Elizabeth M. Quinlan - US grants

Despite the importance of visual experience in the normal and pathological development of the visual cortex, the molecular mechanisms by which visual activity induces long-lasting changes in the function of cortical synapses have not been defined. The highly modifiable synapses between excitatory neurons in the mammalian visual cortex use glutamate as a neurotransmitter. As the primary effectors of synaptic glutamate release, changes in the number and/or function of post-synaptic glutamate receptors (GluRs) are likely to dramatically effect synaptic strength. This proposal uses binocular and monocular deprivation paradigms to regulate the strength of synaptic connections in the rat visual cortex, and tests the hypothesis that an experience-induced increase in synaptic strength is mediated by delivery of GluRs, while a deprivation-induced decrease in synaptic strength is due to the removal of synaptic GluRs. Analysis of GluR levels is performed in synatoneurosomes, a subcellular fraction which is enriched for intact, metabolically active glutamatergic synapses. The power of this procedure is revealed by the observation that levels of synaptoneurosomal GluRs are highly correlated with changes in the physiological and pharmacological response properties of synaptic GluRs, and in fact are highly predictive of changes in synaptic strength. The role of synaptic protein synthesis in the long-term experience- dependent maintenance of synaptic GluR levels will also be examined in vivo, and in isolated metabolically active synaptic profiles in vitro. Bidirectional, activity-dependent control of synaptic GluR composition would represent a novel mechanism for the regulation of synaptic efficacy in the developing visual cortex, and identify potential targets (GluR or GluR trafficking proteins) for pharmacological intervention, especially in the case of severe, therapy-resistant amblyopia.

The ability of the nervous system to learn and remember ultimately depends on the changes in the strength of synapses, the points of contact between individual nerve cells. The majority of research on memory formation has focused on early time points during the encoding of a memory, when the change in synaptic strength is first induced. Therefore, little is known about the mechanisms by which memories are retained over the lifetime of an organism. This obstacle can be overcome by using a multi-disciplinary approach to studying the molecular basis of olfactory discrimination learning in rodents. Olfaction is the primary sensory modality in the rat, and rats quickly learn to associate odor with reward. Olfactory discrimination is a robust animal learning paradigm that results in activation of a large population of synapses in the piriform cortex, thereby offering an invaluable opportunity to combine behavioral, electrophysiological and biochemical methods to study the cellular mechanisms of learning. Olfactory discrimniation learning will be used to test the hypothesis that synapses in the piriform cortex become less "plastic" after learning. The proposed experiments merge the research efforts of two laboratories in very different geographical/political environments. It is predicted that the data gleaned from these experiments have a wide readership, and will help to build bridges between the fields of behavioral neuroscience, synaptic physiology, molecular and cell biology. In addition it will focus research attention on a very important aspect of memory processes, memory retention, which has been previously ignored.

DESCRIPTION (provided by applicant): The ability of experience to regulate the cortical function decreases significantly over the lifetime of an animal. During an early, postnatal critical period, monocular deprivation (MD) induces a shift in the ocular dominance (OD) of binocular neurons through a rapid decrease in the strength of synapses serving the deprived eye. In addition, a slower increase in the strength of synapses serving the non-deprived eye is observed. Recent work, by our lab and others, demonstrates that ocular dominance shifts can also be induced in adults, after the classical critical period, however longer periods of MD are required. In adults, deprivation engages only the slow component, increasing the strength of synapses serving the non-deprived input. This demonstrates that OD plasticity persists into adulthood, and suggests the intriguing possibility that opportunities to regulate OD plasticity may also persist throughout lifetime. Our preliminary experiments tested this hypothesis, and demonstrate that visual deprivation, through dark exposure (DE), reactivates rapid juvenile-like OD plasticity in response to monocular deprivation. The OD shift induced after dark exposure is due to a rapid decrease in the strength of synapses serving the deprived eye, previously only described in juveniles, and a rapid increase in the strength of synapses serving the non-deprived eye, which typically develops slowly in juveniles and adults. The proposed experiments examine the temporal requirements and functional consequences of dark exposure, and use a battery of transgenic and pharmacological manipulations to test the hypothesis that dark exposure decreases inhibition in the visual cortex, allowing a return to a more plastic, juvenile-like state. In addition, we test the hypothesis that DE will increase the success of regaining function in an eye deprived of vision from birth. Such a non-invasive method to restore experience-dependent synaptic plasticity in the mammalian cortex holds great therapeutic potential, as the visual deficit resulting from amblyopia in humans is often irreversible by age 10.

? DESCRIPTION (provided by applicant): During a critical period of early postnatal development, an asymmetry in the quality of visual input to the two eyes shifts ocular preference away from the weaker eye and induces amblyopia, the most common cause of monocular visual deficits in humans. Amblyopia is highly resistant to reversal in adulthood, due in large part to th termination of the critical period of heightened plasticity. Understanding how the enhanced plasticity of the critical period is initiated and terminated over development is fundamental to th development of therapeutic strategies aimed to reactivate plasticity to treat amblyopia in adults, which can be translated to a clinical population and to other critical periods. A popular model for the regulation of the critical period proposes that inhibitory control of plasticity at excitatory synapses is mediated by the maturation of the output of fast-spiking interneurons (FS-INs) that mediate perisomatic inhibition. However, we have shown that ocular dominance plasticity can be induced several months after the maturation of perisomatic inhibition. We propose instead that ocular dominance plasticity is regulated by plasticity upstream of inhibitory output, likely affecting the recruitment of inhibition into functional circuits. In addition we propose that the functional connectivity of Pyr->FS synapses must be retained in a permissive range for ocular dominance plasticity to be expressed, as larger reductions in Pyr->FS connectivity induced by genetic manipulations inhibit the expression of ocular dominance plasticity. Our preliminary analysis of the regulation of excitation from pyramidal neurons onto FS-INs (Pyr->FS) reveals that monocular deprivation during the critical period may functionally disconnect FS-INs from the cortical network by significantly reducing the number of excitatory inputs onto these neurons. Therefore we hypothesize that a novel mechanism of plasticity, deprivation-induced loss of functional Pyr->FS connectivity 1) is an early and obligatory step in the shift in ocular dominance induced by MD, and 2) determines the timing of the critical period. We propose a multidisciplinary set of experiments to test these hypotheses that combine: the expertise of the Quinlan lab in the examination of physiological changes in visual cortex in vivo in response to monocular deprivation; the expertise of the Kirkwood lab in the direct assessment of contribution of changes in single synapses to activity-dependent plasticity in the visual cortex; and the expertise of the Lee lab in the use optogenetic methods to identify foci and mechanisms of activity-dependent changes in synaptic function. Our model for the regulation of the timing of the critical period refutes many widely-held assumptions regarding developmental changes in synaptic plasticity in the mammalian cortex.

Project Summary The decline in synaptic plasticity with age is thought to impose severe constraints on the recovery from amblyopia in adults. Although this developmental loss was previously thought to be irreversible, our previous work established that robust plasticity can be reactivated in the adult visual cortex via visual deprivation by dark exposure. Furthermore, dark exposure followed by instructive visual experience enables complete recovery from severe amblyopia in adulthood. Our previous work assumed that the elimination of visual input during dark exposure was sufficient to reactivate plasticity. However, our preliminary data demonstrate that light reintroduction after dark exposure is responsible for the reactivation of structural and functional plasticity in the adult mouse visual cortex. We propose to show that LRx following dark exposure increases the activity of a key extracellular protease (matrix metalloprotease-9) at thalamic inputs to cortical neuron and a counter- intuitive decrease in the excitability of regular spiking neurons to reactivate robust plasticity in the adult dark- exposed cortex.

Project Summary This application requests funds to support the Biennial International Perceptual Learning Workshop, to be held August 15-20, 2021 in Girdwood, Alaska. Robust improvements in visual perception can be induced by practice on a variety of visual tasks. This visual perceptual learning has the potential to provide powerful insight into the organization and flexibility of the mammalian visual system. The recognition that visual perceptual learning can be clinically applied to improve visual functions with age and disease have intensified recent efforts to optimize and characterize training paradigms and standardize outcome measurements. In addition, the persistence of visual perceptual learning in the adult visual system contributes to a revision of our understanding of how the capacity for plasticity is retained in mature sensory cortices. The overall goal of this international conference is to provide a forum for discussion and debate of recent advances, long-term debates and new directions for the field of perceptual learning. This conference has become an important opportunity for individuals across the globe with expertise in perceptual learning ranging from psychophysics, neurophysiology, functional imaging, computational neuroscience, and perceptual rehabilitation to receive critical commentary of new unpublished work, and to influence and learn from each other. The meeting serves an important need as the field of perceptual learning is highly interdisciplinary and there are no other forums that bring together researchers across the many disciplines that contribute to the field. The meeting also has been successful in enhancing interactions between junior and senior investigators, providing opportunities for new collaborations and influencing subsequent work by identifying the most important issues in the field. The specific goals for the 2021 Visual Perceptual Learning Workshop are: Specific Aim 1: To highlight new and exciting developments on the frontiers of perceptual learning. Specific Aim 2: To promote communication and interactions between meeting attendees Specific Aim 3: To achieve the highest scientific quality and diversity, by optimizing a balance of gender, age, ethnicity, and nationality.

Project Summary/Abstract Amblyopia is induced in model systems by monocular deprivation (MD), which changes the stimulus selectivity of neurons in the primary visual cortex. Prior research utilizing this model established that the changes in neural selectivity induced by MD result from the reorganization of excitatory glutamatergic cortical synapses onto excitatory cortical neurons, which is regulated by an inhibitory GABAergic network composed of parvalbumin positive inhibitory interneurons (PV INs). An emerging consensus is that a permissive level of inhibition from PV IN circuits in cortical layer 2/3 is required for plasticity at downstream excitatory synapses, and that inhibition above or below the permissive range constrains the response to MD. Accordingly, developmental strengthening of inhibition triggers the onset of the critical period; at later stages, the ?permissive? range of inhibition is achieved by reductions the recruitment of PV INs. Here we identify the plasticity of excitation onto layer 2/3 PV INs as a critical locus for the regulation of circuit reorganization in V1. Our preliminary data demonstrate that the initial response to MD is a rapid and transient elimination of excitatory connections made by local pyramidal neurons (Pyr) onto PV INs. Following 1 day of MD, we find that ~50% of local L2/3 Pyrà?PV-IN connections are eliminated. Importantly, synapses from distal L2/3 Pyrs and excitation from layer 4 Pyrs remains unchanged. This all-or-none elimination of specific connections coincides with the loss of synaptic structure, is transient, and returns to control values following 3 days of MD. Our preliminary results also demonstrate that the MD-induced elimination of proximal L2/3 Pyrà?PV INs inputs depends on mGluR5 activation and is inhibited by expression of activity-independent neuronal pentraxin 2 (NPTX2). We propose that the rapid mGluR5 and NPTX2-dependent elimination of local L2/3 Pyrà?PV INs connection is an obligatory initial step for subsequent changes in ocular dominance and spatial acuity induced by MD. Accordingly, we show that accumulation of NPTX2 prevents L2/3 Pyrà?PV IN elimination and ocular dominance plasticity. Conversely, expression of dominant negative NPTX2 in adults reactivates the elimination of L2/3 Pyrà?PV INs and ocular dominance plasticity in response to MD We propose a series of multidisciplinary experiments to test the validity of this model that combine the expertise of the Quinlan lab in the assessment of physiological changes in vivo physiology and the Kirkwood lab in the assessment of changes in single synapses between identified neurons. We will test the hypothesis that the elimination of L2/3 Pyrà?PV INs excitatory synapses is 1) local, transient and confined to a postnatal critical period 2) dependent on mGluR and NPTX2 signaling and 3) an obligatory initial step for subsequent changes in ocular dominance and spatial acuity induced by MD. Our model predicts that the end of the critical period reflects directly the loss of L2/3 Pyrà?PV-IN plasticity, which departs from many widely-held assumptions regarding developmental changes in synaptic plasticity in the mammalian cortex.