Wenzhi Sun

Large-scale morphological survey of mouse retinal ganglion cells

Five hundred twenty ganglion cells in an isolated whole‐mount preparation of the mouse retina were labeled using the “DiOlistic” method (Gan et al. [ 2000 ] Neuron 27:219–225) and were classified according to their morphological properties. Tungsten particles coated with a lipophilic dye (DiI) were propelled into the whole‐mount retina using a gene gun. When a dye‐coated particle contacted the cell membrane, the entire cell was labeled. The ganglion cells were classified into four groups based on their soma size, dendritic field size, and pattern and level of stratification. Broadly monostratified cells were classified into three groups: RGA cells (large soma, large dendritic field), RGB cells (small to medium‐sized soma, small to medium‐sized dendritic field), and RGC cells (small to medium‐sized size soma, medium‐sized to large dendritic field). Bistratified cells were classified as RGD. This study represents the most complete morphological classification of mouse retinal ganglion cells available to date and provides a foundation for further understanding of the correlation of physiology and morphology and ganglion cell function with genetically manipulated animals. J. Comp. Neurol. 451:115–126, 2002.

Identification of ON-OFF direction-selective ganglion cells in the mouse retina

We identified the ON–OFF direction‐selective ganglion cells (DSGCs) in the mouse retina and characterized their physiological, morphological and pharmacological properties. These cells showed transient responses to the onset and termination of a stationary flashing spot, and strong directional selectivity to a moving rectangle. Application of various pharmacological reagents demonstrated that the ON–OFF DSGCs in the mouse retina utilize a similar array of transmitters and receptors to compute motion direction to their counterparts in the rabbit retina. Voltage clamp recording showed that ON–OFF DSGCs in the mouse retina receive a larger inhibitory input when the stimulus is moving in the null direction and a larger excitatory input when the stimulus is moving in the preferred direction. Finally, intracellular infusion of neurobiotin revealed a bistratified dendritic field with recursive dendrites forming loop‐like structures, previously classified as RGD2 by morphology. Overall, the ON–OFF DSGCs in the mouse retina exhibit almost identical properties to their counterparts in the rabbit retina, indicating that the mechanisms for computing motion direction are conserved from mouse to rabbit, and probably also to higher mammals. This first detailed characterization of ON–OFF DSGCs in the mouse retina provides fundamental information for further study of maturation and regulation of the neuronal circuitry underlying computation of direction.

Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of ∼28,000 thalamic boutons and ∼4,000 cortical neurons in layers 1–5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.

Multiplexed aberration measurement for deep tissue imaging in vivo.

We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein–labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue

Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

ON direction-selective ganglion cells in the mouse retina

Two types of ganglion cells (RGCs) compute motion direction in the retina: the ON–OFF direction‐selective ganglion cells (DSGCs) and the ON DSGCs. The ON DSGCs are much less studied mostly due to the low encounter rate. In this study, we investigated the physiology, dendritic morphology and synaptic inputs of the ON DSGCs in the mouse retina. When a visual stimulus moved back and forth in the preferred–null axis, we found that the ON DSGCs exhibited a larger EPSC when the visual stimulus moved in the preferred direction and a larger IPSC in the opposite, or null direction, similar to what has been found in ON–OFF DSGCs. This similar synaptic input pattern is in contrast to other well‐known differences, namely: profile of velocity sensitivity, distribution of preferred directions, and different central projection of the axons. Immunohistochemical staining showed that the dendrites of ON DSGCs exhibited tight cofasciculation with the cholinergic plexus. These findings suggest that cholinergic amacrine cells may play an important role in generating direction selectivity in the ON DSGCs, and that the mechanism for coding motion direction is probably similar for the two types of DSGCs in the retina.

CELF4 Regulates Translation and Local Abundance of a Vast Set of mRNAs, Including Genes Associated with Regulation of Synaptic Function

RNA–binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA–binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%–20% of the transcriptome, with striking specificity for the mRNA 3′ untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Nav1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA–binding proteins play in regulating and shaping the activity of neural circuits.

Layer-specific network oscillation and spatiotemporal receptive field in the visual cortex

A quintessential feature of the neocortex is its laminar organization, and characterizing the activity patterns in different layers is an important step in understanding cortical processing. Using in vivo whole-cell recordings in rat visual cortex, we show that the temporal patterns of ongoing synaptic inputs to pyramidal neurons exhibit clear laminar specificity. Although low-frequency (∼2 Hz) activity is widely observed in layer 2/3 (L2/3), a narrow-band fast oscillation (10–15 Hz) is prominent in layer 5 (L5). This fast oscillation is carried exclusively by excitatory inputs. Moreover, the frequency of ongoing activity is strongly correlated with the spatiotemporal window of visual integration: Neurons with fast-oscillating spontaneous inputs exhibit transient visual responses and small receptive fields (RFs), whereas those with slow inputs show prolonged responses and large RFs. These findings suggest that the neural representation of visual information within each layer is strongly influenced by the temporal dynamics of the local network manifest in spontaneous activity.

Video-rate volumetric functional imaging of the brain at synaptic resolution

Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

Etiology of a genetically complex seizure disorder in Celf4 mutant mice.

Mice deficient for the gene encoding the RNA‐binding protein CELF4 (CUGBP, ELAV‐like family member 4) have a complex seizure phenotype that includes both convulsive and non‐convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence‐like non‐convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch‐clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron‐specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice.