Browsing by Subject "Biology, Neuroscience"

The loss of the ability to walk as the result of neurological injury or disease critically impacts the mobility and everyday lifestyle of millions. The World Heath Organization (WHO) estimates that approximately 1% of the world's population needs the use of a wheelchair to assist their personal mobility. Advances in the field of brain-machine interfaces (BMIs) have recently demonstrated the feasibility of using neuroprosthetics to extract motor information from cortical ensembles for more effective control of upper-limb replacements. However, the promise of BMIs has not yet been brought to bear on the challenge of restoring the ability to walk. A future neuroprosthesis designed to restore walking would need two streams of information flowing between the user's brain and the device. First, the motor control signals would have to be extracted from the brain, allowing the robotic prosthesis to behave in the manner intended by the user. Second, and equally important would be the flow of sensory and proprioceptive information back to the user from the neuroprosthesis. Here, I contribute to the foundation of such a bi-directional brain machine interface for the restoration of walking in a series of experiments in two animal models, designed to show the feasibility of (1) extracting locomotor information from neuronal ensemble activity and (2) sending information back into the brain via cortical microstimulation.

In a set of experiments designed to investigate the extraction of locomotor parameters, I chronically recorded from ensembles of neurons in primary motor (M1) and primary somatosensory (S1) cortices in two adult female rhesus macaques as they walked bipedally, at various speeds, both forward and backward on a custom treadmill. For these experiments, rhesus monkeys were suitable because of their ability to walk bipedally in a naturalistic manner with training. I demonstrate that the kinematics of bipedal walking in rhesus macaques can be extracted from neuronal ensemble recordings, both offline and in real-time. The activity of hundreds of neurons was processed by a series of linear decoders to extract accurate predictions of leg joints in three dimensional space, as well as leg muscle electromyograms (EMGs). Using a multi-layered switching model allowed us to achieve increased extraction accuracy by segregating different behavioral modes of walking.

In a second set of experiments designed to investigate the usage of microstimulation as a potential artificial sensory channel, I instructed two adult female Aotus trivirgatus (owl monkeys) about the location of a hidden food reward using a series of cortical microstimulation patterns delivered to primary somatosensory (S1) cortex. The owl monkeys discriminated these microstimulation patterns and used them to guide reaching movements to one of two targets. Here, owl monkeys were used which were previously implanted with electrode arrays of high longevity and stability. These monkeys were previously trained on a somatosensory cued task, which allowed a quick transition to microstimulation cueing. The owl monkeys learned to interpret microstimulation patterns, and their skill and speed of learning new patterns improved over several months. Additionally, neuronal activity recorded on non-stimulated electrodes in motor (M1), premotor (PMD) and posterior parietal (PP) cortices allowed us to examine the immediate neural responses to single biphasic stimulation pulses as well as overall responses to the spatiotemporal pattern. Using this recorded neuronal activity, I showed the efficacy of several linear classification algorithms during microstimulation.

These results demonstrate that locomotor kinematic parameters can be accurately decoded from the activity of neuronal ensembles, that multichannel microstimulation is a viable information channel for sensorized prosthetics, and that the technical limitations of combining these techniques can be overcome. I propose that bi-directional BMIs integrating these techniques will one day restore the ability to walk to severely paralyzed patients.

Mice have become the preferred model system for studying brain function and disease. With the powerful genetic tools available, mouse models can be created to study the underlying molecular basis of neurobiology in vivo. Just as magnetic resonance imaging is the dominant tool for evaluating the human brain, high-resolution MRI--magnetic resonance microscopy (MRM)--is a useful tool for studying the brain of mouse models. However, the need for high spatial resolution limits the signal-to-noise ratio (SNR) of the MRM images. To address this problem, T1-shortening contrast agents can be used, which not only improve the tissue contrast-to-noise ratio (CNR) but also increase SNR by allowing the MR signal to recover faster between pulses. By "actively staining" the tissue with these T1-shortening agents, MRM can be performed with higher resolution, greater contrast, and shorter scan times. In this work, active staining with T1-shortening agents was used to enhance three types of in vivo mouse brain MRM: (1) angiographic imaging of the neurovasculature, (2) anatomical imaging of the brain parenchyma, and (3) functional imaging of neuronal activity.

For magnetic resonance angiography (MRA) of the mouse, typical contrast agents are not useful because they are quickly cleared by the body and/or extravasate from the blood pool before a high-resolution image can be acquired. To address these limitations, a novel contrast agent--SC-Gd liposomes--has been developed, which is cleared slowly by the body and is too large to extravasate from the blood pool. In this work, MRA protocols were optimized for both the standard technique (time-of-flight contrast) and SC-Gd liposomes. When the blood was stained with SC-Gd liposomes, small vessel CNR improved to 250% that of time-of-flight. The SC-Gd liposomes could also be used to reduce scan time by 75% while still improving CNR by 32%.

For MRM of the mouse brain parenchyma, active staining has been used to make dramatic improvements in the imaging of ex vivo specimens. However for in vivo imaging, the blood-brain barrier (BBB) prevents T1-shortening agents from entering the brain parenchyma. In this work, a noninvasive technique was developed for BBB opening with microbubbles and ultrasound (BOMUS). Using BOMUS, the parenchyma of the brain could be actively stained with the T1-shortening contrast agent, Gd-DTPA, and MRM images could be acquired in vivo with unprecedented resolution (52 x 52 x 100 micrometers3) in less than 1 hour.

Functional MRI (fMRI), which uses blood oxygen level dependant (BOLD) contrast to detect neuronal activity, has been a revolutionary technique for studying brain function in humans. However, in mice, BOLD contrast has been difficult to detect and thus routine fMRI in mice has not been feasible. An alternative approach for detecting neuronal activity uses manganese (Mn2+). Mn2+ is a T1-shortening agent that can enter depolarized neurons via calcium channels. Thus, Mn2+ is a functional contrast agent with affinity for active neurons. In this work, Mn2+ (administered with the BOMUS technique) was used to map the neuronal response to stimulation of the vibrissae. The resultant activation map showed close agreement to published maps of the posterior-lateral and anterior-medial barrel field of the primary sensory cortex.

The use of T1-shortening agents to actively stain tissues of interest--blood, brain parenchyma, or active neurons--will facilitate the use of MRM for studying mouse models of brain development, function, and disease.

Adolescence is a behaviorally well-defined developmental period during which experimentation with illicit drugs such as marijuana is common. While the lasting effects of adolescent marijuana use have been studied in humans and in animal models, relatively little is known about the acute response to marijuana in adolescents. It is known that adolescent rats are more impaired by the psychoactive ingredient in marijuana, delta-9 tetrahydrocannabinol (THC), than adults in a water maze spatial learning task. However, what causes this greater sensitivity to THC-induced learning impairment is not understood. We characterized adolescent (postnatal day 30-35) and adult (postnatal day 70-75) rat cannabinoid CB1 receptor number, distribution, and functional coupling in the hippocampus, the brain which may be the site at which THC impairs spatial learning impairment. Next, we elucidated the time course of hippocampal CB1 receptor desensitization in adolescents and adults in response to daily treatment with 10 mg/kg THC. Finally, we characterized the development of tolerance to the learning impairment caused by THC in adolescent and adult rats by pre-treating them for five days with 10 mg/kg THC, and measuring learning performance in the Morris water maze. Our results indicate that agonist stimulation of the CB1 receptor in adolescent hippocampus produces less functional coupling to G proteins than adults. Also, adolescent hippocampal CB1 receptors desensitize less rapidly in response to 10 mg/kg THC treatment than those in adults. Finally, adolescent rats do not become tolerant to the learning impairment effects of 10 mg/kg THC after five days of pre-treatment, while adults do. We conclude that adolescents may be more impaired by THC than adults as a result of more slowly desensitizing hippocampal CB1 receptors, which may be due to

lesser functional CB1-G protein coupling in adolescents.

Ube3a is a HECT domain E3 ubiquitin ligase originally recognized for its role in degrading p53 in the presence of the human papilloma virus protein E6. Loss of maternal Ube3a expression causes Angelman syndrome, a severe neurodevelopmental disorder characterized by mental retardation, ataxia, epilepsy, lack of speech, and a unique behavioral phenotype that includes a happy demeanor and frequent laughing. However, characterization of the endogenous properties and cellular role for Ube3a has been limited. Over the last few years, an interesting cohort of Ube3a interacting partners and putative substrates were named, though the consequences of these interactions were not thoroughly investigated. These include two Golgi localized proteins - PIST and Golgin-160 - as well as several proteins that can regulate trafficking of proteins at the Golgi apparatus: Src family kinases, ubiquilin, and tuberin. Therefore, we decided to focus on whether Ube3a could regulate Golgi structure or function.

In this dissertation, I will describe a new role for Ube3a at the Golgi apparatus in the regulation of intralumenal ion homeostasis. First, I characterized the expression pattern of endogenous Ube3a and overexpressed Ube3a isoforms by immunostaining and fractionation and demonstrated that although Ube3a has diffuse nuclear/cytoplasmic localization, it also associates with membrane fractions. I also confirmed that Ube3a interacts endogenously with both PIST and Golgin-160. Next, I demonstrated that Golgi morphology is perturbed in a cell line with stable knockdown of Ube3a. I found that the Golgi apparatus in Ube3a knockdown cells is under-acidifed, and that this is the primary defect underlying the disrupted Golgi morphology. Finally, I extended these findings in vivo and examined the morphology of the Golgi apparatus in the brains of Angelman syndrome model mice. The Golgi structures in the visual cortex of these mice appeared disorganized by immunohistochemistry and individual cisternae were significantly distended by electron microscopy, consistent with a defect in ion homeostasis at the Golgi apparatus. These findings define new cellular role for Ube3a at the Golgi apparatus and provide insight into the pathogenesis of Angelman syndrome.

Deep brain stimulation (DBS) electrodes are intended to stimulate specific areas of the brain to treat movement disorders including essential tremor, Parkinson's disease and dystonia. An important goal in the design of next generation DBS electrodes is to minimize the power needed to stimulate specific regions of the brain. A reduction in power consumption will prolong battery life and reduce the size of implanted pulse generator. Electrode geometry is one approach to increase the efficiency of neural stimulation and reduce the power required to produce the level of activation required for clinical efficacy.

We first characterized the impedance of the presently used clinical DBS electrodes in vitro and in vivo. Characterization of the electrode-tissue interface impedance is required to quantify the composition of charge transfer to the brain tissue. The composition of charge transfer was dependent on both the current density and the sinusoidal frequency. The assumption of the DBS electrode being ideally polarizable was not valid under clinical stimulating conditions. This implies that irreversible processes that can cause electrode or tissue damage might occur when high charge injection is required for DBS.

Current density distribution is an important factor in determining patterns of neural excitation, tissue damage and electrode corrosion. We developed a recursive simulation scheme to calculate the current density distribution that incorporates the nonlinear electrode-tissue interface into finite-element based models of electrodes. The current density distributions on the electrode surface were strongly dependent on the sinusoidal frequency. The primary current density distribution without including the electrode-tissue interface can be used to estimate neural excitation, tissue damage and electrode corrosion with rectangular stimulus pulses as most of the signal power is at frequencies where the secondary current density distribution matches closely the primary current density distribution.

We designed and analyzed novel electrode geometries to decrease stimulation thresholds, thus reducing power consumption of implanted stimulators. Our hypothesis was that high-perimeter electrode geometries that increase the variation of current density on the electrode surface will generate larger activating functions for surrounding neurons and thereby increase stimulation efficiency. We investigated three classes of electrodes: segmented cylindrical electrodes, serpentine-perimeter planar electrodes, and serpentine-perimeter cylindrical electrodes. An approach that combined finite element models of potentials and cable models of axonal excitation was used to quantify the stimulation efficiency of electrodes with various geometries. Increasing the electrode perimeter increased the electrode efficiency by decreasing stimulation threshold. Both segmentation and serpentine edges provided means to increase the efficiency of stimulation. Novel cylindrical electrodes that combined segmentation with serpentine edges decreased power consumption by ~20% for axons parallel to the electrode and by ~35% for axons perpendicular to the electrode. These electrode designs could potentially prolong the average battery life of deep brain stimulator by more than one year.

Gustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development.

As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.

Deep brain stimulation (DBS) is an established treatment for movement disorders that has been implanted in more than 40,000 patients worldwide. Despite the successes of DBS, its mechanisms of action are not well understood. Early descriptions of the mechanisms of DBS focused on whether DBS excited or inhibited neurons in the stimulated nucleus. However, changes in the patterns of neuronal activity, and not just changes in the rate of neuronal activity, play a major role in the pathology of movement disorders. Therefore, we hypothesized that the temporal pattern of stimulation might be an important factor in determining the effectiveness of DBS. The purpose of this dissertation was to use temporally irregular patterns of stimulation (non-regular interpulse intervals) to probe the mechanisms of thalamic DBS in suppressing tremor. The clinical tremor measurements reported in this dissertation represent the first tremor data published during stimulation with temporally irregular stimulus trains in human subjects. First, we tested the effects of paired-pulse DBS on tremor suppression in human subjects with essential tremor and on the responses of a computational model of thalamic neurons. DBS was more effective at reducing tremor when pulses were evenly spaced than when there were large differences between intrapair and interpair pulse intervals, suggesting that tremor suppression is dependent on the pattern of DBS and not just the average rate of stimulation. Increasing the difference between the intrapair and interpair intervals in the computational model rendered model neurons more likely to fire synchronous bursts. Second, we quantified the effects of the degree of regularity of temporally random stimulus trains in human subjects with tremor. We pioneered an innovative preparation to conduct these experiments--during surgery to replace the implantable pulse generator--which allowed us to establish a direct connection to implanted DBS leads under stable conditions. Stimulus trains were less effective at relieving tremor as the temporal spacing between stimulus pulses in DBS trains became more irregular. However, the reasons for the decreased efficacy of the temporally irregular stimulus trains was not clear. Third, we evaluated the contributions of `pauses,' `bursts,' and `irregularity, per se' to the inability of irregular stimulus trains to suppress tremor. Stimulus trains with pauses were significantly less effective at suppressing tremor than stimulus trains without pauses, while there were no significant changes in tremor suppression between trains with bursts and those without bursts, or between trains that were irregular and those that were periodic. We also developed a computer-based biophysical model of a thalamic network to simulate the response of thalamic neurons to the same temporal patterns of DBS. Trains that effectively suppressed tremor in human subjects also suppressed fluctuations in transmembrane potential at the frequency associated with burst-driven cerebellar inputs to the thalamus. Both clinical and computational findings indicate that DBS suppresses tremor by masking cerebellar burst-driven input to the thalamus.

The work in this dissertation bridges an important gap between the hypothesis that high-frequency DBS masks pathological activity in the cerebello-thalamo-cortical circuit and the experimentally observed finding that DBS in the subthalamic area suppresses tremor more effectively than DBS in the Vim thalamus proper. We provided experimental and computational evidence that the mechanism of DBS is to mask the burst-driven cerebellar inputs to the thalamus. Hence, the most relevant neuronal targets for effective tremor suppression are the afferent cerebellar fibers that terminate in the thalamus.

Pain is defined by the international association of pain as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Most people have experienced one form of pain or another and although such experiences can be unsavory, pain serves the basic need for the detection of dangerous stimuli that can cause bodily harm. Because pain serves such an essential need, it is important to understand how the nervous system processes and encodes noxious or potentially tissue damaging stimuli. This neural processing is called nociception.

In this study, I use Drosophila larvae as a genetic model organism to study nociception. In response to noxious thermal and mechanical stimuli, Drosophila larvae perform a nociceptive defensive behavior (termed nocifensive) where larvae rotate in a corkscrew like fashion along the long axis causing them to move in a lateral direction. Using this behavior and genetic tools which can manipulate neuronal output, we have identified the sensory neurons which serve as larval nociceptors as class IV multidendritic sensory neurons. Further characterization of these larval nociceptors, has also shown that they are both cholinergic and peptidergic.

After the identifying the larval nociceptors, I next identified several molecular components which are required for larval mechanical nociception. I have found that the degenerin epithelial sodium channel (DEG/ENaC) called pickpocket is required for larval mechanical nociception by using genetic mutants and RNAi knockdwon. In addition, after performing a screen using RNAi to knockdown ion channel transcripts in larval nociceptors, I have identified two other DEG/ENaC channels which are required for larval mechanical nociception. DEG/ENaCs are particularly interesting because they have been identified as candidate mechanotransducers in C. elegans for the gentle touch behavior. I propose that DEG/ENaCs may serve as candidate mechanotransducers in larval mechanical nociception because they are not generally required for neuronal excitability. However, future research will be required to establish their true role in mechanical nociceptive signaling.

In addition to DEG/ENaCs, transient receptor potential (TRP) channels also play a role in nociception. painless, a channel that was first identified in a thermal nociception screen on Drosophila larvae, is required for both thermal and mechanical nociception. The last section shows that multiple isoforms of painless exist and that these different isoforms may play different roles in thermal and mechanical nociception.

Taken together, these results have begun to establish Drosophila larva as a model for studying nociception. I have identified the sensory neurons used as larval nociceptors and shown that DEG/ENaC channels play an important role in larval mechanical nociception.

The proper modification and trafficking of plasma membrane proteins are essential for normal neuronal function, such as dendrite morphogenesis, spine formation and synaptic plasticity. The secretory organelles including endoplasmic reticulum and Golgi apparatus are critical for the trafficking of these molecules as shown in fibroblasts. Although these secretory organelles have been observed in neurons including dendritic branches, their spatial organization and function in protein trafficking, neuronal development and plasticity are not clear yet. Here, I used photobleaching and photoactivation approaches combined with electron microscopy to show that although rapidly diffusing within the continuous network of the somato-dendritic ER, membrane proteins such as nascent AMPA receptors are confined by ER spatial complexity. The spatial range of ER membrane protein mobility becomes progressively confined over neuronal development and is rapidly restricted by synaptic activity. Thus, constrained lateral mobility within the ER provides a novel mechanism for compartmentalized trafficking of nascent receptors throughout dendrites. I also identified an ER protein as a novel microtubule-associated protein regulating dendritic ER spatial complexity, neuronal dendrite elongation and spine formation. Together, these results describe the spatial organization of dendritic ER and its role in regulating membrane protein trafficking, neuronal morphogenesis and postsynaptic functions.

The visual system is thought to represent the trajectory of moving objects in the activity of large populations of cortical neurons that respond preferentially to the direction of stimulus motion. Here I employed in vivo voltage sensitive dye (VSD) imaging to explore how abrupt changes in the trajectory of a moving stimulus impact the population coding of motion direction in ferret primary visual cortex (V1). For motion in a constant direction, the peak of the cortical population response reliably signaled the stimulus trajectory; but for abrupt changes in motion direction, the peak of the population response departed significantly from the stimulus trajectory in a fashion that depended on the size of the direction deviation. For small direction deviation angles, the peak of the active population shifted from values consistent with the initial direction of motion to those consistent with the final direction of motion by progressing smoothly through intermediate directions not present in the stimulus. In contrast, for large direction deviation angles, peak values consistent with the initial motion direction were followed by: a small deviation away from the final motion direction, a rapid 180° jump, and a gradual shift to the final direction. These departures of the population response from the actual trajectory of the stimulus predict specific misperceptions of motion direction that were confirmed by human psychophysical experiments. I conclude that cortical dynamics and population coding mechanisms combine to place constraints on the accuracy with which abrupt changes in direction of motion can be represented by cortical circuits.

The specification of a single axon and multiple dendrites is the first observable event during neuronal morphogenesis and such structural specialization underlies neural connectivity and nervous system function. Numerous intracellular signaling components that are required for axon specification have been described but how such signaling paradigms are initiated by extracellular factor(s) within the embryonic milieu is poorly understood. Here, I describe how transforming growth factor-β (TGF-β), an embryonic morphogen that directs structural plasticity and growth in various cell types, initiates signaling pathways both in vivo and in vitro to fate naïve neurites into axons. Using conditional knockout strategies, I found that cortical neurons lacking the type II TGF-β receptor (TβR2) fail to initiate axons during development, and interestingly, fail to engage radial migration. In cultured neurons, exogenous TGF-β is sufficient to direct the rapid growth and differentiation of an axon and genetic enhancement of receptor activity promotes the formation of multiple axons. The cellular polarization of receptor activity occurs through the interaction of the type-I TGF-β receptor with Par6, a component of the axon-specifying Par3/Par6 polarity complex. Receptor distribution is restricted to axons, and downstream signaling events required for axon specification are triggered when Par6 is phosphorylated by TβR2. Together, these results indicate that TGF-β is the extrinsic cue for neuronal polarity in vivo and directs neuronal polarity by controlling Par6 activity and cellular migration during axon generation.

At excitatory synapses, NMDAR-mediated synaptic plasticity occurs in response to activity inputs by modifying synaptic strength. While comprehensive studies have been focused on the induction and expression mechanisms underlying synaptic plasticity, it is less clear whether and how synaptic plasticity itself can be subjected to regulations. The presence of "plasticity of plasticity", or meta-plasticity, has been proposed as an essential mechanism to ensure a proper working range of plasticity, which may also offer an additional layer of information storage capacity. However, it remains elusive whether and how meta-plasticity occurs at single synapses and what molecular substrates are locally utilized. Here, I develop systems allowing for sustained alterations of individual synaptic inputs. By implementing a history of inactivity at single synapses, I demonstrate that individual synaptic inputs control synaptic molecular composition homosynaptically, while allowing heterosynaptic integration along dendrites. Furthermore, I report that subunit-specific regulation of NMDARs at single synapses mediates a novel form of input-specific metaplasticity. Prolonged suppression of synaptic releases at single synapses enhances synaptic NMDAR-mediated currents and increases the number of functional NMDARs containing NR2B. Interestingly, synaptic NMDAR composition is adjusted by spontaneous glutamate release rather than evoked activity. I also demonstrate that inactivated synapses with more NMDARs containing NR2B acquire a lower induction threshold for long-term synaptic potentiation. Together, these results suggest that at single synapses, spontaneous release primes the synapse by modifying its synaptic state with specific molecular compositions, which in turn determine the synaptic gain in an input-specific manner.

The chemical cues that provide an olfactory portrait of mammalian individuals are in part detected by chemosensory receptors in the vomeronasal organ (VNO). By and large, the pertinent receptor-cue combinations used for olfactory communication are unidentified. Here we identify members of the formyl peptide receptor (FPR) family of G protein coupled receptors as candidate chemosensory receptors in the VNO of mice. We demonstrate that N-formylated mitochondrially encoded peptides presented by the major histocompatibility complex (MHC) molecule H2-M3 stimulate a subset of the VNO sensory neurons (VSNs). We show that one VNO localized FPR, Fpr-rs1, is differentially activated by strain specific variants of N-formylated peptides. We show that N-formylated peptides can function as chemosignals in a strain selective pregnancy block. We propose that this link between self-recognition peptides of the immune system and chemosensory pathways provides a possible molecular means to communicate the nature of an individual's maternal lineage or strain.

Altered dietary availability of the vital nutrient choline during early development leads to persistent changes in brain and behavior throughout adulthood. Prenatal choline supplementation during embryonic days (ED) 12-17 of the rodent gestation period enhances memory capacity and precision and hippocampal plasticity in adulthood, and protects against spatial learning and memory deficits shortly after excitotoxic seizures, whereas prenatal choline deficiency can compromise hippocampal memory and plasticity in adulthood. Recent evidence from our laboratory has determined that lifelong proliferation of newborn neurons in the adult hippocampus, a feature of adult hippocampal plasticity that has been implicated in some aspects of learning and memory, is modulated by early choline availability. Prenatal choline's effects on adult neurogenesis may be one mechanism for diet-induced cognitive changes throughout life and in response to injury, although little is known about the mechanisms underlying how prenatal choline alters adult neurogenesis or the neural mechanisms underlying prenatal choline supplementation's protection against cognitive deficits after seizures. To address these issues, the present set of experiments investigated how prenatal choline availability modulates specific properties of neurogenesis in the adult brain (in the intact brain and in response to injury), as well as hippocampal markers known to change in response to excitotoxin-induced seizures, and sought to relate changes in neurogenesis and in neuropathological markers following injury to changes in performance on spatial learning and memory tasks. Subjects in each experiment were adult offspring from rat dams that received either a control diet or diet supplemented with choline chloride or deficient of choline on ED 12-17. To measure neurogenesis, rats were given injections of the mitotic marker bromodeoxyurdine to label dividing cells in the hippocampus. Prenatal choline supplementation enhanced several properties of basal adult hippocampal neurogenesis (cell division and survival, neural stem/progenitor cell phenotype and proliferative capacity, trophic support), and this increase was associated with improvements in spatial working memory retention in a delayed-matching-to-place water maze task. In contrast, prenatal choline deficiency had little effect on basal adult hippocampal neurogenesis, and no effect on spatial memory performance. Prenatal choline supplementation also enhanced olfactory bulb neurogenesis without altering cell proliferation in the subventricular zone, while prenatal choline deficiency had no effect on either measure, showing for the first time that prenatal choline's effects on adult neurogenesis is similarly expressed in another distinct neurogenic region of the adult brain. Altered prenatal choline availability also modulated the hippocampal response to kainic acid-induced seizures where supplementation attenuated while deficiency had no effect on the injury-induced proliferative response of the dentate gyrus shortly after injury. Prenatal choline supplementation also attenuated other markers of hippocampal neuropathology shortly after seizures and promoted the long-term hippocampal recovery from seizures months after injury, including rescuing declines in adult hippocampal neurogenesis and in spatial memory performance in a standard water maze task. Taken together, these findings demonstrate a robust neuroprotective effect of prenatal choline supplementation that may be driven by enhanced adult hippocampal plasticity and trophic support prior to injury, and shed light on the mechanisms underlying how prenatal choline availability alters adult hippocampal neurogenesis, which may contribute to changes in memory capacity and precision both throughout life and following neural assault.

Pheromones are chemicals from conspecifics that affect innate behavior or hormonal changes. In mammals, the vomeronasal organ (VNO) is thought to play a prominent role in detecting pheromones; the vomeronasal sensory neurons (VSNs) express three families of seven-transmembrane G-protein coupled receptors (GPCRs): the V1Rs, V2Rs, and FPRs, in two molecularly and spatially-distinct regions. In mice, VSNs that express the V2Rs are thought to detect peptide cues, including MHC-presenting peptides, major urinary proteins (MUPs), and exocrine gland-secreting peptides (ESPs). They are thought to be involved in various pheromone-mediated behaviors and physiological changes, such as mating, aggression, and selective pregnancy block. In order to understand how pheromones are detected by the vomeronasal receptors, it is essential to know which receptors are activated by a given chemical. However, identifying cognate ligands for the V2Rs has been challenging, partly because they are poorly localized to the surface of heterologous cells. Here, we show that the calreticulin chaperone family members play a crucial role in trafficking V2Rs. A calreticulin homologue, calreticulin4 is specifically expressed in the VNO, while calreticulin expression level is low. Depleting calreticulin expression in HEK293T cells allows V2Rs to be trafficked to the cell surface, whereas expression of calreticulin4 does not block the trafficking of the V2Rs. Using this knowledge, we have established a heterologous cell system to functionally identify the V2Rs and demonstrate that the ESP family members can differentially activate the V2Rs. We also show the large extracellular domain of the V2Rs plays a crucial role in ligand selectivity. Our results provide a platform to characterize ligand selectivity of the V2Rs and suggest that a unique mechanism involving calreticulins regulates the functional expression of the V2Rs.

To solicit the attention or determine the intentions of another, we use our eyes. While many animals appear to use eyes as an important behavioral cue, for humans, these cues are especially critical. The power of the eyes to attract and direct attention shapes human behavior from an early age and likely serves as a foundation for social skill acquisition, ranging from simple, friendly eye contact to complex, spoken language, even to our almost mystical ability to empathize and "see the world through another's eyes". Humans have transformed our environment through our economic alliances and military competitions, and our individual successes and failures depend critically on social skills built on a foundation of shared attention. When these abilities break down, as in autism, pervasive social awkwardness can challenge the close relationship of individuals with their friends, family, and community. Nonetheless, we know almost nothing about the brain mechanisms that have evolved to process social cues and convert them into a rich experience of shared attention. To investigate this process, we explored the ability of human and nonhuman primates to follow the attention of other individuals. First, we characterized natural gaze-following behavior using a novel telemetric device in socially-interacting prosimian primates, and later in monkeys and humans responding to gaze cues in the lab. Finally, we examined the neuronal responses to gaze cues in a macaque posterior parietal area implicated in attention control--the lateral intraparietal area, LIP. Our findings suggest that gaze-following abilities may be widespread in social primates, relying on conserved, homologous brain pathways; and that they may not be informationally-encapsulated reflexes, but rather are densely interwoven with diverse social processes. Indeed, we found gaze cues influenced neurons in LIP, part of the dorsal frontoparietal attention network. Finally, we report that "mirror" neurons in parietal areas may thus play a role not only in representing perceived bodily actions, but also perceived mental states such as observed attention.

Perception arises from sensory inputs detected by peripheral organs and processed in the brain by complex neuronal circuits required for the integration of external information with internal states such as expectation and attention. Stimulus discrimination requires activation of primary sensory areas in the brain, but expectation is traditionally associated with the activation of higher-order brain areas. Sensory information obtained by tactile organs is represented along the primary areas that comprise the trigeminal thalamocortical pathway. In anesthetized animals, neuronal activity in the somatosensory system has been extensively described over the past century. However, it is still unclear how the different thalamocortical structures contribute to active tactile discrimination and represent relevant features of the stimulus. It is also unknown whether expectation modulates tactile representations in these regions. In this dissertation, I investigated neuronal ensemble activity recorded from freely behaving rats performing a whisker-based tactile discrimination t-+ask. Multielectrode arrays were chronically implanted to record simultaneously from the main stages of the trigeminal thalamocortical pathways involved in whisking: the primary somatosensory cortex (S1), the ventral posterior medial nucleus of the thalamus (VPM), the posterior medial complex (POm) and the zona incerta (ZI). In Chapter 1 I describe the behavior of rats performing the tactile discrimination task, which requires animals to associate two different tactile stimuli with two corresponding choices of spatial trajectory in order for reward to be delivered. I found that both cortical and thalamic neurons are dynamically engaged during execution of the task. The data reveal a very complex mosaic of responses comprising single or multiple periods of inhibition and excitation. Thalamocortical activity was modulated during whisker stimulation as well as after stimulus removal, up until reward delivery. To investigate whether reward expectation plays a role in tactile processing at early processing stages, I also recorded neuronal activity from rats performing a freely-rewarded version of the tactile discrimination task. Comparing data from regularly-rewarded and freely-rewarded sessions, I show in chapter 2 that the activity of single neurons in the primary somatosensory thalamocortical loop is strongly modulated by reward expectation. Stimulus-related information coded by primary thalamocortical neurons is high when a correct association between stimulus and response is crucial for reward, but decreases significantly when the association is irrelevant. These results indicate that tactile processing in primary somatosensory areas of the thalamus and cerebral cortex is directly affected by reward expectation.

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.