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Development of functional organization of the visual circuits in mice

Periodic Reporting for period 3 - CIRCUITASSEMBLY (Development of functional organization of the visual circuits in mice)

Reporting period: 2018-04-01 to 2019-09-30

The key organizing principles that characterize neuronal systems include asymmetric, parallel, and topographic connectivity of the neural circuits. The main aim of my research is to elucidate the key principles underlying functional development of neural circuits by focusing on those organizing principles. I choose mouse visual system as my model since it contains all of these principles and provides access to sophisticated genetic tools to label and manipulate individual cell types. My research is based on the central hypothesis that the mechanisms of brain development cannot be fully understood without first identifying individual functional cell types in adults, and then understanding how the functions of these cell types become established, using cell-type-specific molecular and synaptic mechanisms in developing animals. For this proposal, I have identified several transgenic mouse lines in which specific cell types in a visual center, the superior colliculus, are labeled with Cre recombinase in both developing and adult animals. Here I am taking advantage of these mouse lines to ask fundamental questions about the functional development of neural circuits. First, how are distinct sensory features processed by the parallel topographic neuronal pathways, and
how do they contribute to behavior? Second, what are the molecular and synaptic mechanisms that underlie developmental circuit plasticity for forming parallel topographic neuronal maps in the brain? Third, what are the molecular mechanisms that set up spatially asymmetric circuit connectivity without the need for sensory experience? I predict that my insights into the developmental mechanism of asymmetric, parallel, and topographic connectivity and circuit plasticity will be instructive when studying other brain circuits which contain similar organizing principles.

Impairment of neuronal computation, neuronal connectivity or synaptic physiology would lead to psychiatric and neurological disorders. Efficient treatment of such disorders does not exist, and the disorders burden human society with significant medical and social expenses. Gained mechanistic insight into function and development of nervous system would be very helpful for understanding and developing treatment for such diseases in future.
Aim 1. We have established a resonant scanning 2-photon imaging system in my laboratory, and established a method of in vivo imaging of mouse superior colliculus. First we have characterized dendritic and axonal morphology of Cre-labeled cell types in superior colliculus in 4 transgenic mouse lines. Second we are labeling collicular neurons by GCaMP6s expressed from AAV and performing in vivo 2-photon imaging. To image superior colliculus without damaging overlying cortex, we implant silicon plug over superior colliculus to push aside cortex to secure the field of view of posterior part of superior colliculus. This imaging experiment is still ongoing.

We have done retrograde mono-trans-synaptic tracing from the 4 Cre mouse lines. This is a first demonstration of cell type-to-cell type connectivity pattern between retina and superior colliculus in mammals, providing insight into how information about individual visual features could be processed by dedicated neural circuits in the visual center. Currently we are performing confocal scanning of labelled ganglion cells to reconstruct dendritic structure and classifying into distinct ganglion cell types.

For the investigation of function of cell types in visually-guided behaviour, we have built an automated optomotor and optokinetic reflex assessment box, a looming-dependent escaping behavior assessment box, and prey capture measurement system. We have ablated Cre-labeled collicular cell types with Cre-dependent AAV expressing diphtheria toxin A (DTA) and are testing the effect of cell type ablation on these behaviors.

Aim 2. We have established galvo scanning 2-photon imaging system in my laboratory for neonatal in vivo imaging. We have characterized dendritic and axonal morphology of Cre-labeled cell types in superior colliculus in neonatal transgenic mouse lines by targeting the cell types with transgenically expressed fluorescent markers followed by immunohistochemistry and confocal scanning. We confirmed that in most of the transgenic mouse lines the cell types are already labelled in neonatal periord.

Aim 3. We have established galvo scanning two photon imaging system combined with LED visual stimulation system and microelectrode array spike recording system in my laboratory for exo vivo retinal recordings. We have identified Frmd7 knockout mice in which horizontal optokinetic reflex is lost and retinal direction selectivity is lost along horizontal axis, and this work was published in Neuron last year as my first authored paper (Yonehara et al., 2016). Expression of Frmd7 is localized to starburst amacrine cells in mice and primates, and mutation of Frmd7 leads to congenital nystagmus, accompanied by lack of horizontal optokinetic reflex, in human. We will analyse Frmd7 mice further to understand the molecular mechanisms of circuit assembly of retinal direction-selective circuits.

To investigate cell type transcriptome, I have FACS sorted and collected total RNA from various retinal cell types in wild-type background. Next we have performed RNA sequencing and analyzed the cell-type transcriptome and identified hundreds of cell-type-specific genes. We have screened about 20 mutant mouse lines in which one of identified cell type enriched gene is mutated, and identified one mutant mouse line in which horizontal optomotor reflex is significantly reduced. Next we recorded light-evoked spiking responses from isolated retinas with microelectrode array system which equipped with 26,400 electrodes. Strikingly horizontal direction selectivity was significantly impaired in the mutant retinas, and the phenotype is reminiscent of Frmd7 phenotype. Now we are trying to understand molecular function of the identified gene.
My research on Frmd7 in collaboration with Dr. Botond Roska was the first to link human neurodevelopmental disease to an impairment of single inhibitory neuronal type. This research also suggested that mouse and humans share similar neuronal mechanism that depends on retinal direction selective circuits for gaze stabilization. Understanding of molecular mechanism of Frmd7 and other related genes in the establishment of direction selective circuit would provide us better insight into how our neuronal system develops by forming spatially organized synaptic connectivity and how dysfunction of such synaptic specificity leads to neurological disorders. We may find candidate molecular targets for the genetic restoration of gaze stability in congenital nystagmus patients.

By understanding cell type basis of visual processing and visually guided behaviors mediated by superior colliculus, we expect that we will understand principles underlying how neuronal inputs from retina is processed by central target to evoke ethologically relevant visually guided behaviors. Such insight would add better understanding of how our visual system works and how impairment of specific cell type or circuits could lead to dysfunction of selective symptom in visual disorders such as cerebral visual impairment, which is a leading cause for blindness in children.

By understanding how topographic projection from retina to superior colliculus is established and what is the cell type and synaptic basis for its developmental mechanism, we aim to understand how our brain circuit is shaped by genetic mechanisms and activity-dependent mechanisms. Particularly, we aim to understand molecular basis of activity-dependent synaptic formation. Gained knowledge would provide a hint into how we could restore plasticity of neuronal circuits and ability to learn and memorize, which is impaired in many neurological diseases such as Alzheimer´s disease.