Postsynaptic NMDA Receptor Expression Is Required for Visual Corticocollicular Projection Refinement in the Mouse Superior Colliculus.

Efficient sensory processing of spatial information is facilitated through the organization of neuronal connections into topographic maps of space. In integrative sensory centers, converging topographic maps must be aligned to merge spatially congruent information. The superior colliculus (SC) receives topographically ordered visual inputs from retinal ganglion cells (RGCs) in the eye and layer 5 neurons in the primary visual cortex (L5-V1). Previous studies suggest that RGCs instruct the alignment of later-arriving L5-V1 inputs in an activity-dependent manner. However, the molecular mechanisms underlying this remain unclear. Here, we explored the role of NMDA receptors in visual map alignment in the SC using a conditional genetic knockout approach. We leveraged a novel knock-in mouse line that expresses tamoxifen-inducible Cre recombinase under the control of the Tal1 gene (Tal1CreERT2 ), which we show allows for specific recombination in the superficial layers of the SC. We used Tal1CreERT2 mice of either sex to conditionally delete the obligate GluN1 subunit of the NMDA receptor (SC-cKO) during the period of visual map alignment. We observed a significant disruption of L5-V1 axon terminal organization in the SC of SC-cKO mice. Importantly, retinocollicular topography was unaffected in this context, suggesting that alignment is also disrupted. Time-course experiments suggest that NMDA receptors may play a critical role in the refinement of L5-V1 inputs in the SC. Together, these data implicate NMDA receptors as critical mediators of activity-dependent visual map alignment in the SC.SIGNIFICANCE STATEMENT Alignment of topographic inputs is critical for integration of spatially congruent sensory information; however, little is known about the mechanisms underlying this complex process. Here, we took a conditional genetic approach to explore the role of NMDA receptors in the alignment of retinal and cortical visual inputs in the superior colliculus. We characterize a novel mouse line providing spatial and temporal control of recombination in the superior colliculus and reveal a critical role for NMDA expression in visual map alignment. These data support a role for neuronal activity in visual map alignment and provide mechanistic insight into this complex developmental process.

Retinal input instructs alignment of visual topographic maps.

Sensory information is represented in the brain in the form of topographic maps, in which neighboring neurons respond to adjacent external stimuli. In the visual system, the superior colliculus receives topographic projections from the retina and primary visual cortex (V1) that are aligned. Alignment may be achieved through the use of a gradient of shared axon guidance molecules, or through a retinal-matching mechanism in which axons that monitor identical regions of visual space align. To distinguish between these possibilities, we take advantage of genetically engineered mice that we show have a duplicated functional retinocollicular map but only a single map in V1. Anatomical tracing revealed that the corticocollicular projection bifurcates to align with the duplicated retinocollicular map in a manner dependent on the normal pattern of spontaneous activity during development. These data suggest a general model in which convergent maps use coincident activity patterns to achieve alignment.

A cell-ECM mechanism for connecting the ipsilateral eye to the brain.

Information about features in the visual world is parsed by circuits in the retina and is then transmitted to the brain by distinct subtypes of retinal ganglion cells (RGCs). Axons from RGC subtypes are stratified in retinorecipient brain nuclei, such as the superior colliculus (SC), to provide a segregated relay of parallel and feature-specific visual streams. Here, we sought to identify the molecular mechanisms that direct the stereotyped laminar targeting of these axons. We focused on ipsilateral-projecting subtypes of RGCs (ipsiRGCs) whose axons target a deep SC sublamina. We identified an extracellular glycoprotein, Nephronectin (NPNT), whose expression is restricted to this ipsiRGC-targeted sublamina. SC-derived NPNT and integrin receptors expressed by ipsiRGCs are both required for the targeting of ipsiRGC axons to the deep sublamina of SC. Thus, a cell-extracellular matrix (ECM) recognition mechanism specifies precise laminar targeting of ipsiRGC axons and the assembly of eye-specific parallel visual pathways.

Diverse modes of binocular interactions in the mouse superior colliculus.

The superior colliculus (SC) integrates visual and other sensory information to regulate critical reflexive and innate behaviors, such as prey capture. In the mouse, the vast majority of retinal ganglion cells (RGCs) innervate the SC, including inputs from both the contralateral (contra-RGCs) and ipsilateral (ipsi-RGCs) eye. Despite this, previous studies revealed minimal neuronal responses to ipsilateral stimulation and few binocular interactions in the mouse SC. More recent work suggests that ipsi-RGC function and innervation of the SC are critical for efficient prey capture, raising the possibility that binocular interactions in the mouse SC may be more prevalent than previously thought. To explore this possibility, we investigated eye-specific and binocular influences on visual responses and tuning of SC neurons, focusing on the anteromedial region. Although the majority of SC neurons were primarily driven by contralateral eye stimulation, we observed that a substantial proportion of units were influenced or driven by ipsilateral stimulation. Clustering based on differential responses to eye-specific stimulus presentation revealed five distinct putative subpopulations and multiple modes of binocular interaction, including facilitation, summation, and suppression. Each of the putative subpopulations exhibited selectivity for orientation, and differences in spatial frequency tuning and spatial summation properties were observed between subpopulations. Further analysis of orientation tuning under different ocular conditions supported differential modes of binocular interaction between putative subtypes. Taken together, these data suggest that binocular interactions in the mouse SC may be more prevalent and diverse than previously understood.NEW & NOTEWORTHY The mouse superior colliculus (SC) receives binocular inputs, which inform complex behavioral programs. However, we know surprisingly little about binocular tuning in the rodent SC. Here, we characterize responses to eye-specific presentations of visual stimuli and reveal a previously unappreciated diversity of binocularly modulated neurons in the SC. This foundational work broadens our understanding of visual processing in the SC and sets the stage for future studies interrogating the circuit mechanisms underlying binocular tuning.

A Chrnb3-Cre BAC transgenic mouse line for manipulation of gene expression in retinal ganglion cells.

The mechanisms by which retinal ganglion cells (RGCs) make specific connections during development is an intense area of research and have served as a model for understanding the general principles of circuit wiring. As such, genetic tools allowing for specific recombination in RGCs are critical to further our understanding of the cell-specific roles of different genes during these processes. However, many RGC-specific Cre lines have drawbacks, due to their broad expression in other cell types and/or retinorecipient regions or lack of expression in broad swaths of the retina. Here, we characterize a Cre BAC transgenic line driven by elements of the cholinergic receptor nicotinic beta 3 subunit (Chrnb3). We show that Cre expression is restricted to RGCs in the retina and sparsely expressed in the brain, importantly excluding retinorecipient regions. Furthermore, Chrnb3-Cre mice label a wide variety of RGCs distributed throughout the retina and Cre activity is detected embryonically, shortly following RGC differentiation. Finally, we find that Chrnb3-Cre-labeled RGCs innervate multiple retinorecipient areas that serve both image-forming and nonimage forming functions. Thus, this genetic tool will be of broad use to investigators studying the RGC-specific contributions of genes to visual circuit development.

The Elegance of Sonic Hedgehog: Emerging Novel Functions for a Classic Morphogen.

Sonic Hedgehog (SHH) signaling has been most widely known for its role in specifying region and cell-type identity during embryonic morphogenesis. This mini-review accompanies a 2018 SFN mini-symposium that addresses an emerging body of research focused on understanding the diverse roles for Shh signaling in a wide range of contexts in neurodevelopment and, more recently, in the mature CNS. Such research shows that Shh affects the function of brain circuits, including the production and maintenance of diverse cell types and the establishment of wiring specificity. Here, we review these novel and unexpected functions and the unanswered questions regarding the role of SHH and its signaling pathway members in these cases.

Novel Models of Visual Topographic Map Alignment in the Superior Colliculus.

The establishment of precise neuronal connectivity during development is critical for sensing the external environment and informing appropriate behavioral responses. In the visual system, many connections are organized topographically, which preserves the spatial order of the visual scene. The superior colliculus (SC) is a midbrain nucleus that integrates visual inputs from the retina and primary visual cortex (V1) to regulate goal-directed eye movements. In the SC, topographically organized inputs from the retina and V1 must be aligned to facilitate integration. Previously, we showed that retinal input instructs the alignment of V1 inputs in the SC in a manner dependent on spontaneous neuronal activity; however, the mechanism of activity-dependent instruction remains unclear. To begin to address this gap, we developed two novel computational models of visual map alignment in the SC that incorporate distinct activity-dependent components. First, a Correlational Model assumes that V1 inputs achieve alignment with established retinal inputs through simple correlative firing mechanisms. A second Integrational Model assumes that V1 inputs contribute to the firing of SC neurons during alignment. Both models accurately replicate in vivo findings in wild type, transgenic and combination mutant mouse models, suggesting either activity-dependent mechanism is plausible. In silico experiments reveal distinct behaviors in response to weakening retinal drive, providing insight into the nature of the system governing map alignment depending on the activity-dependent strategy utilized. Overall, we describe novel computational frameworks of visual map alignment that accurately model many aspects of the in vivo process and propose experiments to test them.

Eph and ephrin signaling in the formation of topographic maps.

The axonal connections between the retina and its midbrain target, the superior colliculus (SC), is mapped topographically, such that the spatial relationships of cell bodies in the retina are maintained when terminating in the SC. Topographic map development uses a Cartesian mapping system such that each axis of the retina is mapped independently. Along the nasal-temporal mapping axis, EphAs and ephrin-As, are graded molecular cues required for topographic mapping while the dorsal-ventral axis is mapped in part via EphB and ephrin-Bs. Because both Ephs and ephrins are cell surface molecules they can signal in the forward and reverse directions. Eph/ephrin signaling leads to changes in cytoskeletal dynamics that lead to actin depolymerization and endocytosis guiding axons via attraction and repulsion.

Estrogen receptor-ß regulates mechanical signaling in primary osteoblasts.

Mechanical loading is an important regulator in skeletal growth, maintenance, and aging. Estrogen receptors have a regulatory role in mechanically induced bone adaptation. Estrogen receptor-α (ERα) is known to enhance load-induced bone formation, whereas ERß negatively regulates this process. We hypothesized that ERß regulates mechanical signaling in osteoblasts. We tested this hypothesis by subjecting primary calvarial cells isolated from wild-type and ERß-knockout mice (BERKO) to oscillatory fluid flow in the absence or presence of estradiol (E2). We found that the known responses to fluid shear stress, i.e., phosphorylation of the mitogen-activated protein kinase ERK and upregulation of COX-2 expression, were inhibited in BERKO cells in the absence of E2. Flow-induced increase in prostaglandin E2 (PGE2) release was not altered in BERKO cells in the absence of E2, but was increased when E2 was present. Additionally, immunofluorescence analysis and estrogen response element luciferase assays revealed increased ERα expression and flow- and ligand-induced nuclear translocation as well as transcriptional activity in BERKO cells in both the presence and absence of E2. Taken together, these data suggest that ERß plays both ligand-dependent and ligand-independent roles in mechanical signaling in osteoblasts. Furthermore, our data suggest that one mechanism by which ERß regulates mechanotransduction in osteoblasts may result from its inhibitory effect on ERα expression and function. Targeting estrogen receptors (e.g., inhibiting ERß) may represent an effective approach for prevention and treatment of age-related bone loss.

Polysialylated NCAM and ephrinA/EphA regulate synaptic development of GABAergic interneurons in prefrontal cortex.

A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry.

MINERALS IN THE HUMAN BODY: Erionite and offretite from the Killdeer Mountains, Dunn County, North Dakota, U.S.A.

The carcinogenic potential of erionite has sparked concern about human exposure in areas where it is present in regional bedrock. The Arikaree Formation in western North Dakota contains altered tuffaceous units with authigenic zeolites. We sampled stratigraphic profiles in the Killdeer Mountains, Dunn County, North Dakota, to determine the distribution and chemical composition of zeolites. Powder X-ray diffraction, SEM/EDS and electron microprobe analyses were carried out on sample concentrates. Only samples stratigraphically in or below the distinctive burrowed marker unit were found to contain zeolites. Erionite and offretite were the most common zeolites identified, with offretite being more abundant based on frequency of measured Mg/(Ca+Na) ratios. Intermediate chemical compositions could be natural or due to intimate intergrowths of the two minerals. A better understanding is needed of the potential toxicity across the range of erionite and offretite compositions.

Competition is a driving force in topographic mapping.

Topographic maps are the primary means of relaying spatial information in the brain. Understanding the mechanisms by which they form has been a goal of experimental and theoretical neuroscientists for decades. The projection of the retina to the superior colliculus (SC)/tectum has been an important model used to show that graded molecular cues and patterned retinal activity are required for topographic map formation. Additionally, interaxon competition has been suggested to play a role in topographic map formation; however, this view has been recently challenged. Here we present experimental and computational evidence demonstrating that interaxon competition for target space is necessary to establish topography. To test this hypothesis experimentally, we determined the nature of the retinocollicular projection in Math5 (Atoh7) mutant mice, which have severely reduced numbers of retinal ganglion cell inputs into the SC. We find that in these mice, retinal axons project to the anteromedialj portion of the SC where repulsion from ephrin-A ligands is minimized and where their attraction to the midline is maximized. This observation is consistent with the chemoaffinity model that relies on axon-axon competition as a mapping mechanism. We conclude that chemical labels plus neural activity cannot alone specify the retinocollicular projection; instead axon-axon competition is necessary to create a map. Finally, we present a mathematical model for topographic mapping that incorporates molecular labels, neural activity, and axon competition.

Alignment of multimodal sensory input in the superior colliculus through a gradient-matching mechanism.

The superior colliculus (SC) is a midbrain structure that integrates visual, somatosensory, and auditory inputs to direct head and eye movements. Each of these modalities is topographically mapped and aligned with the others to ensure precise behavioral responses to multimodal stimuli. While it is clear that neural activity is instructive for topographic alignment of inputs from the visual cortex (V1) and auditory system with retinal axons in the SC, there is also evidence that activity-independent mechanisms are used to establish topographic alignment between modalities. Here, we show that the topography of the projection from primary somatosensory cortex (S1) to the SC is established during the first postnatal week. Unlike V1-SC projections, the S1-SC projection does not bifurcate when confronted with a duplicated retinocollicular map, showing that retinal input in the SC does not influence the topography of the S1-SC projection. However, S1-SC topography is disrupted in mice lacking ephrin-As, which we find are expressed in graded patterns along with their binding partners, the EphA4 and EphA7, in both S1 and the somatosensory recipient layer of the SC. Together, these data support a model in which somatosensory inputs into the SC map topographically and establish alignment with visual inputs in the SC using a gradient-matching mechanism.

Wiring subcortical image-forming centers: Topography, laminar targeting, and map alignment.

Efficient sensory processing is a complex and important function for species survival. As such, sensory circuits are highly organized to facilitate rapid detection of salient stimuli and initiate motor responses. For decades, the retina's projections to image-forming centers have served as useful models to elucidate the mechanisms by which such exquisite circuitry is wired. In this chapter, we review the roles of molecular cues, neuronal activity, and axon-axon competition in the development of topographically ordered retinal ganglion cell (RGC) projections to the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN). Further, we discuss our current state of understanding regarding the laminar-specific targeting of subclasses of RGCs in the SC and its homolog, the optic tectum (OT). Finally, we cover recent studies examining the alignment of projections from primary visual cortex with RGCs that monitor the same region of space in the SC.

NMDA Receptor Expression by Retinal Ganglion Cells Is Not Required for Retinofugal Map Formation nor Eye-Specific Segregation in the Mouse.

Retinal ganglion cells (RGCs) project topographically to the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN). Spontaneous activity plays a critical role in retinotopic mapping in both regions; however, the molecular mechanisms underlying activity-dependent refinement remain unclear. Previous pharmacologic studies implicate NMDA receptors (NMDARs) in the establishment of retinotopy. In other brain regions, NMDARs are expressed on both the presynaptic and postsynaptic side of the synapse, and recent work suggests that presynaptic and postsynaptic NMDARs play distinct roles in retinotectal developmental dynamics. To directly test the role of NMDARs expressed by RGCs in retinofugal map formation, we took a conditional genetic knock-out approach to delete the obligate GluN1 subunit of NMDARs in RGCs. Here, we demonstrate reduced GluN1 expression in the retina of Chrnb3-Cre;GluN1flox/flox (pre-cKO) mice without altered expression in the SC. Anatomical tracing experiments revealed no significant changes in termination zone size in the SC and dLGN of pre-cKO mice, suggesting NMDAR function in RGCs is not an absolute requirement for topographic refinement. Further, we observed no change in the eye-specific organization of retinal inputs to the SC nor dLGN. To verify that NMDA induces activity in RGC terminals, we restricted GCaMP5 expression to RGCs and confirmed induction of calcium transients in RGC terminals. Together, these findings demonstrate that NMDARs expressed by RGCs are not required for retinofugal topographic map formation nor eye-specific segregation in the mouse.

Visual subcircuit-specific dysfunction and input-specific mispatterning in the superior colliculus of fragile X mice.

BACKGROUND: Sensory processing deficits are frequently co-morbid with neurodevelopmental disorders. For example, patients with fragile X syndrome (FXS), caused by a silencing of the FMR1 gene, exhibit impairments in visual function specific to the dorsal system, which processes motion information. However, the developmental and circuit mechanisms underlying this deficit remain unclear. Recently, the superior colliculus (SC), a midbrain structure regulating head and eye movements, has emerged as a model for dissecting visual circuit development and function. Previous studies have demonstrated a critical role for activity-dependent processes in the development of visual circuitry in the SC. Based on the known role of the FMR1 gene product in activity-dependent synaptic plasticity, we explored the function and organization of visual circuits in the SC of a mouse model of FXS (Fmr1-/y). METHODS: We utilized in vivo extracellular electrophysiology in combination with computer-controlled visual stimuli to determine the receptive field properties of visual neurons in the SC of control and Fmr1-/y mice. In addition, we utilized anatomical tracing methods to assess the organization of visual inputs to the SC and along the retinogeniculocortical pathway. RESULTS: Receptive fields of visual neurons in the SC of Fmr1-/y mice were significantly larger than those found in control animals, though their shape and structure were unaffected. Further, selectivity for direction of movement was decreased, while selectivity to axis of movement was unchanged. Interestingly, axis-selective (AS) neurons exhibited a specific hyperexcitability in comparison to AS neurons in control SC and to direction-selective (DS) neurons in both control and Fmr1-/y SC. Anatomical tracings revealed that retinocollicular, retinogeniculate, and geniculocortical projections were normally organized in the absence of Fmr1. However, projections from primary visual cortex (V1) to the SC were poorly refined. CONCLUSIONS: Fmr1 is required for the proper development of visual circuit organization and function in the SC. We find that visual dysfunction is heterogeneously manifested in a subcircuit-specific manner in Fmr1-/y mice, consistent with previous studies in human FXS patients. Further, we show a specific alteration of inputs to the SC from V1, but not the retina. Together, these data suggest that Fmr1 may function in distinct ways during the development of different visual subcircuits.

Visual Neurons in the Superior Colliculus Innervated by Islet2+ or Islet2- Retinal Ganglion Cells Display Distinct Tuning Properties.

Throughout the visual system, different subtypes of neurons are tuned to distinct aspects of the visual scene, establishing parallel circuits. Defining the mechanisms by which such tuning arises has been a long-standing challenge for neuroscience. To investigate this, we have focused on the retina's projection to the superior colliculus (SC), where multiple visual neuron subtypes have been described. The SC receives inputs from a variety of retinal ganglion cell (RGC) subtypes; however, which RGCs drive the tuning of different SC neurons remains unclear. Here, we pursued a genetic approach that allowed us to determine the tuning properties of neurons innervated by molecularly defined subpopulations of RGCs. In homozygous Islet2-EphA3 knock-in (Isl2EA3/EA3) mice, Isl2+ and Isl2- RGCs project to non-overlapping sub-regions of the SC. Based on molecular and anatomic data, we show that significantly more Isl2- RGCs are direction-selective (DS) in comparison with Isl2+ RGCs. Targeted recordings of visual responses from each SC sub-region in Isl2EA3/EA3 mice revealed that Isl2- RGC-innervated neurons were significantly more DS than those innervated by Isl2+ RGCs. Axis-selective (AS) neurons were found in both sub-regions, though AS neurons innervated by Isl2+ RGCs were more tightly tuned. Despite this segregation, DS and AS neurons innervated by Isl2+ or Isl2- RGCs did not differ in their spatial summation or spatial frequency (SF) tuning. Further, we did not observe alterations in receptive field (RF) size or structure of SC neurons innervated by Isl2+ or Isl2- RGCs. Together, these data show that innervation by Isl2+ and Isl2- RGCs results in distinct tuning in the SC and set the stage for future studies investigating the mechanisms by which these circuits are built.

Stochastic Interaction between Neural Activity and Molecular Cues in the Formation of Topographic Maps.

Topographic maps in visual processing areas maintain the spatial order of the visual world. Molecular cues and neuronal activity both play critical roles in map formation, but their interaction remains unclear. Here, we demonstrate that when molecular- and activity-dependent cues are rendered nearly equal in force, they drive topographic mapping stochastically. The functional and anatomical representation of azimuth in the superior colliculus of heterozygous Islet2-EphA3 knockin (Isl2(EphA3/+)) mice is variable: maps may be single, duplicated, or a combination of the two. This heterogeneity is not due to genetic differences, since map organizations in individual mutant animals often differ between colliculi. Disruption of spontaneous waves of retinal activity resulted in uniform map organization in Isl2(EphA3/+) mice, demonstrating that correlated spontaneous activity is required for map heterogeneity. Computational modeling replicates this heterogeneity, revealing that molecular- and activity-dependent forces interact simultaneously and stochastically during topographic map formation.