Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

Adult hippocampal neurogenesis—a unique form of plasticity in the dentate gyrus (DG)—is regulated by experience, and when manipulated can have specific effects on behavior. Different methods have been used over the years to study new neurons’ functional role in the hippocampus, many of which focus on ablating neurogenesis. While ablation methods can test the necessity of adult-born granule cells (abGCs) for behavior, these techniques remove all abGCs from the circuit and thus do not allow one to determine which properties of abGCs are required for behavior. Such information is required to understand the mechanism of their action. Thus, new strategies are needed to determine what properties of young abGCs allow them to distinguish themselves from their mature counterparts and uniquely impact behavior. Recent hypotheses have suggested that the enhanced synaptic plasticity exhibited by 4–6-week-old abGCs allows them to uniquely contribute to hippocampal circuit function, and thus behavior. The primary goal of this thesis was to explore the contribution young abGCs’ heightened synaptic plasticity makes to hippocampal function. This was achieved using a transgenic mouse approach that allowed for the conditional deletion of NR2B from abGCs. Overall, iNR2BNes mice generated the same number of new neurons in adulthood as control mice at baseline. These neurons survived and matured with only a slight reduction in dendritic complexity. However, a potentially important electrophysiological property of these neurons—their enhanced synaptic plasticity—had been eliminated. From an electrophysiological standpoint, iNR2BNes mice resemble mice with ablated neurogenesis, while from all other neurogenic standpoints examined they most closely resemble wild-type mice. Consequently, these mice provided a novel model to test the extent to which young abGCs’ enhanced plasticity contributes to hippocampal-dependent behaviors. The results reveal that eliminating NR2B-containing NMDA receptors from abGCs does not alter baseline anxiety or antidepressant (AD)-like behavior. However, iNR2BNes mice differed from controls in measures of cognitive function. These mice were able to learn in the contextual fear conditioning test, but were impaired in the more difficult contextual fear discrimination test. Mice also exhibited a decreased novelty exploration phenotype that impaired their performance in the novel object recognition test. Together, these results indicate that the NR2B-dependent heightened plasticity exhibited by 4–6-week-old abGCs is necessary for responses to novelty and fine contextual discrimination, but does not contribute to baseline anxiety or emotionality. AD treatment increases levels of adult neurogenesis in the hippocampus, and these newborn neurons have been shown to be necessary for some of the behavioral effects of ADs seen in rodents. In addition, the maturation timeline of adult neurogenesis correlates with the onset of behavioral responses to ADs. ADs also enhance a neurogenesis-dependent form of long-term potentiation (LTP) in the DG evoked by medial perforant path stimulation under intact GABAergic tone called ACSF-LTP. Thus, a potential mechanism by which abGCs may contribute to AD behavioral efficacy is by providing extra plastic units to the DG circuit. This theory was tested by once again using the mouse line in which NR2B can be conditionally deleted from abGCs in the DG. Here, we found that deletion of the NR2B subunit significantly attenuated a neurogenesis-dependent behavioral response to fluoxetine in the novelty suppressed feeding test, and additionally blocked fluoxetine’s ability to enhance young abGCs’ maturation and subsequent integration into the hippocampal network. This suggests that eliminating abGCs’ enhanced plasticity decreases their ability to influence DG output resulting in an AD response that is less robust than seen in control mice. Control experiments revealed the specificity of this effect, as NR2B deletion did not impact the effect of fluoxetine in a neurogenesis-independent behavioral assay (tail suspension test) or in an assay that was insensitive to fluoxetine in this strain of mice (elevated plus maze). Our efforts to isolate the contribution of abGCs’ unique physiology from the neurogenic effects of fluoxetine were not entirely successful as the results presented here also revealed slight group differences in neurogenesis between control mice and mice lacking NR2B in young neurons. Yet, this data still supports the idea that fluoxetine increases the ability of abGCs to participate in DG output by increasing the chance that new neurons will be activated during DG stimulation. This may be achieved either by increasing their overall number, increasing their potential to make synaptic connections, or increasing their ability to strengthen their connections. However, due to the close link between activity and maturation that appears to be enhanced with fluoxetine treatment, a different approach with greater temporal resolution is needed to separate the neurogenic effects of fluoxetine from the physiological contribution abGCs make to hippocampal output. With this in mind, a mouse line in which abGCs could be temporally inhibited was also generated. Cellular and behavioral characterization of mice conditionally expressing hM4Di—a mutated muscarinic acetylcholine receptor that is insensitive to endogenous acetylcholine, but can be activated by the biologically inert, highly bioavailable compound, clozapine N-oxide (CNO)—has begun. Results show that acute CNO treatment in mice expressing this designer receptor exclusively activated by a designer drug (DREADD) in DG granule cells can impair encoding of contextual fear memory. Chronically treating these mice had an anxiogenic effect in the open field test, but otherwise anxiety and emotionality in these mice were comparable to controls. Chronic CNO treatment in mice expressing hM4Di in young abGCs effectively decreased these cells’ dendritic complexity, but did not alter proliferation or early survival. Thus, hM4Di DREADDs represent a novel tool that can be used to modulate activity of neurons in a temporally restricted manner, allowing for both acute and chronic manipulations of hippocampal granule cells. The experiments put forth in this thesis will highlight the importance of abGCs enhanced plasticity. The utility as well as potential pitfalls of the mouse models used here to test theories of abGC function will also be explored. Hopefully this analysis will provide an improved framework in which future experiments can be developed with the aim of uncovering novel insights into the hippocampal circuitry that underlies learning and memory and discovering new strategies for the treatment of neurological and psychiatric disorders.

Switching between a state of fear and safety is a critical aspect of adaptive behavior. Aversive and non-aversive associations must be formed quickly and reliably but remain malleable as these associations change dynamically. When these associations become biased towards aversive associations by traumatic and stressful circumstances, as in PTSD, fear generalization and impaired fear extinction arise. These changes are associated with reduced activity in the medial prefrontal cortex (mPFC) and enhanced activity in the basolateral amygdala (BLA). It has been hypothesized that the mPFC mediates top-down control of the BLA to signal safety. It has previously been demonstrated that synchronous activity within the mPFC-BLA circuit is strongly engaged during fear conditioning, but it is unknown how activity in this circuit changes to mediate aversive discrimination. We investigated how the mPFC and BLA cooperate to mediate successful discrimination between aversive and non-aversive stimuli both for learned and innately-valent associations. Extracellular elecrophysiological recordings were obtained simultaneously form the mPFC and BLA in mice during innate anxiety, fear discrimination, and fear extinction. Local field potentials were recorded in both structures along with single unit recordings from the BLA. We discovered that fear was associated with enhanced theta-frequency synchrony and theta-gamma coupling within the mPFC-BLA circuit. On the other hand, safety was associated with predominant mPFC-to-BLA directionality of synchronous information flow and enhanced fast gamma frequency activity in both structures. Interestingly, gamma oscillations in the BLA were strongly coupled to theta frequency activity arising in the mPFC. This data is consistent with entrainment of inhibitory circuits in the BLA by mPFC input to mediate safety.

The ventral hippocampus (vHPC), medial prefrontal cortex (mPFC), and basolateral amygdala (BLA) are each required for the expression of anxiety-like behavior. Yet the role of each individual element of the circuit is unclear. The projection from the vHPC to the mPFC has been implicated in anxiety-related neural synchrony and spatial representations of aversion. The role of this projection was examined using multi-site neural recordings combined with optogenetic terminal inhibition. Inhibition of vHPC input to the mPFC disrupted anxiety and mPFC representations of aversion, and reduced theta synchrony in a pathway-, frequency- and task-specific manner. Moreover, bilateral, but not unilateral, inhibition altered physiological correlates of anxiety in the BLA, mimicking a safety-like state. These results reveal a specific role for the vHPC-mPFC projection in anxiety-related behavior and the spatial representation of aversive information within the mPFC. Moreover, these data suggested that theta-frequency input from the vHPC plays a causal role in anxiety-like behavior. Next, it was investigated whether optogenetic stimulation of the vHPC-mPFC at a theta frequency was sufficient to increase anxiety. Stimulating the vHPC input to the mPFC with a sinusoidal light pattern at 8 Hz significantly increased anxiety behavior. The anxiogenic effect of vHPC terminal stimulation was frequency- (8 Hz but not 20 Hz) and pattern- (sinusoids but not pulses) specific. To understand how pulses and sinusoidal light modulate mPFC neurons differentially, mPFC pyramidal neurons were recorded both in vitro and in vivo while stimulating vHPC terminals with the same sinusoidal or pulsatile patterns. In vitro, sinusoidal stimulation increased the rate of spontaneous EPSCs, while pulses evoked strong, stimulus-locked EPSCs. In vivo, sinusoidal stimulation of vHPC terminals increased the phase-locking of mPFC single unit spiking to the optical stimulation pattern without changing overall firing rates. Together, these results suggest that sinusoidal stimulation at 8 Hz enhances theta-frequency activity in mPFC neurons as well as anxiety-related behavior. Moreover, they suggest that theta-frequency components of neural activity play a privileged role in vHPC-mPFC communication and hippocampal-dependent forms of anxiety.

It is a major challenge to understand functional neuronal circuits across the whole brain. Existing methods for observing neuronal activity represent a major bottleneck in addressing biological problems. In our lab, we developed Swept Confocally Aligned Planar Excitation (SCAPE) microscopy, which offers the ability to image a large 3D volume (e.g. 1000x800x250um) at speeds exceeding 10 volumes per second. Used with different genetically encoded fluorescent indicators, SCAPE enables us to observe neuronal activity across the whole brain of different small animal models, or a much larger volume of intact cortex/tissue compared to traditional approaches. The unique single objective design and flexible system layout of SCAPE makes it simple to image different samples without complex sample preparation and restraint. During this thesis work, I collaborated with biology and neuroscience labs to develop and optimize a range of novel in-vivo/in-vitro neuroimaging applications using SCAPE microscopy. In particular, my research has focused on using SCAPE to image freely crawling Drosophila Melanogaster larvae, intact mouse olfactory epithelium, head fixed behaving adult Drosophila, larval zebrafish brain and beating heart, and the neuronal system of behaving C. elegans, all in collaboration with experts in these models from Columbia University and other research institutions. I also developed and optimized different sample preparations and experimental procedures to take full advantage of the high-speed 3D imaging capabilities and flexibility of SCAPE microscopy. Finally, I optimized computational and image analysis techniques for large scale 5D SCAPE imaging datasets, including 3D cell tracking, large scale 3D data motion correction/registration, and cellular level neuronal activity extraction with different dimensionality reduction methods. The experiments I have performed in different animal models have enriched the long-term development of SCAPE by providing valuable feedback for system improvement and dissemination, and pushing the SCAPE design towards a more interchangeable platform with diverse capabilities suitable for routine uses by our collaborators and the wider neuroscience community.

The field of neuroscience has recently seen optogenetics emerge as a highly utilized and powerful method of non-invasive neural activation and inhibition. This thesis seeks to enhance the optogenetic toolbox through the design, construction, and evaluation of a number of hardware and software modules for research in Caenorhabditis elegans neuroscience. In the first aim, we combine optogenetics, microfluidics, and automated image processing, to create a system capable of high-throughput analysis of synaptic function. In the second aim, we develop a multi-modal illumination system for the manipulation of optogenetic reagents. The system is capable of multi-spectral illumination in definable patterns, with the ability to dynamically alter the intensity, color, and shape of the illumination. The illumination system is controlled by a set of software programs introduced in aim three, and is demonstrated through a set of experiments in aim four where we selectively activate and inhibit specific neural nodes expressing optogenetic reagents in freely moving C. elegans. With the ability to target specific nodes in a freely moving animal, we can correlate specific neural states to behaviors allowing for the dissection of neural circuits. Taken together, the developed technologies for optogenetic researchers will allow for experimentation with previously unattainable speed, precision and flexibility.

Population-wide synchronized rhythmic bursts of electrical activity are present in a variety of neural circuits. The proposed general mechanisms for rhythmogenesis are often attributed to intrinsic and synaptic properties. For example, the recurrent excitation through excitatory synaptic connections determines burst initiation, and the slower kinetics of ionic currents or synaptic depression results in burst termination. In such theories, a slow recovery process is essential for the slow dynamics associated with bursting. This thesis presents a new hypothesis that depends on the connectivity pattern among neurons rather than a slow kinetic process to achieve the network-wide bursting. The thesis begins with an introduction of bursts of electrical activity in a purely excitatory neural network and existing theories explaining this phenomenon. It then covers the small-world approach, which is applied to modify the network structure in the simulation, and the Morris-Lecar (ML) neuron model, which is used as the component cells in the network. Simulation results of the dependence of bursting activity on network connectivity, as well as the inherent network properties explaining this dependence are described. This work shows that the network-wide bursting activity emerges in the small-world network regime but not in the regular or random networks, and this small-world bursting primarily results from the uniform random distribution of long-range connections in the network, as well as the unique dynamics in the ML model. Both attributes foster progressive synchronization in firing activity throughout the network during a burst, and this synchronization may terminate a burst in the absence of an obvious slow recovery process. The thesis concludes with possible future work.

What changes in our brain when we learn? This is perhaps the most intriguing question of science in this century. In an attempt to learn more about the inner workings of neural circuitry, I studied cultured 2-dimensional networks of neurons on multi-electrode arrays (MEAs). MEAs are ideal tools for studying long-term neural ensemble activity because many individual cells can be studied continuously for months, through electrical stimulation and recording. One of the most prominent patterns of activity observed in these cultures is network-wide spontaneous bursting, during which most of the active electrodes in the culture show elevated firing rates. We view the persistence of spontaneous bursting in vitro as a sign of arrested development due to deafferentation. Substituting distributed electrical stimulation for afferent input transformed the activity in dissociated cultures from bursting to more dispersed spiking, reminiscent of activity in the adult brain. Burst suppression reduced the variability in neural responses making it easier to induce and detect functional plasticity caused by tetanic stimulation. This suggests that spontaneous bursts interfere with the effects of external stimulation and that a burst-free environment leads to more stable connections and predictable effects of tetanization. Moreover, our culture models continuously receive input stimulation in the form of background electrical stimulation, and so better resemble the intact brain than isolated (non-continuously stimulated) cultures. The proportion of GABAergic neurons in the cultures was significantly increased (p<1e-2, paired t-test) after burst-quieting for 2 days, suggesting that burst suppression operated through the homeostatic control of inhibitory neurotransmitter levels. We also studied the role of spontaneous bursts as potential carriers of information in the network by clustering these spatiotemporally diverse bursts. Spontaneous burst clusters were stable over hours and tetanic stimulation significantly reorganized the distribution of the clusters. In summary, this body of work explores the rules of network-level functional plasticity and provides the input (electrical stimulation) output (spatiotemporal patterns) mappings for behavioral studies in embodied hybrid systems. The results of this study may also have clinical implications in the development of sensory prostheses and treatment of diseases of aberrant network activity such as epilepsy.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 91-106).

Thesis (Ph. D.)--Oregon State University, 2006. / Blank pages 143 and 159 not microfilmed. Includes bibliographical references. Also available online (PDF file) by a subscription to the set or by purchasing the individual file.