Photochemical control of neuronal activity: methods and clinical application

by Ivan Tochitsky

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Richard Kramer, Chair

Mammalian nervous systems are incredibly complex, with almost 100 billion neurons making up the human brain. Neurons in the brain primarily communicate with one another in one of two ways - electrically, via the flow of ions across the cell membrane, or chemically by releasing and detecting a variety of signaling molecules. In order to understand the function of the nervous system, we need to be able to manipulate it with high spatial and temporal precision. Conventional electrical or chemical stimuli do not allow for such precise control. Thus, a new and orthogonal stimulus modality had to be utilized in order to facilitate the study of the nervous system. The emerging field of optogenetics uses light as such a stimulus since light can be delivered only to a small part of the nervous system, or even a single neuron, and the illumination can be controlled with millisecond time resolution. Optogenetic techniques involve the expression of light-sensitive proteins from microbes in genetically targeted populations of neurons, rendering those neurons sensitive to light. Recent advances in optogenetics have greatly advanced our understanding of the function of the nervous system both in healthy organisms, and in the context of disease. Optogenetics is a powerful technique for investigating neural networks, but this approach primarily studies the function of the nervous system at a system rather than molecular level. The vast complexity of the human brain is created not only by the large number of individual neurons and the intricate connections between them, but also by the dizzying variety of proteins found in the cell membranes of these neurons. These proteins sense and respond to the release of chemical signaling molecules from neighboring cells, or changes in ion concentrations that alter the cell's membrane potential, allowing for the generation and propagation of electrical signals.

We have combined the powers of synthetic chemistry and genetics to develop novel optopharmacological or optochemical genetic methods which enable precise optical control of neuronal function at the molecular level. These strategies involves the generation of light-sensitive "photoswitch" molecules that selectively target a population of either genetically engineered or endogenous membrane proteins - including receptors sensing chemical stimuli, or ion channels responding to electrical potential changes in the cell. The addition of a photoswitch compound to a neuron expressing the target protein makes that protein, and, by extension, the neuron, sensitive to light. We first applied this strategy to generate light regulated neuronal nicotinic acetylcholine receptors, which are a group of proteins that respond to the chemical neurotransmitter acetylcholine. These receptors modulate the activity of other neurons in different parts of the brain and are also sensitive to nicotine, an addictive chemical found in tobacco products. The function of acetylcholine receptors in the brain and their role in nicotine addiction, neuropsychiatric and neurodegenerative disorders is not fully understood, in large part because it quite difficult to chemically manipulate individual receptors without affecting others. Making light-sensitive, genetically targeted acetylcholine receptors should thus greatly advance our understanding of those receptors' function.

The main rationale for making proteins or neurons light-sensitive is to facilitate the study of the healthy nervous system as well as its malfunction in disease. There are, however, several human diseases where optical methods for controlling neuronal activity could directly provide a clinical benefit. Degenerative blinding diseases such as retinitis pigmentosa or age-related macular degeneration leave the retinas of affected patients either partly or completely insensitive to light by causing the death of light-detecting photoreceptor cells in the eye. Light responses can be restored to a blind retina by making some or all of the remaining retinal neurons sensitive to light. This can be achieved via the expression of light sensitive microbial opsins or engineered receptors in retinal neurons that are not normally light sensitive. Both of these approaches have restored some visual perception to blind mice suffering from retinitis pigmentosa. However, in order to use either optogenetic or optochemical genetic tools in the clinic, the mutant proteins must be artificially expressed in the patient's retina, which requires the use of viral gene therapy. Gene therapy has potential health risks, so we decided to develop a treatment for blinding diseases that would only involve a light-sensitive chemical, without the need for gene therapy or invasive surgery.

To that end, we have developed an optopharmacological therapy for vision restoration by creating photoswitch molecules that block and unblock endogenous voltage-gated ion channels in a light-dependent manner, allowing us to control almost any neuron with light. The first photoswitch tested, called AAQ, restored electrical retinal light responses, the pupillary light reflex, as well as other simple visual behaviors in blind mice. In order to optimize this treatment for clinical use, we generated a compound called DENAQ with improved light sensitivity and persistence in the eye, which responds to broad spectrum white light, similar to what people encounter in natural visual scenes. Furthermore, DENAQ acts selectively on retinas suffering from photoreceptor cell death, but leaves healthy retinas unaffected. This selectivity raises the possibility that we may be able to treat not only patients who are completely blind, but also those suffering from partial vision loss, by restoring light sensitivity only to the parts of the retina experiencing photoreceptor degeneration. The promising preliminary results from animal studies suggest that our optopharmacological strategy for vision restoration may eventually be used in the clinic, in addition to helping researchers understand the function of the nervous system in its normal state and in disease.

GABAA receptors (GABAARs) mediate fast synaptic inhibition in the mammalian brain and are essential for balancing excitation and sharpening our sensory perceptions. GABAARs are plastic and respond to changes in neuronal activity by strengthening or weakening inhibitory neurotransmission. This inhibitory plasticity responds to many of the same signals that mediate glutamatergic excitatory plasticity and works in tandem with or in contraposition to excitation to form the cellular basis of memories. GABAARs are added to or dispersed from synapses to modulate synaptic strength, and this is achieved by lateral diffusion in the plane of the membrane. By tethering GABAARs with synaptic scaffold proteins, primarily gephyrin, the inhibitory synapse can accumulate GABAARs dependent on the potency of this interaction. The scaffolds themselves are highly plastic and can agglomerate or wash away in coordination with GABAARs. An additional element of this process is the availability of extrasynaptic GABAARs able to diffuse into synapses, a supply that is tightly controlled by endo- and exocytosis of receptors from internal endosomal reservoirs and which is important for both acute plasticity and long-term homeostasis. The result is inhibitory synapses that are able to respond to activity changes with structural rearrangements on the scale of minutes to days.

Thus far, GABAAR mobility has been primarily interrogated with single particle tracking of quantum dots and FRAP in the context of neuronal cultures, where dendrite morphology and molecular composition have been radically altered from in vivo conditions. The rise of new optogenetic techniques, which provide great spatial and temporal precision, affords an opportunity to study mobility in more natural and intact conditions such as the acute slice preparation. Here we extend the optogenetic strategy to GABAARs, creating a light-regulated GABAAR (LiGABAR) by conjugating a photo-switchable tethered ligand (PTL) onto a mutant receptor containing a cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500-nm and 380-nm light, respectively, resulting in photo-switchable receptor antagonism. We created a transgenic knock-in α1 photoswitch-ready mouse (α1 PhoRM) to express the mutant receptor at endogenous levels and distributions.

We make use of LiGABAR technology to pioneer a novel electrophysiological technique for measuring GABAAR mobility, which we call CREAP. We use this method to functionally probe GABAAR mobility in the context of acute brain slices and find that α1-containing receptors are a highly mobile population GABAARs that constitute a fluid portion of inhibitory synapses.

This dissertation addresses two non-genetic therapeutic approaches for treating inherited retinal diseases such as retinitis pigmentosa and age related retinal degeneration. The first approach utilizes a light sensitive molecule Azobenzene-acrylomide-quaternary-amonium (AAQ) to restore light sensitivity to retinal degenerated (rd1) mice. AAQ molecules permeate through the cell membrane and photosensitize voltage-gated K^{+ words} channels in response to different wavelengths of light. Consequently, we achieved robust photosensitization of most retinal cells ex-vivo upon application of AAQ on rd1 mouse retinal explants. We then characterized the effect of AAQ on mouse behavior and restored a pupillary light reflex and light elicited behavior in these blind rd1 mice.

The third chapter addresses improvements to the AAQ therapeutic approach. We utilize Di-ethyl-amine-azobenzene quaternary ammonium (DeNAQ) that also confers light sensitivity on RGCs by photosensitizing voltage-gated channels in response to 480 nm light and silencing the channels in the dark (trans form). The property of DeNAQ being sensitive in the visible range is one of the major advantages of this therapeutic. The quick relaxation of DeNAQ in the dark is also an advantage, since only one wavelength is required for photosensitization. In addition to the red-shifted properties we found that DeNAQ can confer light sensitivity to retinal cells for several weeks and restores light elicited behavioral responses.

The fourth chapter addresses the question of whether the physiological and anatomical changes that accompany retinal degeneration lead to functional changes in the retinal ganglion cells (RGCs). We used a membrane impermeable Quarternary-azobenzene-quaternary molecule (QAQ) molecule that can only enter the cells and render them light sensitive through large pore openings, those seen during pore dilation of P2X and TRP channels. We characterized the path of entry of QAQ using pharmacology and found that photosensitization of RGCs could be reduced by adding P2XR antagonists. In agreement with other studies suggesting that pore dilation has been associated with neurodegenerative processes, we show that QAQ can only confer light sensitivity to RGCs from rd1 and not from wildtype (WT) mice. We also demonstrate that QAQ does not photosensitize RGCs in glaucoma mouse models, suggesting that this physiological change in permeability is specific to retinal degenerating disease.

The therapeutic approaches addressed in this thesis apply to a variety of retinal disease genotypes. The high promiscuity of AAQ delivery allows us to optically stimulate any cell type in the retina. Additionally, the red-shifted molecule DeNAQ, combined with its ability to target only diseased tissue, offers a promising clinical alternative for vision restoration. Finally, using another photosensitive molecule, QAQ, we study the physiological impact of retinal degeneration on pore-dilating ion channel activity. Understanding the sources of physiological changes could help prevent further damage caused by degenerative retinal diseases.

The development of novel techniques utilizing the advantages of light has created an optical revolution for neuroscience research. Controlling and probing neuronal function with light has provided unprecedented insights by being able to manipulate many neurons simultaneously in intact circuits and living organisms.

In my dissertation research, I used novel optical methods to probe the cellular permeability of sensory neuron populations. Primary nociceptive afferents detect, modulate and integrate many pain-related stimuli. Sensory information is transmitted from the periphery through the dorsal root ganglia along labeled lines coding for sensory modalities and receptive fields. Recently, our understanding of sensory processing has been expanded by the discovery of neuronal interactions both on the level of peripheral nerve endings and in the spinal cord. Here I report a crosstalk between injured and intact neuronal population after peripheral nerve injury. This communication is revealed at the somata of sensory neurons distant from the site of injury and correlates with permeability changes of both injured and intact nociceptor populations after injury. I devised a sensitive probe to measure the activity of nociceptive cells in intact tissue using a photoswitch molecule. Transient Receptor Potential (TRP) ion channel are a major class of molecular sensors detecting painful stimuli. I show that TRPA1, a member of the TRP family, is necessary for this crosstalk which is limited to petidergic, slow conducting C fibers. In addition, electrical activity of nociceptive fibers alone is sufficient to mimic similar permeability changes as nerve injury. Injury induced hyperexcitability of sensory fibers and TRP channel dysfunction have been implicated with conditions of pain hypersensitivity, chronic and neuropathic pain. I therefore propose that this crosstalk might contribute to adaptions of pain signaling in various disease states.

Photoswitch molecules have found many experimental applications in neuroscience research. However, the exact mechanism by which they function remains unclear. Therefore, another part of my research focused on studying interactions of the photoswitchable compound QAQ with the model potassium channel Shaker. I investigated the affinity of both QAQ isomers to the open Shaker channel and surprisingly found that block is not voltage-dependent. Further, I analyzed the effects of use-dependent inhibition and report that QAQ does not associate with closed channels. However, once channels are opened, QAQ does interfere with the kinetics of the gate slowing down the closing of the channel.

Neural dendrites continually prove to harness more computational complexity than previously thought. The voltage-gated ion channels distributed throughout a dendritic tree are key determinants of dendritic excitability and computation. However, little is known about the specific functional impacts of voltage-gated excitability in discrete dendritic regions. Recently, an optical revolution in neuroscience has yielded a vast array of optical tools for functional interrogation of neurons and neural circuits. One such tool, Quarternary-ammonium Azobenzene Quarternary-ammonium (QAQ), is an optically-controllable small-molecule drug that affects voltage-gated ion channels. In its trans conformation, which is photo-inducible with green light, QAQ directly blocks all voltage-gated ion channels tested, but rapidly un-blocks those channels when converted to its cis form with near-ultraviolet light (Mourot et al. 2012). It does not photo-bleach, and can be robustly photoswitched back and fourth to either block or unblock channels in a matter of milliseconds. QAQ is a promising tool to control voltage-gated excitability in neural dendrites with the spatiotemporal precision of light.

In this thesis we use QAQ to rapidly, reversibly, and locally control voltage-gated ion channel activity in neural dendrites using targeted light. A wealth of experimental evidence using traditional pharmacology is already available about specific voltage-gated ion channels in CA1 pyramidal cells, so we first apply QAQ via a patch-pipette to CA1 pyramidal cells and confirm that it works as expected in a whole-cell. We find that trans-QAQ blocks somatic action potentials, blocks dendritic calcium activity, and enhances EPSP summation. These are all processes driven by QAQ-sensitive voltage-gated ion channel types that either boost (sodium and calcium channels) or dampen (potassium channels) intrinsic excitability.

We then investigate the level of spatial control we can achieve with QAQ using dendritic calcium imaging. Indeed, for up to three seconds after photo-switching the molecule, control is extremely precise. With this knowledge, we use local block of voltage-gated ion channels and calcium imaging to confirm and extend previous findings that voltage-gated calcium channel activity is relatively uniform throughout the apical dendritic tree of CA1 pyramidal cells. Finally, we specifically photo-control voltage-gated ion channels in the apical dendrites of CA1 neurons to experimentally probe whether dendrite-specific voltage-gated excitability affects the degree of action potential back-propagation. We find that dendritic voltage-gated ion channels determine whether a CA1 pyramidal neuron will undergo strong or weak back-propagation, a notion that has only previously been modeled.

The retina offers unique insight into synaptic activity in the central nervous system. It is the only part of the central nervous system that can be visualized without advanced imaging or invasive methods. Its highly organized structure has turned it into a main target for neuroscientific studies, and while it has a simplistic anatomical organization its synaptic functions have only been partially elucidated. Zebrafish have been utilized to study vision since they have a cone-rich retina, high acuity vision and they are a convenient model to work with and manipulate genetically. Furthermore, zebrafish can absorb drugs and small molecules directly from their liquid environment thereby facilitating chemical manipulation of their nervous system. For these reasons we have chosen to use zebrafish to generate tools that improve our understanding of retinal synaptic activity in the outer plexiform layer of the retina, and in horizontal cells (HCs) in particular. Five genetically modified strains of zebrafish (4 of which were developed at the Kramer lab) are presented in this dissertation. Two strains have been modified using the introduction of foreign receptors, FaNaC and PSAM-GlyR, allowing controlled and reversible depolarization and hyperpolarization of HCs, respectively. Using these modified strains, we show that by changing the membrane potential of HCs we can manipulate neuronal function upstream, and downstream to the HC synapse. Using three additional strains, we explore the change in pH at different locations in the HC to cone synapse during lateral inhibition. Those locations include the AMPA receptor on HCs, and synaptophysin and the voltage gated calcium channel on the photoreceptor side. By combining our chemogenic tool FaNaC to depolarize HCs, and a genetically introduced pHluorin to detect pH changes, we reaffirm the hypothesis that pH is the signal which mediates lateral inhibition in the outer retina. Using the three pH probes, we show that the change in pH is highly localized nearby photoreceptor voltage-gated calcium channels. In conclusion, while HCs form a wide interconnected network that spans the entire retina, their communication with individual photoreceptors through acidification of the synaptic cleft is spatially constrained. Moreover, slight deviations in HC membrane potential greatly alter their effects on other retinal neurons including photoreceptors, bipolar cells and retinal ganglion cells.

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent in wild-type animals with intact rods and cones. While RGCs survive following photoreceptor death, they exhibit physiological changes, leading to heightened spontaneous activity and permissiveness to azobenzene photoswitch-reanimation. Here it is shown show that P2X receptors mediate photoswitch entry into RGCs where they associate with upregulated HCN channels, enabling light to control action potential firing. This remodeling is limited to morphologically identified OFF-RGCs, enabling selective and specific chemical photosensitization. This selectivity is not only for OFF-RGCs within a degenerated retina, but also displays selectivity within a focally degenerated retina, where photoreceptor death is non-uniform. Retinoic acid (RA), an important developmental morphogen, is also shown to be the signal that triggers pathophysiological retinal remodeling. Blocking RA signaling reduces remodeling and enhances light sensitivity in degenerating retinas. Enhancing RA signaling mimics pathophysiological remodeling in healthy retinas. A genetically-encoded fluorescent reporter shows increased RA signaling during degeneration. Identification of RA as the local, degeneration-dependent initiator of remodeling presents a new therapeutic opportunity for boosting low-level vision and performance of retinal prosthetics during degenerative blindness. Understanding the changes that occur in a degenerating retina and the signal for these changes has broad implications for disease-specific drug delivery.

Retinal photoreceptors are the primary sensory neurons responsible for vertebrate vision. The photoreceptor converts light into a voltage signal that modulates neurotransmitter release at the photoreceptor synapse. Synaptic release from photoreceptors stimulates downstream cells and ultimately provides our brain with an image of the visual world. Hence, converting the voltage signal into a synaptic signal at the photoreceptor synapse is the crucial first step in visual perception. Here I present research conducted for this dissertation that elucidates multiple mechanisms that shape synaptic transmission from cone photoreceptors.