Early Development of the Thalamo-Pallial Stage of the Tectofugal Visual Pathway in the Chicken (Gallus gallus).

The tectofugal pathway is a highly conserved visual pathway in all amniotes. In birds and mammals, retinorecipient neurons located in the midbrain roof (optic tectum/superior colliculus) are the source of ascending projections to thalamic relays (nucleus rotundus/caudal pulvinar), which in turn project to specific pallial regions (visual dorsal ventricular ridge [vDVR]/temporal cortex) organized according to a columnar recurrent arrangement of interlaminar circuits. Whether or to which extent these striking hodological correspondences arise from comparable developmental processes is at present an open question, mainly due to the scarcity of data about the ontogeny of the avian tectofugal system. Most of the previous developmental studies of this system in birds have focused on the establishment of the retino-tecto-thalamic connectivity, overlooking the development of the thalamo-pallial-intrapallial circuit. In this work, we studied the latter in chicken embryos by means of immunohistochemical assays and precise ex vivo crystalline injections of biocytin and DiI. We found that the layered organization of the vDVR as well as the system of homotopic reciprocal connections between vDVR layers were present as early as E8. A highly organized thalamo-vDVR projection was also present at this stage. Our immunohistochemical assays suggest that both systems of projections emerge simultaneously even earlier. Combined with previous findings, these results reveal that, in striking contrast with mammals, the peripheral and central stages of the avian tectofugal pathway develop along different timelines, with a tecto-thalamo-intrapallial organization arising before and possibly independently of the retino-isthmo-tectal circuit.

A blinking focal pattern of re-entrant activity in the avian tectum.

Re-entrant connections are inherent to nervous system organization; however, a comprehensive understanding of their operation is still lacking. In birds, topographically organized re-entrant signals, carried by axons from the nucleus-isthmi-parvocellularis (Ipc), are distinctly recorded as bursting discharges across the optic tectum (TeO). Here, we used up to 48 microelectrodes regularly spaced on the superficial tectal layers of anesthetized pigeons to characterize the spatial-temporal pattern of this axonal re-entrant activity in response to different visual stimulation. We found that a brief luminous spot triggered repetitive waves of bursting discharges that, appearing from initial sources, propagated horizontally to areas representing up to 28° of visual space, widely exceeding the area activated by the retinal fibers. In response to visual motion, successive burst waves started along and around the stimulated tectal path, tracking the stimulus in discontinuous steps. When two stimuli were presented, the burst-wave sources alternated between the activated tectal loci, as if only one source could be active at any given time. Because these re-entrant signals boost the retinal input to higher visual areas, their peculiar dynamics mimic a blinking "spotlight," similar to the internal searching mechanism classically used to explain spatial attention. Tectal re-entry from Ipc is thus highly structured and intrinsically discontinuous, and higher tectofugal areas, which lack retinotopic organization, will thus receive incoming visual activity in a sequential and piecemeal fashion. We anticipate that analogous re-entrant patterns, perhaps hidden in less bi-dimensionally organized topographies, may organize the flow of neural activity in other parts of the brain as well.

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.

Parallel organization of the avian sensorimotor arcopallium: Tectofugal visual pathway in the pigeon (Columba livia).

The sensory-motor division of the avian arcopallium receives parallel inputs from primary and high-order pallial areas of sensory and vocal control pathways, and sends a prominent descending projection to ascending and premotor, subpallial stages of these pathways. While this organization is well established for the auditory and trigeminal systems, the arcopallial subdivision related to the tectofugal visual system and its descending projection to the optic tectum (TeO) has been less investigated. In this study, we charted the arcopallial area displaying tectofugal visual responses and by injecting neural tracers, we traced its connectional anatomy. We found visual motion-sensitive responses in a central region of the dorsal (AD) and intermediate (AI) arcopallium, in between previously described auditory and trigeminal zones. Blocking the ascending tectofugal sensory output, canceled these visual responses in the arcopallium, verifying their tectofugal origin. Injecting PHA-L into the visual, but not into the auditory AI, revealed a massive projection to tectal layer 13 and other tectal related areas, sparing auditory, and trigeminal ones. Conversely, CTB injections restricted to TeO retrogradely labeled neurons confined to the visual AI. These results show that the AI zone receiving tectofugal inputs sends top-down modulations specifically directed to tectal targets, just like the auditory and trigeminal AI zones project back to their respective subpallial sensory and premotor areas, as found by previous studies. Therefore, the arcopallium seems to be organized in a parallel fashion, such that in spite of expected cross-modal integration, the different sensory-motor loops run through separate subdivisions of this structure.

"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.

Axon terminals from the nucleus isthmi pars parvocellularis control the ascending retinotectofugal output through direct synaptic contact with tectal ganglion cell dendrites.

The optic tectum in birds and its homologue the superior colliculus in mammals both send major bilateral, nontopographic projections to the nucleus rotundus and caudal pulvinar, respectively. These projections originate from widefield tectal ganglion cells (TGCs) located in layer 13 in the avian tectum and in the lower superficial layers in the mammalian colliculus. The TGCs characteristically have monostratified arrays of brush-like dendritic terminations and respond mostly to bidimensional motion or looming features. In birds, this TGC-mediated tectofugal output is controlled by feedback signals from the nucleus isthmi pars parvocellularis (Ipc). The Ipc neurons display topographically organized axons that densely ramify in restricted columnar terminal fields overlapping various neural elements that could mediate this tectofugal control, including the retinal terminals and the TGC dendrites themselves. Whether the Ipc axons make synaptic contact with these or other tectal neural elements remains undetermined. We double labeled Ipc axons and their presumptive postsynaptic targets in the tectum of chickens (Gallus gallus) with neural tracers and performed an ultrastructural analysis. We found that the Ipc terminal boutons form glomerulus-like structures in the superficial and intermediate tectal layers, establishing asymmetric synapses with several dendritic profiles. In these glomeruli, at least two of the postsynaptic dendrites originated from TGCs. We also found synaptic contacts between retinal terminals and TGC dendrites. These findings suggest that, in birds, Ipc axons control the ascending tectal outflow of retinal signals through direct synaptic contacts with the TGCs.

Anatomical organization of the visual dorsal ventricular ridge in the chick (Gallus gallus): Layers and columns in the avian pallium.

The dorsal ventricular ridge (DVR) is one of the main components of the sauropsid pallium. In birds, the DVR is formed by an inner region, the nidopallium, and a more dorsal region, the mesopallium. The nidopallium contains discrete areas that receive auditory, visual, and multisensory collothalamic projections. These nidopallial nuclei are known to sustain reciprocal, short-range projections with their overlying mesopallial areas. Recent findings on the anatomical organization of the auditory DVR have shown that these short-range projections have a columnar organization that closely resembles that of the mammalian neocortex. However, it is unclear whether this columnar organization generalizes to other areas within the DVR. Here we examine in detail the organization of the visual DVR, performing small, circumscribed deposits of neuronal tracers as well as intracellular fillings in brain slices. We show that the visual DVR is organized in three main laminae, the thalamorecipient nucleus entopallium; a dorsally adjacent nidopallial lamina, the intermediate nidopallium; and a contiguous portion of the ventral mesopallium, the mesopallium ventrale. As in the case of the auditory DVR, we found a highly topographically organized system of reciprocal interconnections among these layers, which was formed by dorsoventrally oriented, discrete columnar bundles of axons. We conclude that the columnar organization previously demonstrated in the auditory DVR is not a unique feature but a general characteristic of the avian sensory pallium. We discuss these results in the context of a comparison between sauropsid and mammalian pallial organization.

Zebrin II Expression in the Cerebellum of a Paleognathous Bird, the Chilean Tinamou (Nothoprocta perdicaria).

Zebrin II (ZII) is a glycolytic enzyme expressed in cerebellar Purkinje cells. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with a high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). To date, ZII expression studies are limited to neognathous birds: pigeons (Columbiformes), chickens (Galliformes), and hummingbirds (Trochilidae). These previous studies divided the avian cerebellum into 5 transverse regions based on the pattern of ZII expression. In the lingular region (lobule I) all Purkinje cells are ZII+. In the anterior region (lobules II-V) there are 4 pairs of ZII+/- stripes. In the central region (lobules VI-VIII) all Purkinje cells are ZII+. In the posterior region (lobules VIII-IX) there are 5-7 pairs of ZII+/- stripes. Finally, in the nodular region (lobule X) all Purkinje cells are ZII+. As the pattern of ZII stripes is quite similar in these disparate species, it appears that it is highly conserved. However, it has yet to be studied in paleognathous birds, which split from the neognaths over 100 million years ago. To better understand the evolution of cerebellar compartmentation in birds, we examined ZII immunoreactivity in a paleognath, the Chilean tinamou (Nothoprocta perdicaria). In the tinamou, Purkinje cells expressed ZII heterogeneously such that there were sagittal ZII+ and ZII- stripes of Purkinje cells, and this pattern of expression was largely similar to that observed in neognathous birds. For example, all Purkinje cells in the lingular (lobule I) and nodular (lobule X) regions were ZII+, and there were 4 pairs of ZII+/- stripes in the anterior region (lobules II-V). In contrast to neognaths, however, ZII was expressed in lobules VI-VII as a series of sagittal stripes in the tinamou. Also unlike in neognaths, stripes were absent in lobule IXab, and all Purkinje cells expressed ZII in the tinamou. The differences in ZII expression between the tinamou and neognaths could reflect behavior, but the general similarity of the expression patterns across all bird species suggests that ZII stripes evolved early in the avian phylogenetic tree.

The isthmic nuclei providing parallel feedback connections to the avian tectum have different neurochemical identities: Expression of glutamatergic and cholinergic markers in the chick (Gallus gallus).

Retinal inputs to the optic tectum (TeO) triggered by moving stimuli elicit synchronized feedback signals from two isthmic nuclei: the isthmi parvocelullaris (Ipc) and isthmi semilunaris (SLu). Both of these nuclei send columnar axon terminals back to the same tectal position receiving the retinal input. The feedback signals from the Ipc seem to act as an attentional spotlight by selectively boosting the propagation of retinal inputs from the tectum to higher visual areas. Although Ipc and SLu nuclei are widely considered cholinergic because of their immunoreactivity for choline acetyltransferase (ChAT), contradictory findings, including the expression of the vesicular glutamate transporter 2 (VGluT2) mRNA in Ipc neurons, have raised doubts about the purely cholinergic nature of this nucleus. In this study, in chicks, we revise the neurochemical identity of the isthmic nuclei by using in situ hybridization assays for VGluT2 along with three cholinergic markers: the vesicular acetylcholine transporter (VAChT), the high-affinity choline transporter (CHT1) and ChAT. We found that neurons in the SLu showed strong mRNA expression of all three cholinergic markers, whereas the expression of VAChT mRNA in the Ipc was undetectable in our essays. Instead, Ipc neurons exhibited a strong expression of VGluT2 mRNA. Immunohistochemistry assays showed VGluT2 immunoreactivity in the TeO codistributing with anterogradely labeled Ipc axon-terminal boutons, further supporting a glutamatergic function for the Ipc nucleus. Therefore, our results strongly suggest that, in the chick, whereas the feedback from the SLu to the TeO is indeed cholinergic, the feedback from the Ipc has a marked glutamatergic component.

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.

Functional implications of species differences in the size and morphology of the isthmo optic nucleus (ION) in birds.

In birds, there is a retinofugal projection from the brain to the retina originating from the isthmo optic nucleus (ION) in the midbrain. Despite a large number of anatomical, physiological and histochemical studies, the function of this retinofugal system remains unclear. Several functions have been proposed including: gaze stabilization, pecking behavior, dark adaptation, shifting attention, and detection of aerial predators. This nucleus varies in size and organization among some species, but the relative size and morphology of the ION has not been systematically studied. Here, we present a comparison of the relative size and morphology of the ION in 81 species of birds, representing 17 different orders. Our results show that several orders of birds, besides those previously reported, have a large, well-organized ION, including: hummingbirds, woodpeckers, coots and allies, and kingfishers. At the other end of the spectrum, parrots, herons, waterfowl, owls and diurnal raptors have relatively small ION volumes. ION also appears to be absent or unrecognizable is several taxa, including one of the basal avian groups, the tinamous, which suggests that the ION may have evolved only in the more modern group of birds, Neognathae. Finally, we demonstrate that evolutionary changes in the relative size and the cytoarchitectonic organization of ION have occurred largely independent of phylogeny. The large relative size of the ION in orders with very different lifestyles and feeding behaviors suggest there is no clear association with pecking behavior or predator detection. Instead, our results suggest that the ION is more complex and enlarged in birds that have eyes that are emmetropic in some parts of the visual field and myopic in others. We therefore posit that the ION is involved in switching attention between two parts of the retina i.e. from an emmetropic to a myopic part of the retina.

Attentional capture? Synchronized feedback signals from the isthmi boost retinal signals to higher visual areas.

When a salient object in the visual field captures attention, the neural representation of that object is enhanced at the expense of competing stimuli. How neural activity evoked by a salient stimulus evolves to take precedence over the neural activity evoked by other stimuli is a matter of intensive investigation. Here, we describe in pigeons (Columba livia) how retinal inputs to the optic tectum (TeO, superior colliculus in mammals), triggered by moving stimuli, are selectively relayed on to the rotundus (Rt, caudal pulvinar) in the thalamus, and to its pallial target, the entopallium (E, extrastriate cortex). We show that two satellite nuclei of the TeO, the nucleus isthmi parvocelullaris (Ipc) and isthmi semilunaris (SLu), send synchronized feedback signals across tectal layers. Preventing the feedback from Ipc but not from SLu to a tectal location suppresses visual responses to moving stimuli from the corresponding region of visual space in all Rt subdivisions. In addition, the bursting feedback from the Ipc imprints a bursting rhythm on the visual signals, such that the visual responses of the Rt and the E acquire a bursting modulation significantly synchronized to the feedback from Ipc. As the Ipc feedback signals are selected by competitive interactions, the visual responses within the receptive fields in the Rt tend to synchronize with the tectal location receiving the "winning" feedback from Ipc. We propose that this selective transmission of afferent activity combined with the cross-regional synchronization of the areas involved represents a bottom-up mechanism by which salient stimuli capture attention.