The nucleus pretectalis principalis: A pretectal structure hidden in the mammalian thalamus.

A defining feature of the amniote tecto-fugal visual pathway is a massive bilateral projection to the thalamus originating from a distinct neuronal population, tectal ganglion cells (TGCs), of the optic tectum/superior colliculus (TeO/SC). In sauropsids, the thalamic target of the tecto-fugal pathway is the nucleus rotundus thalami (Rt). TGCs axons collateralize en route to Rt to target the nucleus pretectalis principalis (PT), which in turn gives rise to bilateral projection to the TeO. In rodents, the thalamic target of these TGCs afferents is the caudal division of the pulvinar complex (PulC). No pretectal structures in receipt of TGC collaterals have been described in this group. However, Baldwin et al. (Journal of Comparative Neurology, 2011;519(6):1071-1094) reported in the squirrel a feedback projection from the PulC to the SC. Pulvino-tectal (Pul-T) cells lie at the caudal pole of the PulC, intermingled with the axonal terminals of TGCs. Here, by performing a combination of neuronal tracing, immunohistochemistry, immunofluorescence, and in situ hybridization, we characterized the pattern of projections, neurochemical profile, and genoarchitecture of Pul-T cells in the diurnal Chilean rodent Octodon degus. We found that Pul-T neurons exhibit pretectal, but not thalamic, genoarchitectonical markers, as well as hodological and neurochemical properties that match specifically those of the avian nucleus PT. Thus, we propose that Pul-T cells constitute a pretectal cell population hidden within the dorsal thalamus of mammals. Our results solve the oddity entailed by the apparent existence of a noncanonic descending sensory thalamic projection and further stress the conservative character of the tectofugal pathway.

Transcriptome of the Wistar audiogenic rat (WAR) strain following audiogenic seizures.

The Wistar Audiogenic Rat (WAR) is a model whose rats are predisposed to develop seizures following acoustic stimulation. We aimed to establish the transcriptional profile of the WAR model, searching for genes that help in understanding the molecular mechanisms involved in the predisposition and seizures expression of this strain. RNA-Seq of the corpora quadrigemina of WAR and Wistar rats subjected to acoustic stimulation revealed 64 genes differentially regulated in WAR. We validated twelve of these genes by qPCR in stimulated and naive (non-stimulated) WAR and Wistar rats. Among these, Acsm3 was upregulated in WAR in comparison with both control groups. In contrast, Gpr126 and Rtel1 were downregulated in naive and stimulated WAR rats in comparison with the Wistar controls. Qdpr was upregulated only in stimulated WAR rats that exhibited audiogenic seizures. Our data show that there are genes with differential intrinsic regulation in the WAR model and that seizures can alter gene regulation. We identified new genes that might be involved in the epileptic phenotype and comorbidities of the WAR model.

The effects of repetitive transcranial magnetic stimulation in an animal model of tinnitus.

Tinnitus (phantom auditory perception associated with hearing loss) can seriously affect wellbeing. Its neural substrate is unknown however it has been linked with abnormal activity in auditory pathways. Though no cure currently exists, repetitive transcranial magnetic stimulation (rTMS) has been shown to reduce tinnitus in some patients, possibly via induction of cortical plasticity involving brain derived neurotrophic factor (BDNF). We examined whether low intensity rTMS (LI-rTMS) alleviates signs of tinnitus in a guinea pig model and whether this involves changes in BDNF expression and hyperactivity in inferior colliculus. Acoustic trauma was used to evoke hearing loss, central hyperactivity and tinnitus. When animals developed tinnitus, treatment commenced (10 sessions of 10 minutes 1 Hz LI-rTMS or sham over auditory cortex over 14 days). After treatment ceased animals were tested for tinnitus, underwent single-neuron recordings in inferior colliculus to assess hyperactivity and samples from cortex and inferior colliculus were taken for BDNF ELISA. Analysis revealed a significant reduction of tinnitus after LI-rTMS compared to sham, without a statistical significant effect on BDNF levels or hyperactivity. This suggests that LI-rTMS alleviates behavioural signs of tinnitus by a mechanism independent of inferior colliculus hyperactivity and BDNF levels and opens novel therapeutic avenues for tinnitus treatment.

The use of fluorescein sodium in the biopsy and gross-total resection of a tectal plate glioma.

Intravenous administration of fluorescein sodium fluoresces glioma burden tissue and can be visualized using the surgical microscope with a specialized filter. Intraoperative guidance afforded through the use of fluorescein may enhance the fidelity of tissue sampling, and increase the ability to accomplish complete resection of tectal lesions. In this report the authors present the case of a 19-year-old man with a tectal anaplastic pilocytic astrocytoma in which the use of fluorescein sodium and a Zeiss Pentero surgical microscope equipped with a yellow 560 filter enabled safe complete resection. In conjunction with neurosurgical navigation, added intraoperative guidance provided by fluorescein may be beneficial in the resection of brainstem gliomas.

Endocannabinoid signaling mechanisms in the substantia nigra pars reticulata modulate GABAergic nigrotectal pathways in mice threatened by urutu-cruzeiro venomous pit viper.

The substantia nigra pars reticulata (SNpr) is rich in γ-aminobutyric acid (GABA)-ergic neurons and connected to the mesencephalic tectum (MT) structures, such as the superior colliculus and dorsal periaqueductal gray matter. The SNpr presents a high density of cannabinoid receptors (CBRs), suggesting a possible regulatory role that is played by endocannabinoids (eCBs) in the ventral mesencephalon. The present study investigated the involvement of SNpr eCB mechanisms in nigrotectal pathways in the expression of defensive behavior associated with instinctive fear and panic reactions in mice that are confronted with the venomous Viperidae snake Bothrops alternatus. The localization of CB1 receptors (CB1RS) and synaptophysin glycoprotein in the SNpr was also evaluated. Administration of the GABAA receptor antagonist bicuculline in the MT increased defensive responses to the snake that are related to panic, such as freezing and non-oriented escape reactions, sometimes toward the snake itself. Mice that were pretreated with anandamide (5 or 50pmol) in the SNpr, followed by an injection of physiological saline or bicuculline in the MT, exhibited significant decreases in the expression of alertness, freezing, and escape responses. Immunofluorescence showed the presence of fibers that were rich in CB1RS and synaptophysin in the SNpr, indicating that these receptors appear to be located mainly in presynaptic terminals in the striatonigral pathway. These findings suggest that eCB mechanisms in the SNpr facilitate the activity of nigrotectal GABAergic pathways, modulating the activity of striatonigral links during the elaboration and organization of innate fear and panic-like responses in threatening situations.

Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons.

Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2-containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi-like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2-positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections.

Hunting increases phosphorylation of calcium/calmodulin-dependent protein kinase type II in adult barn owls.

Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is "off" in adults.

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.

Role of En2 in the tectal laminar formation of chick embryos.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

Inhibition of dendritic L-type calcium current by memantine in frog tectum.

UNLABELLED: The aim of the study was to explore the effects of memantine on responses elicited in the frog tectum by the bursts of spikes of moderate strength of a single retina ganglion cell and to gain an insight about the effect of memantine on the L-type Ca(2+) current. MATERIAL AND METHODS: The experiments were performed in vivo on adult frogs (Rana temporaria). An individual retina ganglion cell (or its retinotectal fiber) was stimulated by current pulses delivered through a multichannel stimulating electrode positioned on the retina. Responses to the discharge of a single retinal ganglion cell were recorded in the tectum by an extracellular carbon-fiber microelectrode positioned in the terminal arborization of the retinotectal fiber in the tectum layer F. The solution of memantine (1-amino-3,5-dimethyladamantane) hydrochloride (30 or 45 µM) was applied onto the surface of the tectum by perfusion at a rate of 0.4 mL/min. RESULTS: Memantine (30-45 µM) largely inhibited the L-type Ca(2+) channel-mediated slow negative wave and late discharges seen in the tectum responses without any effect on fast synaptic retinotectal transmission. CONCLUSIONS: Our results suggest that the neuroprotective effect of memantine could arise not only through the inhibition of the NMDA receptor current but also through the suppression of the L-type Ca(2+) current.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Frog retinal ganglion cells projecting to the tectum layer F release acetylcholine as co-mediator.

Neurons may release more than one classical neurotransmitter (co-mediator). It has been demonstrated in a recent study that a burst of action potentials in frog retina ganglion cells induces an after-burst increase (phasic potentiation) of the retinotectal transmission that lasts tens of seconds. This increase is mediated by presynaptic nicotinic acetylcholine receptors that are activated by the endogenous acetylcholine released during the burst of action potentials of the retinotectal fiber. The objective of the present study was to find out the origin of acetylcholine release. We show that reduction of the retinotectal transmission to the subthreshold level by application of moderate concentrations of kynurenic acid or CNQX had no effect on the phasic nicotinic potentiation of the retinotectal transmission. This demonstrates that the retinotectal terminals are the source of acetylcholine - responsible for the phasic potentiation of retinotectal transmission. The acetylcholine is thus co-released with glutamate.

Prion protein (PrP) deposits in the tectum of experimental Gerstmann-Sträussler-Scheinker disease following intraocular inoculation.

The abnormal misfolded isoform of prion protein (PrPd; "d" for disease) is considered as a surrogate marker for infectivity in the transmissible spongiform encephalopathies (TSEs) or prion diseases, including Creutzfeldt-Jakob disease (CJD). In this experiment, we used intraocular inoculation to study PrPd deposition in the visual system of the brain of mice infected with the Fujisaki (K.Fu) strain of Gerstmann-Sträussler-Scheinker (GSS) disease. We report here that PrPd is deposited in the superior colliculus following contralateral intraocular inoculation and thus follows neuronal connections when it spreads into the brain. Until 26 weeks postinoculation, no PrPd-specific immunostaining was observed in the brain. At 27 weeks postinoculation, PrPd targeted to the contralateral superior colliculus as delicate granular synaptic deposits located in the superficial part of this structure. As already reported, a few spongiform vacuoles were visible in the same area by conventional H and E staining. In several other sections, vacuoles were visible but no PrPd staining could be detected.

Organization of the cholinergic systems in the brain of two lungfishes, Protopterus dolloi and Neoceratodus forsteri.

Lungfishes (dipnoans) are currently considered the closest living relatives of tetrapods. The organization of the cholinergic systems in the brain has been carefully analyzed in most vertebrate groups, and major shared characteristics have been described, although traits particular to each vertebrate class have also been found. In the present study, we provide the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two representative species of lungfishes, the African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry against the enzyme choline acetyltransferase (ChAT). Distinct groups of ChAT immunoreactive (ChAT-ir) cells were observed in the basal telencephalon, habenula, isthmic nucleus, laterodorsal tegmental nucleus, cranial nerve motor nuclei, and the motor column of the spinal cord, and these groups seem to be highly conserved among vertebrates. In lungfishes, the presence of a cholinergic cell group in the thalamus and the absence of ChAT-ir cells in the tectum are variable traits, unique to this group and appearing several times during evolution. Other characters were observed exclusively in Neoceratodus, such as the presence of cholinergic cells in the suprachiasmatic nucleus, the pretectal region and the superior raphe nucleus. Cholinergic fibers were found in the medial pallium, basal telencephalon, thalamus and prethalamus, optic tectum and interpeduncular nucleus. Comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that the cholinergic systems in lungfishes largely resemble those of amphibians and other tetrapods.

Assembly of lamina-specific neuronal connections by slit bound to type IV collagen.

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.

Early expression of axon guidance molecules in the embryonic chick mesencephalon and pretectum.

Early axon tracts in the developing vertebrate brain are established along precise paths. Yet, little is known about axon guidance processes at early stages of rostral brain development. Using whole mount in situ hybridisation in combination with immunohistochemistry, we have analysed the expression patterns of Slits, Netrins, Semaphorins and the respective receptors during the formation of the early axon scaffold, particularly focusing on the pretectal-mesencephalic boundary. Many of these guidance molecules are expressed in close correlation with the growing tracts, and the nuclei of the corresponding neurons often express the respective receptors. The expression patterns of Slits and Netrins implicate them with the positioning of the longitudinal tracts along the dorsoventral axis, while Semaphorins could provide guidance at specific choice points. Our study provides a catalogue of gene expression for future studies on axon guidance mechanisms in the early brain.

Graded levels of FGF protein span the midbrain and can instruct graded induction and repression of neural mapping labels.

Graded guidance labels are widely used in neural map formation, but it is not well understood which potential strategy leads to their graded expression. In midbrain tectal map development, FGFs can induce an entire midbrain, but their protein distribution is unclear, nor is it known whether they may act instructively to produce graded gene expression. Using a receptor-alkaline phosphatase fusion probe, we find a long-range posterior > anterior FGF protein gradient spanning the midbrain. Heparan sulfate proteoglycan (HSPG) is required for this gradient. To test whether graded FGF concentrations can instruct graded gene expression, a quantitative tectal explant assay was developed. Engrailed-2 and ephrin-As, normally in posterior > anterior tectal gradients, showed graded upregulation. Moreover, EphAs, normally in anterior > posterior countergradients, showed coordinately graded downregulation. These results provide a mechanism to establish graded mapping labels and more generally provide a developmental strategy to coordinately induce a structure and pattern its cell properties in gradients.

Eph/ephrin gradients in the retinotectal system of Rana pipiens: developmental and adult expression patterns.

Eph/ephrin-receptor/ligand A and B families play a variety of roles during CNS development, including patterning the retinotectal projection. However, the alignment of their expression gradients with developing retinotectal maps and gradients of cellular development is not well understood in species whose midbrain tecta undergo a protracted anterior to posterior development. By using anatomical tracing methods and (3)H-thymidine neuronography, we have mapped the retinotectal projection and the spatiotemporal progression of tectal cellular development onto Eph/ephrin expression patterns in the tectum of larval Rana pipiens, as studied by means of in situ affinity analysis with fusion proteins. EphA expression is maximal in anterior tectum (and temporal retina); ephrin-A expression is maximal at the posterior pole (and nasal retina). EphB expression is graded in the early larva, where it is maximal in the posterior tectum just anterior to the posterior pole (and in the ventral retina). Tectal EphB expression becomes uniform at later stages and remains so in the adult, although its retinal expression remains maximal ventrally. In the early larva, EphA, EphB, and ephrin-A protein gradients are parallel to each other and align with the temporonasal axis of the retinal projection. The early EphB expression maximum overlaps the boundary between the mantle layer of newly postmitotic cells and the posterior, epithelial region of cell proliferation, suggesting that the expression maximum is associated with the initial migrations of the postmitotic cells. Ephrin-B expression was detected in the olfactory bulb and dorsal retina at all ages, but not in the tectum.

Apc1-mediated antagonism of Wnt/beta-catenin signaling is required for retino-tectal pathfinding in the zebrafish.

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/beta-catenin pathway. We examined the effects of an Apc1 loss-of-function mutation on retino-tectal axon pathfinding in zebrafish. In apc mutants, the retina is disorganized and optic nerves portray pathfinding defects at the optic chiasm and do not project properly to the tectum. Wild-type cells, transplanted into mutant retinae, acquire retinal ganglion cell fate and project axons that cross at the mispositioned optic chiasm and extend to the contralateral tectum, suggesting a function of apc1 in axon pathfinding. These defects are caused mainly by stabilization of beta-catenin. These data demonstrate that Apc1 function is required for correct patterning of the retina and proper retinal ganglion axon projections.

Developmental species differences in brain cell cycle rates between northern bobwhite quail (Colinus virginianus) and parakeets (Melopsittacus undulatus): implications for mosaic brain evolution.

Adult brains differ among species in the proportional sizes of their major subdivisions. For example, the telencephalon occupies 71% of the entire brain in parakeets (Melopsittacus undulatus) but only 54% in quail (Colinus virginianus). In contrast, the tectum is smaller in parakeets than in quail. To determine whether these differences in brain region size arise because of species differences in cell cycle rates, parakeet and quail embryos were collected at various stages of development (HH24-HH37) and stained with antibodies against proliferating cell nuclear antigen (PCNA), which labels all dividing cells, and phosphorylated histone-3 (pH3), which labels M-phase cells. Analysis of pH3+ cell densities and pH3+/PCNA+ cell ratios were used to compare cell cycle rates across stages and species. Cumulative labeling with bromodeoxyuridine (BrdU) was also used to compare cell cycle rates at stages 24 and 28 in quail. We found that telencephalic cell cycle rates lengthen with age in both species, but that they lengthen significantly later in parakeets than in quail. This species difference in cell cycle rates explains, at least partly, why adult parakeets have a proportionately larger telencephalon. Tectal cell cycle rates also remain elevated for a prolonged period of time in parakeets compared to quail. This seems paradoxical at first, given that the parakeet's adult tectum is relatively small. However, the tectum is initially much smaller but then grows more extensively in parakeets than in quail. Thus, species differences in adult brain proportions can be traced back to species differences in cell cycle kinetics.