prominent retinal target in all amniotes. In birds, it is in receipt of a dense and topographically organized retinal projection. The GLv is also the target of substantial and topographically organized projections from the optic tectum and the visual wulst (hyperpallium). Tectal and retinal afferents terminate homotopically within the external GLv-neuropil. Efferents from the GLv follow a descending course through the tegmentum and can be traced into the medial pontine nucleus. At present, the cells of origin of the Tecto-GLv projection are only partially described. Here we characterized the laminar location, morphology, projection pattern, and neurochemical identity of these cells by means of neural tracer injections and intracellular fillings in slice preparations and extracellular tracer injections in vivo. The Tecto-GLv projection arises from a distinct subset of layer 10 bipolar neurons, whose apical dendrites show a complex transverse arborization at the level of layer 7. Axons of these bipolar cells arise from the apical dendrites and follow a course through the optic tract to finally form very fine and restricted terminal endings inside the GLv-neuropil. Double-label experiments showed that these bipolar cells were choline acetyltransferase (ChAT)-immunoreactive. Our results strongly suggest that Tecto-GLv neurons form a pathway by which integrated tectal activity rapidly feeds back to the GLv and exerts a focal cholinergic modulation of incoming retinal inputs.

Does nocturnality drive binocular vision? Octodontine rodents as a case study.

Binocular vision is a visual property that allows fine discrimination of in-depth distance (stereopsis), as well as enhanced light and contrast sensitivity. In mammals enhanced binocular vision is structurally associated with a large degree of frontal binocular overlap, the presence of a corresponding retinal specialization containing a fovea or an area centralis, and well-developed ipsilateral retinal projections to the lateral thalamus (GLd). We compared these visual traits in two visually active species of the genus Octodon that exhibit contrasting visual habits: the diurnal Octodon degus, and the nocturnal Octodon lunatus. The O. lunatus visual field has a prominent 100° frontal binocular overlap, much larger than the 50° of overlap found in O. degus. Cells in the retinal ganglion cell layer were 40% fewer in O. lunatus (180,000) than in O. degus (300,000). O. lunatus has a poorly developed visual streak, but a well developed area centralis, located centrally near the optic disk (peak density of 4,352 cells/mm(2)). O. degus has a highly developed visual streak, and an area centralis located more temporally (peak density of 6,384 cells/mm(2)). The volumes of the contralateral GLd and superior colliculus (SC) are 15% larger in O. degus compared to O. lunatus. However, the ipsilateral projections to GLd and SC are 500% larger in O. lunatus than in O. degus. Other retinorecipient structures related to ocular movements and circadian activity showed no statistical differences between species. Our findings strongly suggest that nocturnal visual behavior leads to an enhancement of the structures associated with binocular vision, at least in the case of these rodents. Expansion of the binocular visual field in nocturnal species may have a beneficial effect in light and contrast sensitivity, but not necessarily in stereopsis. We discuss whether these conclusions can be extended to other mammalian and non-mammalian amniotes.

Bilateral and ipsilateral ascending tectopulvinar pathways in mammals: a study in the squirrel (Spermophilus beecheyi).

The mammalian pulvinar complex is a collection of dorsal thalamic nuclei related to several visual and integrative processes. Previous studies have shown that the superficial layers of the superior colliculus project to multiple divisions of the pulvinar complex. Although most of these works agree about the existence of an ipsilateral tectopulvinar projection arising from the stratum griseum superficialis, some others report a bilateral projection originating from this same tectal layer. We investigated the organization of the tectopulvinar projections in the Californian ground squirrel using cholera toxin B (CTb). We confirmed previous studies showing that the caudal pulvinar of the squirrel receives a massive bilateral projection originating from a specific cell population located in the superficial collicular layers (SGS3, also called the "lower SGS" or "SGSL"). We found that this projection shares striking structural similarities with the tectorotundal pathway of birds and reptiles. Morphology of the collicular cells originating this projection closely corresponds to that of the bottlebrush tectal cells described previously for chickens and squirrels. In addition, we found that the rostral pulvinar receives an exclusively ipsilateral projection from a spatially separate population of collicular cells located at the base of the stratum opticum, deeper than the cells projecting to the caudal pulvinar. These results strongly support, at a structural level, the homology of the pathway originating in the SGS3 collicular cells upon the caudal pulvinar with the tectorotundal pathway of nonmammalian amniotes and contribute to clarifying the general organization of the tectopulvinar pathways in mammals.