Zhuo Hua Pan

Ectopic Expression of a Microbial-Type Rhodopsin Restores Visual Responses in Mice with Photoreceptor Degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Structure and function of GABAC receptors: a comparison of native versus recombinant receptors

In less than a decade our knowledge of the GABA(C) receptor, a new type of Cl(-)-permeable ionotropic GABA receptor, has greatly increased based on studies of both native and recombinant receptors. Careful comparison of properties of native and recombinant receptors has provided compelling evidence that GABA receptor rho-subunits are the major molecular components of GABA(C) receptors. Three distinct rho-subunits from various species have been cloned and the pattern of their expression in the retina, as well as in various brain regions, has been established. The pharmacological profile of GABA(C) receptors has been refined and more specific drugs have been developed. Molecular determinants that underlie functional properties of the receptors have been assigned to specific amino acid residues in rho-subunits. This information has helped determine the subunit composition of native receptors, as well as the molecular basis underlying subtle variations among GABA(C) receptors in different species. Finally, GABA(C) receptors play a unique functional role in retinal signal processing via three mechanisms: (1) slow activation; (2) segregation from other inhibitory receptors; and (3) contribution to multi-neuronal pathways.

T-Type Ca2+ Channels Mediate Neurotransmitter Release in Retinal Bipolar Cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.

Ectopic Expression of Multiple Microbial Rhodopsins Restores ON and OFF Light Responses in Retinas with Photoreceptor Degeneration

By expressing channelrhodopsin-2 (ChR2) in inner retinal neurons, previous studies have demonstrated restoration of ON responses in the retina after the death of rod and cone photoreceptors. In this study, we report that the expression of halorhodopsin (HaloR), a light-driven chloride pump, can effectively restore OFF responses in inner retinal neurons of mice with retinal degeneration. We show that HaloR-expressing retinal ganglion cells respond to light with rapid hyperpolarization and suppression of spike activity. After termination of the light stimulus, their membrane potential exhibits a rapid rebound overshoot with robust sustained or transient spike firing. Furthermore, we show that coexpression of ChR2/HaloR in retinal ganglion cells can produce ON, OFF, and even ON–OFF responses, depending on the wavelength of the light stimulus. Our results suggest that the expression of multiple microbial rhodopsins such as ChR2 and HaloR is a possible strategy to restore both ON and OFF light responses in the retina after the death of rod and cone photoreceptors.

Evaluation of AAV-Mediated Expression of Chop2-GFP in the Marmoset Retina

PURPOSE Converting inner retinal neurons to photosensitive cells by expressing channelrhodopsin-2 (ChR2) offers a novel approach for treating blindness caused by retinal degenerative diseases. In the present study, the recombinant adeno-associated virus serotype 2 (rAAV2)-mediated expression and function of a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP) (Chop2-GFP) were evaluated in the inner retinal neurons in the common marmoset Callithrix jacchus. METHODS rAAV2 vectors carrying ubiquitous promoters were injected into the vitreous chamber. Expression of Chop2-GFP and functional properties of ChR2 were examined by immunocytochemical and electrophysiological methods 3 months after injection. RESULTS The percentage of Chop2-GFP-expressing cells in the ganglion cell layer was found to be retinal region- and animal age-dependent. The highest percentage was observed in the far-peripheral region. Chop2-GFP expression was also found in the foveal and parafoveal region. In the peripheral retina in young animals with high viral concentrations, the expression of Chop2-GFP was observed in all major classes of retinal neurons, including all major types of ganglion cells. The morphologic properties of Chop2-GFP-positive cells were normal for at least 3 months, and ChR2-mediated light responses were demonstrated by electrophysiological recordings. CONCLUSIONS The rAAV2-mediated expression of ChR2 was observed in the inner retinal neurons in the marmoset retina through intravitreal delivery. The marmoset could be a valuable nonhuman primate model for developing ChR2-based gene therapy for treating blinding retinal degenerative diseases.

Evidence for coassembly of mutant GABACρ1 with GABAAγ2S, glycine α1 and glycine α2 receptor subunits in vitro

Functional coassembly of γ‐aminobutyric acid (GABA)Cρ1 subunits with GABAA (α1, β2, and γ2S) or glycine (α1, α2, and β) subunits was examined using two‐electrode voltage‐clamp recordings in the Xenopus laevis oocyte expression system. To facilitate this study, we took advantage of the unique gating and pharmacological properties of two mutant ρ1 subunits, ρ1(T314A) and ρ1(T314A/L317A). When the ρ1(T314A) subunit was coexpressed with GABA γ2S, glycine α1 or glycine α2 subunits, GABA response properties were different from those of homomeric ρ1(T314A) receptors. Additionally, the sensitivity of heteromeric ρ1(T314A) and γ2S receptors to picrotoxinin (PTX) blockade of GABA‐evoked responses was altered compared to that of homomeric ρ1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when ρ1(T314A) subunits were coexpressed with wild‐type ρ1 subunits. When ρ1(T314A/L317A) subunits were coexpressed with GABA γ2S, glycine α1 or glycine α2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric ρ1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA ρ1(T314A/L317A)/γ2S heteromeric receptors displayed an additional component. Coinjection of wild‐type ρ1 with γ2S cRNAs at a ratio of 1 : 1 resulted in a > 10‐fold reduction in GABA‐evoked current. Furthermore, coexpression of wild‐type ρ1 and γ2S subunits was found to shift the GABA dose–response curve. Our results provide functional evidence that the GABACρ1 subunit can coassemble with the GABAAγ2S subunit, and, at least in its mutated form, ρ1 can also form heteromeric receptors with glycine α1 or α2 subunits in vitro.

Characterization of transgenic mouse lines expressing Cre-recombinase in the retina

The mammalian retina consists of five major classes of neuronal cells, as well as glial cells, and it contains more than 50 cell types. The ability to manipulate gene expression in specific cell type(s) in the retina is important for understanding the molecular mechanisms of retinal function and diseases. The Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in vivo in a cell- and tissue-specific fashion. The key to this tool is the availability of Cre mouse lines with cell- or tissue-type specific expression of Cre recombinase. To date, a large number of Cre-transgenic mouse lines have been generated to target Cre recombinase expression to specific neuronal and glial cell populations in the central nervous system; however, information about the expression patterns of Cre recombinase lines in the retina is largely lacking. In this study, we examined and characterized the expression patterns of Cre recombinase in the retinas of 15 Cre-transgenic mouse lines. Significant Cre-induced recombination or expression of Cre recombinase was observed in the majority of these lines. In particular, we found several Cre lines in which the Cre-induced recombination was found to target exclusively or predominantly a single type or class of retinal cells, including bistratified retinal ganglion cells, starburst amacrine cells, rod bipolar cells, and Müller glial cells. In other lines, the Cre-induced recombination was found in several retinal cell types. These Cre lines provide a valuable resource for retinal research.

Two Types of Cone Bipolar Cells Express Voltage-Gated Na+ Channels in the Rat Retina

Two groups of retinal cone bipolar cells (CBCs) in rats were found to express voltage-gated Na+ channels. The axon terminals of the first group stratify in sublamina 2 of the inner plexiform layer (IPL) and partially overlap with the OFF-cholinergic band. This group was identified as type 3 CBCs. The axon terminals of the second group stratify in sublamina 3 of the IPL, slightly distal to the ON-cholinergic band. Cells of this second group resemble type 5 CBCs. In addition, we observed another group of ON-type CBCs with terminal stratification similar to that of the second group. However, this latter group did not show any Na+ current, instead exhibiting a large hyperpolarization-activated cyclic nucleotide-gated cation current, suggesting the existence of two subclasses of physiologically distinct type 5 CBCs. Both groups of Na+-expressing bipolar cells were capable of generating a rapid tetrodotoxin-sensitive action potential as revealed by current injection. Multiple spike-like potentials were also observed in some of these cells. Results of this study provide valuable insights into the function of voltage-gated Na+ channels of retinal bipolar cells in retinal processing.

Heterogeneous expression of voltage-dependent Na+ and K+ channels in mammalian retinal bipolar cells.

Retinal bipolar cells show heterogeneous expression of voltage-dependent Na+ and K+ currents. We used whole-cell patch-clamp recordings to investigate the possible roles of these currents in the response properties of bipolar cells in rats. Isolated bipolar cells showed robust spontaneous regenerative activity, but the regenerative potential of rod bipolar cells reached a more depolarized level than that of cone bipolar cells. In both isolated cells and cells in retinal slices, the membrane depolarization evoked by current injection was apparently capped. The evoked membrane potential was again more depolarized in rod bipolar cells than in cone bipolar cells. Application of tetraethylammonium and 4-aminopyridine shifted the spontaneous regenerative potential as well as the evoked potential to a more depolarized level. In addition, a subclass of cone bipolar cells showed a prominent spike in the initial phase of the voltage response when the cells were depolarized from a relatively negative membrane potential. The spike was mediated mainly by tetrodotoxin-sensitive Na+ current. The presence of the spike sped up the response kinetics and enhanced the peak membrane potential. Results of this study raise the possibility that voltage-dependent K+ currents may play a role in defining different membrane operating ranges of rod and cone bipolar cells and that voltage-dependent Na+ currents may enhance the response kinetics and amplitude of certain cone bipolar cells.

rAAV-Mediated Subcellular Targeting of Optogenetic Tools in Retinal Ganglion Cells In Vivo

Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system.