Browsing by Subject "Neurobiology"

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

Forebrain cholinergic projection systems innervate the entire cortex and hippocampus. These cholinergic systems are involved in a wide range of cognitive and behavioral functions, including learning and memory, attention, and sleep-waking modulation. However, the in vivo physiological mechanisms of cholinergic functions, particularly their fast dynamics and the consequent modulation on the hippocampus and cortex, are not well understood. In this dissertation, I investigated these issues using a number of convergent approaches.

First, to study fast acetylcholine (ACh) dynamics and its interaction with field potential theta oscillations, I developed a novel technique to acquire second-by-second electrophysiological and neurochemical information simultaneously with amperometry. Using this technique on anesthetized rats, I discovered for the first time the tight in vivo coupling between phasic ACh release and theta oscillations on fine spatiotemporal scales. In addition, with electrophysiological recording, putative cholinergic neurons in medial setpal area (MS) were found with firing rate dynamics matching the phasic ACh release.

Second, to further elucidate the dynamic activities and physiological functions of cholinergic neurons, putative cholinergic MS neurons were identified in behaving rats. These neurons had much higher firing rates during rapid-eye-movement (REM) sleep, and brief responses to auditory stimuli. Interestingly, their firing promoted theta/gamma oscillations, or small-amplitude irregular activities (SIA) in a state-dependent manner. These results suggest that putative MS cholinergic neurons may be a generalized hippocampal activation/arousal network.

Third, I investigated the hypothesis that ACh enhances cortical and hippocampal immediate-early gene (IEG) expression induced by novel sensory experience. Cholinergic transmission was manipulated with pharmacology or lesion. The resultant cholinergic impairment suppressed the induction of arc, a representative IEG, suggesting that ACh promotes IEG induction.

In conclusion, my results have revealed that the firing of putative cholinergic neurons promotes hippocampal activation, and the consequent phasic ACh release is tightly coupled to theta oscillations. These fast cholinergic activities may provide exceptional opportunities to dynamically modulate neural activity and plasticity on much finer temporal scales than traditionally assumed. By the subsequent promotion of IEG induction, ACh may further substantiate its function in neural plasticity and memory consolidation.

Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis.

We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons.

To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway.

These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.

Sequential activity in brain networks has been observed across multiple species, neural structures, and behavioral contexts. This activity consists of groups of neurons that are active at different times in a reproducible manner, and in many contexts appears to be internally generated, proceeding autonomously in the absence of ongoing sensory input. Such activity underlies a number of motor behaviors and working memory tasks. This activity displays diverse temporal characteristics, with properties that depend on the neural area, and on the timescale of observation, and experimental evidence suggests that it forms gradually over the course of task learning and behavioral training. Understanding how single neuron and network mechanisms give rise to such activity remains a fundamental challenge in neuroscience.

Learning in the brain is believed to occur through activity-dependent modifications of synaptic connectivity. We explore a class of synaptic plasticity rules, temporally asymmetric Hebbian learning rules, and investigate whether their application in network models can account for the diversity of temporal characteristics. These Hebbian rules transform a sequence of input patterns into synaptic weight updates. After learning, recalled sequential activity is reflected in the transient correlation of network activity with each of the stored input patterns. We use mean field-theory to develop a low-dimensional description of the network dynamics and compute the storage capacity of these networks. On short timescales, we find that the temporal statistics of this activity are consistent with experimental observations in multiple brain structures. Nonlinearities in the learning rule alter the degree of temporal sparsity during retrieval. On long timescales, we find that modification of synaptic connectivity due to noise, storage of other patterns, or rehearsal of learned patterns, produces sequential activity with highly labile dynamics.

In many experimental settings, the speed of sequential activity can be flexibly controlled depending on task context. We find that adding heterogeneity in the learning rule across synapses allows retrieval speed to be controlled through changes in the external input drive. Speed changes are accompanied by a temporal rescaling of activity dynamics, similar to neural dynamics observed in cortex.

Glia make up roughly half of all cells in the mammalian nervous system and play a major part in nervous system development, function and disease. Although research in the past few decades have shed light on their morphological and functional diversity, there is still much to be known about key aspects of their development such as the generation of glial diversity and the factors governing proper morphogenesis. In the work presented here I started with a forward genetic screen using amphid sheath (AMsh) glia in the model organism C. elegans and uncovered various factors that govern different aspects of glial development including glial fate specification, migration and growth. First, I identified the function of the proneural gene lin-32/Atoh1 in repressing an AMsh glial fate. Gliogenesis is a fundamental process during nervous system development, and generating the appropriate number of specific glial cell types is required for proper nervous system function. lin-32 loss of function mutants possess additional AMsh glia beyond the normal pair. Interestingly, the ectopic AMsh cells at least partially arise from cells originally fated to become CEPsh glia, suggesting that lin-32 may be involved in the specification of specific glial subtypes. I also found that lin-32 acts in parallel with two other proneural transcription factors cnd-1/NeuroD1 and ngn-1/Neurog1 in negatively regulating an AMsh glia fate. Furthermore, expression of murine Atoh1 fully rescues lin-32 mutant phenotypes, suggesting potential functional conservation during glial fate specification. Next, I found that AMsh glial migration is regulated by vitamin B12 through isoform-specific expression of PTP-3/LAR PTPR (Leukocyte-common antigen related receptor-type tyrosine-protein phosphatase). The uptake of diet-supplied vitamin B12 in the intestine was found to be critical for the expression of a long isoform of PTP-3 (PTP-3A) in neuronal and glial cells. The expression of PTP-3A then cell-autonomously regulates glial migration and synapse formation through interaction with an extracellular matrix protein NID-1/Nidogen 1. Together, my findings demonstrate that isoform-specific regulation of PTP-3/LAR PTPR expression mediates vitamin B12-dependent neuronal and glial development. These results may also help inform our understanding of neurodevelopmental and degenerative disorders linked to vitamin B12 deficiency. Finally I identified a pathway that regulates AMsh cell growth involving the conserved cis Golgi membrane protein eas-1/GOLT1B. Coordination of cell growth is essential for the development of the brain, but the molecular mechanisms underlying the regulation of glial size are poorly understood. My research shows that that eas-1 inhibits a conserved E3 ubiquitin ligase rnf-145/RNF145, which in turn promotes nuclear activation of sbp-1/ SREBP, an important regulator of sterol and fatty acid synthesis, to restrict cell growth. At early developmental stages, rnf-145 in the cis Golgi network inhibits sbp-1 activation to promote the growth of glia, and when animals reach the adult stage this inhibition is released through an eas-1-dependent shuttling of rnf-145 from the cis Golgi to the trans Golgi network to stop glial growth. Furthermore, I identified long chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA), as downstream products of the eas-1-rnf-145-sbp-1 pathway that functions to prevent the overgrowth of glia. These findings reveal a novel and potentially conserved mechanism underlying glial size control. Taken together, my research reveals several pathways that regulate different stages of AMsh glial development. Since many of these pathways are conserved, study of C. elegans glial development may also help inform our understanding of glial biology in vertebrate systems.

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to

generate defined motor outputs, or behavior. This integration requires developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown.

Here we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless. During development, the putative chromatin modulator Alhambra (Alh) represses the expression of both fru and of specific olfactory receptors, helping to coordinate and establish both the sensory and circuit identities of the ORNs involved in sex-specific behaviors. In contrast, the maintenance of fru expression and thus the identities of

these ORNs in adults utilize signaling from olfactory receptors through Cam Kinases and the histone acetyl transferase p300/CBP. Our results highlight feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs.These mechanisms might underlie innate and adaptable aspects of odor-guided sex- specific behaviors.

Negotiating the complex decisions that we encounter daily requires coordinated neu-

ronal activity. The enormous variety of decisions we make, the intrinsic complexity

of the situations we encounter, and the extraordinary flexibility of our behaviors

suggest the existence of intricate neural mechanisms for negotiating contexts and

making choices. Further evidence for this prediction comes from the behavioral al-

terations observed in illness and after injury. Both clinical and scientific evidence

suggest that decision signals are carried by electrical neuronal activity and influenced

by neuromodulatory chemicals. This dissertation addresses the function of two puta-

tive contributors to decision-making: neuronal activity in posterior cingulate cortex

and modulatory effects of serotonin. I found that posterior cingulate neurons respond

phasically to salient events (informative cues; intentional saccades; and reward deliv-

ery) across multiple contexts. In addition, these neurons signal heuristically guided

choices across contexts in a gambling task. These observations suggest that posterior

cingulate neurons contribute to the detection and integration of salient information

necessary to transform event detection to expressed decisions. I also found that

lowering levels of the neuromodulator serotonin increased the probability of making

risky decisions in both monkeys and mice, suggesting that this neurotransmitter con-

tributes to preference formation across species. These results suggest that posterior

cingulate cortex and serotonin each contribute to decision formation. In addition, the

unique serotonergic pro jections to posterior cingulate cortex, as well as the frequent

implication of altered serotonergic and posterior cingulate function in psychiatric dis-

orders, suggest that the confluence of cingulate and serotonergic activity may offer

key insights into normal and pathological mechanisms of decision making.

After every eye movement, the brain must realign the visual and auditory reference frames in order to co-locate sights and sounds. Exactly where, when, and how such visual-auditory spatial integrations occur is not fully understood. We recently discovered that the eardrum oscillates beginning a few milliseconds before saccades and continuing until well into ensuing periods of fixation (Gruters et al., 2018)(Gruters, Murphy et al PNAS 2018). Information about at least the horizontal direction and length of saccades appear to be reflected in the phase and magnitude of these eye movement-related eardrum oscillations (EMREO).

Here, we sought to assess the full spatial characteristics of this signal for saccade parameters in both vertical and horizontal dimensions. Concurrently we sought to validate that independent estimations of vertical and horizontal saccade parameter contributions can be linearly combined to predict EMREO waveforms for saccades in all directions – a fundamental assumption of current analyses.

We found that EMREOs depend on both horizontal and vertical saccade components, varying predominantly with eye displacement, but modulated by absolute (initial or final) position as well. In toto, EMREO appear to represent combinations of these saccade parameters such that any saccade corresponds to a specific eardrum oscillation that contains a linear combination of the vertical and horizontal saccade parameters. Regressions in both the time and frequency domain create a fuller picture of the spatial information contained in EMREO. These results demonstrate that detailed information about the relationship between visual and auditory reference frames is present in the earliest stage of the auditory pathway. They also demonstrate that this information is mapped linearly and can therefore be recovered with a small set of basis components.

Future work delving into the relationship between EMREO and the transduction of incoming sounds will be needed to ascertain their effects on the processing of auditory spatial locations in relation to the visual scene. While the frequency and magnitude of EMREO suggest that they may be related to middle ear muscle contractions, the underlying mechanisms that generate them are unknown.

Estradiol modulates the use of spatial navigation strategies in female rats. The presence of circulating estradiol enhances learning on tasks that require the use of a hippocampus-dependent place strategy and impairs learning on tasks that require the use of a dorsal striatum-dependent response strategy. When either strategy may be used successfully, estradiol biases females to use a place strategy. While this behavioral effect has been well-described in the young adult female rat, little is known about the mechanisms in the brain that underlie it or how it changes across age. The experiments in this dissertation examined how age, previous experience, and hormonal condition affect the ability of estradiol to modulate learning during explicit training of place and response tasks, as well as navigation strategy use during ambiguous navigation tasks. Age highly influenced the ability of estradiol to influence strategy use. While female rats could use place and response strategies to navigate by postnatal day (PD) 21, estradiol did not bias them to use a response strategy until PD26, just before puberty. In adulthood, previous navigation experience and estradiol interacted to influence navigation strategy use on a series of experiences to an ambiguous navigation task. And, estradiol impaired learning during explicit response training but did not affect place learning. In middle age, estradiol further impaired response learning but still did not affect place learning. Long-term hormone deprivation, however, was detrimental to acquisition of a place task but did not affect response learning. These experiments also examined the effects of estradiol on activity, plasticity, and reliability of neuronal ensembles in several subregions of the hippocampus and striatum during spatial navigation using cellular and molecular techniques that take advantage of the kinetics of the immediate-early genes c-fos and Arc. Increased activation and plasticity during active exploration across several subregions of the hippocampus and striatum reflected similar inputs to these neural systems and similar effects of exploration. However, estradiol modulated the plasticity and reliability of neuronal ensembles in the hippocampus and striatum specifically during goal-directed spatial navigation. Estradiol increased plasticity in CA1 of all behaviorally-trained rats, but only place strategy users displayed high reliability in this plasticity across training and probe trials on a navigation task. Estradiol prevented increase in plasticity and reliability in the dorsolateral striatum displayed by low estradiol response strategy users. These experiments reveal how several factors, including age, influence estradiol's modulation of spatial navigation strategy use and suggest functional mechanisms by which this modulation occurs.

Targeted stimulation of the brain has the potential to treat mental illnesses. The objective of this work is to develop methodology that enables scientists to design stimulation methods based on the electrophysiological dynamics. We first develop several factor models that characterize aspects of the dynamics relevant to these illnesses. Using a novel approach, we can then find a single predictive factor of the trait of interest. To improve the quality of the associated loadings, we develop a method for removing concomitant variables that can dominate the observed dynamics. We also develop a novel inference technique that increases the relevance of the predictive loadings. Finally, we demonstrate the efficacy of our methodology by finding a single factor responsible for social behavior. This factor is stimulated in new subjects and modifies behavior in the new individuals. These results indicate that our methodology has high potential in developing future cures of mental illness.

The retina signals visual information to the brain with the parallel channels of different retinal ganglion cell (RGC) types, whose signals ultimately lead to visual perception. Between cloudy nights and sunny days, the retina must combat the trillion-fold change in mean light intensity to successfully convey visual information. Critically, the nature of both signal and noise in RGC populations is altered across this broad range of light levels, creating a rich problem of how visual messages are encoded by the retina and transmitted to the brain. This thesis addresses these topics using large-scale multielectrode array recordings of RGC populations in different light conditions. In Chapter 2, I characterize how retinal signaling is altered over a wide range of light intensities. Chapter 3 investigates how adaptation impacts visual encoding of different RGC types. My results suggest that although retinal computations change substantially over light conditions, there are some elements of visual encoding that are invariant to light adaptation. Finally, Chapter 4 examines adaptation-induced changes in the structure of correlated activity and the subsequent impact on processing retinal output. The findings of this chapter clarify the nature of RGC responses crucial for downstream readout across light levels. Overall, this work identifies aspects of RGC activity that are important for encoding visual information and decoding retinal output from starlight to sunlight.

Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.

In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.

The ultimate goal of the nervous systems of all animals is conceptually simple: Manipulate the external environment to maximize one's own survival and reproduction. The myriad means animals employ in pursuit of this goal are astoundingly complex, but constrained by common factors. For example, to ensure survival, all animals must acquire the necessary nutrients to sustain metabolism. Similarly, social interaction of some form is necessary for mating and reproduction. For some animals, the required social interaction goes far beyond that necessary for mating. Humans and many other primates exist in complex social environments, the navigation of which are essential for adaptive behavior. This dissertation is concerned with processes of transforming sensory stimuli regarding both nutritive and social information into motor commands pursuant to the goals of survival and reproduction. Specifically, this dissertation deals with these processes in the rhesus macaque. Using a task in which monkeys make decisions simultaneously weighing outcomes of fruit juices and images of familiar conspecifics, I have examined the neurophysiology of social and nutritive factors as they contribute to choice behavior; with the ultimate goal of understanding how these disparate factors are weighed against each other and combined to produce coherent motor commands that result in adaptive social interactions and the successful procurement of resources. I began my investigation in the lateral intraparietal cortex, a well-studied area of the primate brain implicated in visual attention, oculomotor planning and control, and reward processing. My findings indicate the lateral intraparietal cortex represents social and nutritive reward information in a common neural currency. That is, the summed value of social and nutritive outcomes is proportional to the firing rates of parietal neurons. I continued my investigation in the striatum, a large and functionally diverse subcortical nuclei implicated in motor processing, reward processing and learning. Here I find a different pattern of results. Striatal neurons generally encoded information about either social outcome or juice rewards, but not both, with a medial or lateral bias in the location of social or juice information encoding neurons, respectively. In further contrast to the lateral intraparietal cortex, the firing rates of striatal neurons coding social and nutritive outcome information is heterogeneous and not directly related to the value of the outcome. This dissertation represents a few incremental steps toward understanding how social information and the drive toward social interaction are incorporated with other motivators to influence behavior. Understanding this process is a necessary step for elucidating, treating, and preventing pathologies