Intersectional, anterograde transsynaptic targeting of neurons receiving monosynaptic inputs from two upstream regions.

A brain region typically receives inputs from multiple upstream areas. However, currently, no method is available to selectively dissect neurons that receive monosynaptic inputs from two upstream regions. Here, we developed a method to genetically label such neurons with a single gene of interest in mice by combining the anterograde transsynaptic spread of adeno-associated virus serotype 1 (AAV1) with intersectional gene expression. Injections of AAV1 expressing either Cre or Flpo recombinases and the Cre/Flpo double-dependent AAV into two upstream regions and the downstream region, respectively, were used to label postsynaptic neurons receiving inputs from the two upstream regions. We demonstrated this labelling in two distinct circuits: the retina/primary visual cortex to the superior colliculus and the bilateral motor cortex to the dorsal striatum. Systemic delivery of the intersectional AAV allowed the unbiased detection of the labelled neurons throughout the brain. This strategy may help analyse the interregional integration of information in the brain.

Functional quantitative susceptibility mapping (fQSM) of rat brain during flashing light stimulation.

Functional magnetic resonance imaging (fMRI) based on the blood oxygenation level-dependent (BOLD) contrast has become an indispensable tool in neuroscience. However, the BOLD signal is nonlocal, lacking quantitative measurement of oxygenation fluctuation. This preclinical study aimed to introduced functional quantitative susceptibility mapping (fQSM) to complement BOLD-fMRI to quantitatively assess the local susceptibility and venous oxygen saturation (SvO2). Rats were subjected to a 5 Hz flashing light and the different inhaled oxygenation levels (30% and 100%) were used to observe the venous susceptibility to quantify SvO2. Phase information was extracted to produce QSM, and the activation responses of magnitude (conventional BOLD) and the QSM time-series were analyzed. During light stimulation, the susceptibility change of fQSM was four times larger than the BOLD signal change in both inhalation oxygenation conditions. Moreover, the responses in the fQSM map were more restricted to the visual pathway, such as the lateral geniculate nucleus and superior colliculus, compared with the relatively diffuse distributions in the BOLD map. Also, the calibrated SvO2 was approximately 84% (88%) when the task was on, 83% (87%) when the task was off during 30% (and during 100%) oxygen inhalation. This is the first fQSM study in a small animal model and increases our understanding of fQSM in the brains of small animals. This study demonstrated the feasibility, sensitivity, and specificity of fQSM using light stimulus, as fQSM provides quantitative clues as well as localized information, complementing the defects of BOLD-fMRI. In addition to neural activity, fQSM also assesses SvO2 as supplementary information while BOLD-fMRI dose not. Accordingly, the fQSM technique could be a useful quantitative tool for functional studies, such as longitudinal follow up of neurodegenerative diseases, functional recovery after brain surgery, and negative BOLD studies.

Circuits That Mediate Expression of Signaled Active Avoidance Converge in the Pedunculopontine Tegmentum.

An innocuous sensory stimulus that reliably signals an upcoming aversive event can be conditioned to elicit locomotion to a safe location before the aversive outcome ensues. The neural circuits that mediate the expression of this signaled locomotor action, known as signaled active avoidance, have not been identified. While exploring sensorimotor midbrain circuits in mice of either sex, we found that excitation of GABAergic cells in the substantia nigra pars reticulata blocks signaled active avoidance by inhibiting cells in the pedunculopontine tegmental nucleus (PPT), not by inhibiting cells in the superior colliculus or thalamus. Direct inhibition of putative-glutamatergic PPT cells, excitation of GABAergic PPT cells, or excitation of GABAergic afferents in PPT, abolish signaled active avoidance. Conversely, excitation of putative-glutamatergic PPT cells, or inhibition of GABAergic PPT cells, can be tuned to drive avoidance responses. The PPT is an essential junction for the expression of signaled active avoidance gated by nigral and other synaptic afferents.SIGNIFICANCE STATEMENT When a harmful situation is signaled by a sensory stimulus (e.g., street light), subjects typically learn to respond with active or passive avoidance responses that circumvent the threat. During signaled active avoidance behavior, subjects move away to avoid a threat signaled by a preceding innocuous stimulus. We identified a part of the midbrain essential to process the signal and avoid the threat. Inhibition of neurons in this area eliminates avoidance responses to the signal but preserves escape responses caused by presentation of the threat. The results highlight an essential part of the neural circuits that mediate signaled active avoidance behavior.

A collicular visual cortex: Neocortical space for an ancient midbrain visual structure.

Visual responses in the cerebral cortex are believed to rely on the geniculate input to the primary visual cortex (V1). Indeed, V1 lesions substantially reduce visual responses throughout the cortex. Visual information enters the cortex also through the superior colliculus (SC), but the function of this input on visual responses in the cortex is less clear. SC lesions affect cortical visual responses less than V1 lesions, and no visual cortical area appears to entirely rely on SC inputs. We show that visual responses in a mouse lateral visual cortical area called the postrhinal cortex are independent of V1 and are abolished upon silencing of the SC. This area outperforms V1 in discriminating moving objects. We thus identify a collicular primary visual cortex that is independent of the geniculo-cortical pathway and is capable of motion discrimination.

In vivo effect of acute exposure to interleukin-6 on the developing visual system.

Interleukin-6 (IL-6) is involved in different processes of the central nervous system. Our aims were to investigate the effect of IL-6 on retinotectal topography and on different signaling pathways. Rats were submitted to an intravitreous injection of either IL-6 (50 ng/ml) or PBS (vehicle) at postnatal day 10 (PND10). At PND11 or PND14, different groups were processed for western blot, histochemistry or immunofluorescence analysis. IL-6 treatment leads to an increase in pSTAT-3 levels in the retina and a disruption in the retinotectal topographic map, suggesting that a transient increase in interleukin-6 levels may impact neural circuitry development.

Sheets of human retinal progenitor transplants improve vision in rats with severe retinal degeneration.

Loss of photoreceptors and other retinal cells is a common endpoint in retinal degenerate (RD) diseases that cause blindness. Retinal transplantation is a potential therapy to replace damaged retinal cells and improve vision. In this study, we examined the development of human fetal retinal sheets with or without their retinal pigment epithelium (RPE) transplanted to immunodeficient retinal degenerate rho S334ter-3 rats. Sheets were dissected from fetal human eyes (11-15.7 weeks gestation) and then transplanted to the subretinal space of 24-31 d old RD nude rats. Every month post surgery, eyes were imaged by high-resolution spectral-domain optical coherence tomography (SD-OCT). SD-OCT showed that transplants were placed into the subretinal space and developed laminated areas or rosettes, with clear development of plexiform layers first seen in OCT at 3 months post surgery. Several months later, as could be expected by the much slower development of human cells compared to rat cells, transplant photoreceptors developed inner and later outer segments. Retinal sections were analyzed by immunohistochemistry for human and retinal markers and confirmed the formation of several retinal subtypes within the retinal layers. Transplant cells extended processes and a lot of the cells could also be seen migrating into the host retina. At 5.8-8.6 months post surgery, selected rats were exposed to light flashes and recorded for visual responses in superior colliculus, (visual center in midbrain). Four of seven rats with transplants showed responses to flashes of light in a limited area of superior colliculus. No response with the same dim light intensity was found in age-matched RD controls (non-surgery or sham surgery). In summary, our data showed that human fetal retinal sheets transplanted to the severely disturbed subretinal space of RD nude rats develop mature photoreceptors and other retinal cells, integrate with the host and induce vision improvement.

Stress Accelerates Defensive Responses to Looming in Mice and Involves a Locus Coeruleus-Superior Colliculus Projection.

Defensive responses to threatening stimuli are crucial to the survival of species. While expression of these responses is considered to be instinctive and unconditional, their magnitude may be affected by environmental and internal factors. The neural circuits underlying this modulation are still largely unknown. In mice, looming-evoked defensive responses are mediated by the superior colliculus (SC), a subcortical sensorimotor integration center. We found that repeated stress caused an anxiety-like state in mice and accelerated defensive responses to looming. Stress also induced c-fos activation in locus coeruleus (LC) tyrosine hydroxylase (TH)+ neurons and modified adrenergic receptor expression in SC, suggesting a possible Th::LC-SC projection that may be involved in the accelerated defensive responses. Indeed, both anterograde and retrograde neural tracing confirmed the anatomical Th::LC-SC projection and that the SC-projecting TH+ neurons in LC were activated by repeated stress. Optogenetic stimulation of either LC TH+ neurons or the Th::LC-SC fibers also caused anxiety-like behaviors and accelerated defensive responses to looming. Meanwhile, chemogenetic inhibition of LC TH+ neurons and the infusion of an adrenergic receptor antagonist in SC abolished the enhanced looming defensive responses after repeated stress, confirming the necessity of this pathway. These findings suggest that the Th::LC-SC pathway plays a key role in the sophisticated adjustments of defensive behaviors induced by changes in physiological states.

Role of the visual experience-dependent nascent proteome in neuronal plasticity.

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity.

Frontal Eye Field Inactivation Diminishes Superior Colliculus Activity, But Delayed Saccadic Accumulation Governs Reaction Time Increases.

Stochastic accumulator models provide a comprehensive framework for how neural activity could produce behavior. Neural activity within the frontal eye fields (FEFs) and intermediate layers of the superior colliculus (iSC) support such models for saccade initiation by relating variations in saccade reaction time (SRT) to variations in such parameters as baseline, rate of accumulation of activity, and threshold. Here, by recording iSC activity during reversible cryogenic inactivation of the FEF in four male nonhuman primates, we causally tested which parameter(s) best explains concomitant increases in SRT. While FEF inactivation decreased all aspects of ipsilesional iSC activity, decreases in accumulation rate and threshold poorly predicted accompanying increases in SRT. Instead, SRT increases best correlated with delays in the onset of saccade-related accumulation. We conclude that FEF signals govern the onset of saccade-related accumulation within the iSC, and that the onset of accumulation is a relevant parameter for stochastic accumulation models of saccade initiation.SIGNIFICANCE STATEMENT The superior colliculus (SC) and frontal eye fields (FEFs) are two of the best-studied areas in the primate brain. Surprisingly, little is known about what happens in the SC when the FEF is temporarily inactivated. Here, we show that temporary FEF inactivation decreases all aspects of functionally related activity in the SC. This combination of techniques also enabled us to relate changes in SC activity to concomitant increases in saccadic reaction time (SRT). Although stochastic accumulator models relate SRT increases to reduced rates of accumulation or increases in threshold, such changes were not observed in the SC. Instead, FEF inactivation delayed the onset of saccade-related accumulation, emphasizing the importance of this parameter in biologically plausible models of saccade initiation.

Panicolytic-like effects caused by substantia nigra pars reticulata pretreatment with low doses of endomorphin-1 and high doses of CTOP or the NOP receptors antagonist JTC-801 in male Rattus norvegicus.

RATIONALE: Gamma-aminobutyric acid (GABA)ergic neurons of the substantia nigra pars reticulata (SNpr) are connected to the deep layers of the superior colliculus (dlSC). The dlSC, in turn, connect with the SNpr through opioid projections. Nociceptin/orphanin FQ peptide (N/OFQ) is a natural ligand of a Gi protein-coupled nociceptin receptor (ORL1; NOP) that is also found in the SNpr. Our hypothesis is that tectonigral opioid pathways and intranigral orphanin-mediated mechanisms modulate GABAergic nigrotectal connections. OBJECTIVES: Therefore, the aim of this work was to study the role of opioid and NOP receptors in the SNpr during the modulation of defence reactions organised by the dlSC. METHODS: The SNpr was pretreated with either opioid or NOP receptor agonists and antagonists, followed by dlSC treatment with bicuculline. RESULTS: Blockade of GABAA receptors in the dlSC elicited fear-related defensive behaviour. Pretreatment of the SNpr with naloxone benzoylhydrazone (NalBzoH), a µ-, δ-, and κ1-opioid receptor antagonist as well as a NOP receptor antagonist, decreased the aversive effect of bicuculline treatment on the dlSC. Either µ-opioid receptor activation or blockade by SNpr microinjection of endomorphin-1 (EM-1) and CTOP promoted pro-aversive and anti-aversive actions, respectively, that modulated the defensive responses elicited by bicuculline injection into the dlSC. Pretreatment of the SNpr with the selective NOP receptor antagonist JTC801 decreased the aversive effect of bicuculline, and microinjections of the selective NOP receptor agonist NNC 63-0532 promoted the opposite effect. CONCLUSIONS: These results demonstrate that opioid pathways and orphanin-mediated mechanisms have a critical role in modulating the activity of nigrotectal GABAergic pathways during the organisation of defensive behaviours.

Simultaneous GCaMP6-based fiber photometry and fMRI in rats.

BACKGROUND: Understanding the relationship between neural and vascular signals is essential for interpretation of functional MRI (fMRI) results with respect to underlying neuronal activity. Simultaneously measuring neural activity using electrophysiology with fMRI has been highly valuable in elucidating the neural basis of the blood oxygenation-level dependent (BOLD) signal. However, this approach is also technically challenging due to the electromagnetic interference that is observed in electrophysiological recordings during MRI scanning. NEW METHOD: Recording optical correlates of neural activity, such as calcium signals, avoids this issue, and has opened a new avenue to simultaneously acquire neural and BOLD signals. RESULTS: The present study is the first to demonstrate the feasibility of simultaneously and repeatedly acquiring calcium and BOLD signals in animals using a genetically encoded calcium indicator, GCaMP6. This approach was validated with a visual stimulation experiment, during which robust increases of both calcium and BOLD signals in the superior colliculus were observed. In addition, repeated measurement in the same animal demonstrated reproducible calcium and BOLD responses to the same stimuli. COMPARISON WITH EXISTING METHOD(S): Taken together, simultaneous GCaMP6-based fiber photometry and fMRI recording presents a novel, artifact-free approach to simultaneously measuring neural and fMRI signals. Furthermore, given the cell-type specificity of GCaMP6, this approach has the potential to mechanistically dissect the contributions of individual neuron populations to BOLD signal, and ultimately reveal its underlying neural mechanisms. CONCLUSIONS: The current study established the method for simultaneous GCaMP6-based fiber photometry and fMRI in rats.

PSA-NCAM expression in the teleost optic tectum is related to ecological niche and use of vision in finding food.

In this study, tangential migration and neuronal connectivity organization were analysed in the optic tectum of seven different teleosts through the expression of polysialylated neural cell adhesion molecule (PSA-NCAM) in response to ecological niche and use of vision. Reduced PSA-NCAM expression in rainbow trout Oncorhynchus mykiss optic tectum occurred in efferent layers, while in pike Esox lucius and zebrafish Danio rerio it occurred in afferent and efferent layers. Zander Sander lucioperca and European eel Anguilla anguilla had very low PSA-NCAM expression in all tectal layers except in the stratum marginale. Common carp Cyprinus carpio and wels catfish Silurus glanis had the same intensity of PSA-NCAM expression in all tectal layers. The optic tectum of all studied fishes was also a site of tangential migration with sustained PSA-NCAM and c-series ganglioside expression. Anti-c-series ganglioside immunoreactivity was observed in all tectal layers of all analysed fishes, even in layers where PSA-NCAM expression was reduced. Since the optic tectum is indispensable for visually guided prey capture, stabilization of synaptic contact and decrease of neurogenesis and tangential migration in the visual map are an expected adjustment to ecological niche. The authors hypothesize that this stabilization would probably be achieved by down-regulation of PSA-NCAM rather than c-series of ganglioside.

AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors.

To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits.

Restoration of Visual Function by Enhancing Conduction in Regenerated Axons.

Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury.

Nicotine-induced plasticity in the retinocollicular pathway: Evidence for involvement of amyloid precursor protein.

During early postnatal development retinocollicular projections undergo activity-dependent synaptic refinement that results in the formation of precise topographical maps in the visual layers of the superior colliculus (SC). Amyloid Precursor Protein (APP) is a widely expressed transmembrane glycoprotein involved in the regulation of several aspects of neural development, such as neurite outgrowth, synapse formation and plasticity. Stimulation of cholinergic system has been found to alter the expression and processing of APP in different cell lines. Herein, we investigated the effect of nicotine on the development of retinocollicular pathway and on APP metabolism in the SC of pigmented rats. Animals were submitted to intracranial Elvax implants loaded with nicotine or phosphate-buffered saline (vehicle) at postnatal day (PND) 7. The ipsilateral retinocollicular pathway of control and experimental groups was anterogradely labeled either 1 or 3 weeks after surgery (PND 14 or PND 28). Local nicotine exposure produces a transitory sprouting of uncrossed retinal axons outside their main terminal zones. Nicotine also increases APP content and its soluble neurotrophic fragment sAPPα. Furthermore, nicotine treatment upregulates nicotinic acetylcholine receptor α7 and ß2 subunits. Taken together, these data indicate that nicotine disrupts the ordering and topographic mapping of axons in the retinocollicular pathway and facilitates APP processing through the nonamyloidogenic pathway, suggesting that sAPPα may act as a trophic agent that mediates nicotine-induced morphological plasticity.

GnRH suppresses excitability of visual processing neurons in the optic tectum.

Animals change their behavior in response to sensory cues in the environment as well as their physiological status. For example, it is generally accepted that their sexual behavior is modulated according to seasonal environmental changes or the individual's maturational/reproductive status, and neuropeptides have been suggested to play important roles in this process. Some behavioral modulation arises from neuropeptide modulation of sensory information processing in the central nervous system, but the neural mechanisms still remain unknown. Here we focused on the neural basis of neuropeptide modulation of visual processing in vertebrates. The terminal nerve neurons that contain gonadotropin-releasing hormone 3 (TN-GnRH3 neurons) are suggested to modulate reproductive behavior and have massive projections to the optic tectum (OT), which plays an important role in visual processing. In the present study, to examine whether GnRH3 modulates retino-tectal neurotransmission in the OT, we analyzed the effect of GnRH3 electrophysiologically and morphologically. We found that field potentials evoked by optic tract fiber stimulation, which represent retino-tectal neurotransmission, were modulated postsynaptically by GnRH3. Whole cell recording from postsynaptic neurons in the retino-tectal pathway suggested that GnRH3 activates large-conductance Ca(2+)-activated K(+) (BK) channels and thereby suppresses membrane excitability. Furthermore, our improved morphological analysis using fluorescently labeled GnRH peptides showed that GnRH receptors are localized mainly around the cell bodies of postsynaptic neurons. Our results indicate that TN-GnRH3 neurons modulate retino-tectal neurotransmission by suppressing the excitability of projection neurons in the OT, which underlies the neuromodulation of behaviorally relevant visual information processing by the neuropeptide GnRH3.

Parvalbumin-immunoreactive cells in the superior colliculus in dog: distribution, colocalization with GABA, and effect of monocular enucleation.

Parvalbumin (PV) is thought to play a major role in buffering intracellular calcium. We studied the distribution, morphology of PV-immunoreactive (IR) cells, and the effect of enucleation on the PV distribution in the superior colliculus (SC) in dog (Canis familiaris) and compared PV labeling to that of calbindin D28K (CB) and GABA. These cells formed three laminar tiers in the dog SC; 1) the upper superficial gray layer (SGL), 2) the lower optic layer (OL) and the upper intermediate gray layer, and 3) the deep layer. The third tier was not very distinct when compared with the other two tiers. The distribution of PV-IR cells is thus complementary to that of CB-IR tiers. Our present data on the distribution of PV-IR cells within the superficial layers are strikingly different from those in previously studied mammals, which show PV-IR cells within the lower SGL and upper OL. However, there were no distinct differences in distribution within the deep layers compared with that of previously studied mammals. PV-IR cells in the SC varied dramatically in morphology and size, and included round/oval, vertical fusiform, stellate, horizontal and pyriform cells. Two-color immunofluorescence revealed quantitatively that 11.67% of the PV-IR cells colocalized with GABA. Monocular enucleation appeared to have no effect on the distribution of PV-IR cells in the contralateral SC. Similar to CB, these data suggest that retinal projection may not control the expression of PV in the dog SC. These results provide important information for delineating similarities and differences in the neurochemical architecture of the visual system.

Macaque dorsolateral prefrontal cortex does not suppress saccade-related activity in the superior colliculus.

Of the many functions ascribed to the dorsolateral prefrontal cortex (DLPFC), the ability to override automatic stimulus-driven behavior is one of the most prominent. This ability has been investigated extensively with the antisaccade task, which requires suppression of saccades toward suddenly appearing visual stimuli. Convergent lines of evidence have supported a model in which the DLPFC suppresses unwanted saccades by inhibiting saccade-related activity in the ipsilateral superior colliculus (SC), a midbrain oculomotor structure. Here, we carried out a direct test of this inhibitory model using unilateral cryogenic deactivation of the DLPFC within the caudal principal sulcus (cPS) and simultaneous single-neuron recording of SC saccade-related neurons in monkeys performing saccades and antisaccades. Contrary to the inhibition model, which predicts that attenuation of inhibition effected by unilateral cPS deactivation should result in activity increases in ipsilateral and decreases in contralateral SC, we observed a delayed onset of saccade-related activity in the ipsilateral SC, and activity increases in the contralateral SC. These effects were mirrored by increased error rates of ipsiversive antisaccades, and reaction times of contraversive saccades. These data challenge the inhibitory model and suggest instead that the primary influence of the DLPFC on the SC is excitatory.

RNA-binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system.

Local synthesis of ß-actin is required for attractive turning responses to guidance cues of growth cones in vitro but its functional role in axon guidance in vivo is poorly understood. The transport and translation of ß-actin mRNA is regulated by the RNA-binding protein, Vg1RBP (zipcode-binding protein-1). To examine whether Vg1RBP plays a role in axon navigation in vivo, we disrupted Vg1RBP function in embryonic Xenopus laevis retinal ganglion cells by expressing a dominant-negative Vg1RBP and by antisense morpholino knockdown. We found that attractive turning to a netrin-1 gradient in vitro was abolished in Vg1RBP-deficient axons but, surprisingly, the long-range navigation from the retina to the optic tectum was unaffected. Within the tectum, however, the branching and complexity of axon terminals were significantly reduced. High-resolution time-lapse imaging of axon terminals in vivo revealed that Vg1RBP-GFP-positive granules accumulate locally in the axon shaft immediately preceding the emergence a filopodial-like protrusion. Comparative analysis of branch dynamics showed that Vg1RBP-deficient axons extend far fewer filopodial-like protrusions than control axons and indicate that Vg1RBP promotes filopodial formation, an essential step in branch initiation. Our findings show that Vg1RBP is required for terminal arborization but not long-range axon navigation and suggest that Vg1RBP-regulated mRNA translation promotes synaptic complexity.

Viral vector-mediated selective and reversible blockade of the pathway for visual orienting in mice.

Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT) tagged with enhanced green fluorescent protein (EGFP) under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7-17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5-9 weeks later, the daily administration of doxycycline (Dox) was initiated. Visual orienting responses toward the left side were impaired 1-4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission also causes behavioral effects in rodents, and that the crossed tecto-reticular pathway clearly controls visual orienting behaviors.