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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.

The midbrain is an important subcortical area involved in distinct functions such as multimodal integration, movement initiation, bottom-up, and top-down attention. Our group is particularly interested in cellular computation of multisensory integration. We focus on the visual part of the avian midbrain, the optic tectum (TeO, counterpart to mammalian superior colliculus). This area has a layered structure with the great advantage of distinct input and output regions. In chicken, the TeO is organized in 15 layers where visual input targets the superficial layers while auditory input terminates in deeper layers. One specific cell type, the Shepherd's crook neuron (SCN), extends dendrites in both input regions. The characteristic feature of these neurons is the axon origin at the apical dendrite. The molecular identity of this characteristic region and thus, the site of action potential generation are of particular importance to understand signal flow and cellular computation in this neuron. We present immunohistochemical data of structural proteins (NF200, Ankyrin G, and Myelin) and ion channels (Pan-Nav , Nav 1.6, and Kv 3.1b). NF200 is strongly expressed in the axon. Ankyrin G is mainly expressed at the axon initial segment (AIS). Myelination starts after the AIS as well as the distribution of Nav channels on the axon. The subtype Nav 1.6 has a high density in this region. Kv 3.1b is restricted to the soma, the primary neurite and the axon branch. The distribution of functional molecules in SCNs provides insight into the information flow and the integration of sensory modalities in the TeO of the avian midbrain.

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.

Cocaine- and amphetamine-regulated transcript peptides (CARTp) are neuropeptides that act as neurotransmitters in the brain of vertebrates. The expression of CARTp has been characterized in teleosts, amphibians, and several mammalian species, but comparative data in reptiles and birds are nonexistent. In this study, we show the distribution of immunoreactivity against CART peptides (CARTp-ir) in the brains of two bird species: the pigeon (Columba livia) and zebra finch (Taeniopygia guttata). We found CARTp-ir cells and terminals in the brains of both, but no major differences between the two species. As in mammals, teleost fish, and amphibians, CARTp-ir terminals and cells were abundant in subpallial regions, particularly the striatum and nucleus accumbens. We also found CARTp-ir cells and terminals in the hypothalamus, and a large number of CARTp-ir terminals in the substantia nigra, ventral tegmental area, periaqueductal gray, parabrachial nucleus, and dorsal vagal complex. However, in contrast to other vertebrates, CARTp-ir was not found in the olfactory bulb. In addition there was almost no CARTp-ir in the pallium or the hippocampal formation, and little CARTp-ir in the cerebellum. The conserved expression of CARTp in the subpallium, hypothalamus, and dorsal vagal complex of birds suggests that some of the functions of CARTp, such as regulation of food intake and interactions with the social control network and mesolimbic reward system, are conserved among vertebrates. J. Comp. Neurol. 524:3747-3773, 2016. © 2016 Wiley Periodicals, Inc.

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.

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.

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.

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.

Microwire arrays are a powerful tool for the exertion of localized thermal stress on cellular networks. Combining microwire arrays with a set of orthogonal axon-guiding microchannels on-chip allows for the positioning of neurites, as well as control over their polarity. In this paper, we present a new fabrication approach, based on standard clean room fabrication and sacrificial layer etching, for the integration of microwire arrays into neurite guiding structures. The system permits the application of strong temperature gradients, enabling localized thermal stimulation inside microchannels.

Pit vipers (Crotalinae) have a specific sensory system that detects infrared radiation with bilateral pit organs in the upper jaw. Each pit organ consists of a thin membrane, innervated by three trigeminal nerve branches that project to a specific nucleus in the dorsal hindbrain. The known topographic organization of infrared signals in the optic tectum prompted us to test the implementation of spatiotopically aligned sensory maps through hierarchical neuronal levels from the peripheral epithelium to the first central site in the hindbrain, the nucleus of the lateral descending trigeminal tract (LTTD). The spatial organization of the anatomical connections was revealed in a novel in vitro whole-brain preparation of the western diamondback rattlesnake (Crotalus atrox) that allowed specific application of multiple neuronal tracers to identified pit-organ-supplying trigeminal nerve branches. After adequate survival times, the entire peripheral and central projections of fibers within the pit membrane and the LTTD became visible. This approach revealed a morphological partition of the pit membrane into three well-defined sensory areas with largely separated innervations by the three main branches. The peripheral segregation of infrared afferents in the sensory epithelium was matched by a differential termination of the afferents within different areas of the LTTD, with little overlap. This result demonstrates a topographic organizational principle of the snake infrared system that is implemented by maintaining spatially aligned representations of environmental infrared cues on the sensory epithelium through specific neuronal projections at the level of the first central processing stage, comparable to the visual system.