In order to guide behavior, humans and animals must flexibly evaluate the motivational significance of stimuli in the environment. We sought to determine if, in different contexts, neurons in the amygdala and orbitofrontal cortex (OFC) indeed rescale their calculation of the motivational significance of stimuli that predict rewards. We used a "contrast revaluation" task in which the reward associated with one stimulus is held constant while other rewards within a particular context (or block of trials) change. This manipulation modulates the relative significance of the reward associated with one stimulus without changing its absolute amount. We recorded the activity of individual neurons in the amygdala and OFC of two monkeys while they performed the contrast revaluation task. On every trial, a monkey viewed one of two conditioned stimuli (CSs; distinct fractal patterns), each predictive of a different reward amount. CSs were novel for every experiment. Unconditioned stimulus (US, liquid reward) delivery followed CS presentation and a brief temporal gap (trace interval). The task consisted of three trial blocks, with switches between blocks occurring without warning. The presentation of CS2 predicted either a small (first and third blocks) or large US (second block). The presentation of CS1 predicted delivery of a medium US in all blocks. Thus CS1 corresponded to the "better" trial type in blocks 1 and 3, but not 2. Anticipatory licking behavior indicated that the monkey adapted its behavior depending upon the relative amount of expected reward. Although the reward amount associated with CS1 remained constant throughout the experiment, anticipatory licking decreased in block 2 and increased in block 3 - the blocks in which CS1 trials had become relatively less (block 2) and more (block 3) valuable. Strikingly, many individual amygdala and OFC neurons also modulated their responses to CS1 depending upon the block. Because this CS predicts the exact same reward in each block, these neurons cannot simply represent the sensory properties of a US associated with a CS. This finding demonstrates that amygdala and OFC neurons are often sensitive to the relative motivational significance of a CS, and not just to the sensory properties of its associated US or to the absolute value of the specific reward. Neurons in both the OFC and amygdala encode the relative value of CS1 but OFC neurons significantly encode relative value earlier than amygdala neurons. Cells in the amygdala and OFC code different properties during different time intervals during the trial and are consistent in valence when they code multiple properties. This implies that neurons are tracking state value: the overall motivational value of an organism's internal and external environment across time and sensory stimuli. Neurons that code relative value during the CS-trace interval and during reinforcement are also consistent in the valence that they code further supporting that these cells track state value. The neurons code with the same sign and strength whether the neuron is representing the relative value of the reward with no sensory input of the reward during CS or trace interval, or actually experiencing the reward during the US interval. Further, amygdala and OFC neural activity was correlated with the animal's behavioral performance, suggesting that these neurons could form the basis for animal's behavioral adaptation during contrast revaluation. These neural representations could also support behavior in other situations requiring flexible and adaptive evaluation of the motivational significance of stimuli.

Schizophrenia is highly heritable yet there are few confirmed, causal mutations. In human genetic studies, we discovered CNVs impacting RAPGEF6 and RAPGEF2. Behavioral analysis of a mouse modeling Rapgef6 deletion determined that amygdala function was the most impaired behavioral domain as measured by reduced fear conditioning and anxiolysis. More disseminated behavioral functions such as startle and prepulse inhibition were also reduced, while locomotion was increased. Hippocampal-dependent spatial memory was intact, as was prefrontal cortex function on a working memory task. Neural activation as measured by cFOS levels demonstrated a reduction in hippocampal and amygdala activation after fear conditioning. In vivo neural morphology assessment found CA3 spine density and primary dendrite number were reduced in knock out animals but additional hippocampal measurements were unaffected. Furthermore, amygdala spine density and prefrontal cortex dendrites were not changed. Considering all levels of analysis, the Rapgef6 mouse was most impaired in hippocampal and amygdala function, brain regions implicated in schizophrenia pathophysiology at a variety of levels. The exact cause of Rapgef6 pathology has not yet been determined, but the dysfunction appears to be due to subtle spine density changes as well as synaptic hypoactivity. Continued investigation may yield a deeper understanding of amygdala and hippocampal pathophysiology, particularly contributing to negative symptoms, as well as novel therapeutic targets in schizophrenia.

Schizophrenia is a debilitating, life-long illness with a still-unknown, complex etiology and, currently, no cure. Many studies have implicated the hippocampus and the parahippocampal region as a place of both primary pathology in the disease and as regions correlated to symptom severity. To better understand the pathophysiology of the region and potentially uncover mechanisms of the disease, the appropriate choice of an animal model is essential. The "MAM E17" model of hippocampal pathology shows anatomical, neurophysiological, and behavioral changes relevant to schizophrenia. Because of these wide-ranging disease-relevant changes, we aimed to relate anatomical to neurophysiological phenotypes in this model. We also performed experiments to assess the feasibility and validity of transferring the MAM E17 model to the mouse in order enable future studies of the genetic basis of the vulnerability or resilience to MAM. In adult offspring of rats exposed to to methylazoxymethanol (MAM) at embryonic day 17 (E17), we found changes in regional hippocampal anatomy and subicular pyramidal cell morphology with homology to abnormalities reported in schizophrenia. Specifically, we found a decrease in dendritic spine density in specific regions of the dendrite of ventral subicular neurons. At the neurophysiological level, we observed abnormalities in afferent-evoked synaptic responses in the ventral subiculum. These changes were not however, accompanied by changes in in vivo spontaneous spike activity in subicular neurons . In the mouse, MAM was found to have much less impact on brain development, as observed at the gross morphological level. However, these mice showed an increased sensitivity to some psychostimulants and a weak trend for metabolic abnormalities relevant to schizophrenia. We conclude from the rat studies that prenatal disruption of brain development by MAM at E17 in the rat, a manipulation that leads to a profile of gross anatomical and cognitive deficits relevant to schizophrenia, also leads to "dysconnectivity" between the ventral subiculum and its inputs. While further work is needed to understand this, we speculate that this synaptic dysconnectivity may contribute to the cognitive deficits in this model and, further, may model an aspect of hippocampal pathophysiology in schizophrenia. A better understanding of these circuits could point to new strategies for treating this disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized clinically by progressive dementia and histopathologically by amyloid plaques and neurofibrillary tangles. The primary molecular culprit in AD is the amyloid-beta (Abeta) peptide, aggregates of which are the main components of the plaques. Numerous studies have implicated soluble Abeta oligomers as the predominant neurotoxic species, although the underlying mechanisms that lead to cognitive failure are not fully understood. In this thesis, I demonstrate that post-translational modification with the small ubiquitin-like modifier (SUMO) is required for normal synaptic and cognitive function but can be impaired by Abeta oligomers. I discovered that SUMOylation was significantly reduced in brain tissue from AD patients and a transgenic mouse model of AD. While neuronal activation normally induced upregulation of SUMOylation, this effect was impaired by Abeta and in the transgenic mice. Abeta is also a known potent disruptor of synaptic function. However, enhancing SUMOylation via transduction of its conjugating enzyme, Ubc9, rescued Abeta-induced deficits in synaptic plasticity and memory. I further demonstrate that inhibition of SUMOylation can directly cause such deficits, similar to Abeta. Overall, the data establish SUMO as a novel regulator of synaptic plasticity and cognition and point to SUMOylation impairments as an underlying factor in AD pathology. In addition to the pathological effects of Abeta, the normal physiological functions of this peptide, which is produced in the brain throughout life, remain unclear. A previous study in our lab demonstrated that physiologically-relevant (low picomolar) amounts of Abeta can enhance synaptic plasticity and memory. Astrocytes, as crucial glial support cells with roles in modulating synaptic transmission, are likely cellular candidates for participating in this type of physiological Abeta signaling. To test this hypothesis, primary cultures of murine astrocytes were exposed to exogenous picomolar Abeta peptides while undergoing calcium imaging. Upon addition of 200 pM Abeta peptides, the percentage of astrocytes exhibiting spontaneous oscillatory calcium transients increased significantly. The periodicities of these transients were analyzed, and it was found that both the frequency and amplitude of the transients were enhanced after Abeta exposure. These effects were dependent on calcium influx and alpha7 nicotinic acetylcholine receptors (alpha7-nAChRs), as the potentiation was blocked by a pharmacological alpha7 inhibitor and in cultures from an alpha7 knockout mouse strain. In addition to spontaneous signaling, evoked intercellular calcium waves were also analyzed. After picomolar Abeta exposure, no significant changes were found in several wave parameters, including spatial and temporal spread, propagation speed and maximum signal intensity. These results indicate that at physiologically-relevant concentrations, Abeta peptides enhance spontaneous astrocyte calcium signaling via astrocytic alpha7-nAChRs. Since astrocyte-mediated "gliotransmission" has been found to have multiple neuromodulatory roles, Abeta peptides may have a normal physiological function in regulating this type of neuron-glia signaling. These studies illustrate the diverse effects of Abeta peptides, which are dependent on the concentration and conformation state. Ultimately, knowledge of both normal Abeta physiology as well as Abeta pathology are necessary to truly understand Alzheimer's disease and enable development of effective therapeutics.

Albinism is a heterogeneous disorder that occurs when one of several genetic defects causes a disruption in melanin synthesis in the hair, skin, and eyes. Every form of albinism results in hypopigmentation in the retinal pigment epithelium (RPE), the monolayer of epithelial cells surrounding the neural retina. All albino mammals that have been studied display a variety of optic abnormalities, one of which is a reduction in the ipsilateral retinal ganglion cell (RGC) axon projection. This thesis addresses the question of how the disruption of melanin synthesis in the RPE leads to abnormal chiasmatic decussation by using a mouse model that has a mutation in the gene encoding tyrosinase, an enzyme required for melanin synthesis. Previously, our lab has shown that Zic2, a zinc finger transcription factor that is expressed in ventrotemporal RGCs from E14.5-E17.5, directs the ipsilateral projection. Zic2 regulates several programs of gene expression important for axon guidance and synaptic connectivity. Zic2 is expressed in fewer RGCs in the albino retina, coincident with the decrease in the ipsilateral RGCs and indicating that cell subtype specification is altered in the VT retina of albino mice. This thesis further characterizes perturbations in RGC genesis and specification in the albino mouse. Analysis of retinogeniculate targeting in the albino mouse revealed a population of contralateral VT RGCS that forms an abnormal patch of terminals in the dorsal lateral geniculate nucleus (dLGN) of the mouse, suggestive of misspecification of RGCs in this region. Further, as revealed by expression of Islet1/2 as a marker of differentiated RGCs, the number of differentiated RGCs in the VT retina is lower in the albino compared to pigmented retina, parallel to the reduction of Zic2+ cells. The decrease in Zic2+ and Islet1/2+ cells was explained by birthdating studies, which demonstrated a delay in the wave of RGC production in the albino VT retina at the time when the ipsilateral projection is established. Thus, this thesis provides a link between neurogenesis and specification in the albino retina. To further elucidate the role of melanin synthesis in VT RGC specification, I tested the effects of L-Dopa treatment of embryonic mice on retinal development and found that L-Dopa ameliorated the defects associated with the reduced ipsilateral projection in the albino mouse by regulating cell proliferation and production. The studies in this thesis contribute to an understanding of the mechanism that underlies the disruption of binocular pathways in the albino visual system, and should illuminate how the pigment pathway in the RPE contributes to development of the neural retina in wild type mice.

Mutations in superoxide dismutase-1 (SOD1) cause a familial form of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder. Transgenic mutant SOD1 rodents capture the hallmarks of this disease, which is characterized by a progressive loss of motor neurons. Studies in chimeric and conditional transgenic mutant SOD1 mice indicate that non-neuronal cells, such as astrocytes, play an important role in motor neuron degeneration. Consistent with this non-cell autonomous scenario are the demonstrations that wild-type primary and embryonic stem cell-derived motor neurons selectively degenerate when cultured in the presence of either mutant SOD1-expressing astrocytes or medium conditioned with such mutant astrocytes. The work in this thesis rests on the use of an unbiased genomic strategy that combines RNA-Seq and "reverse gene engineering" algorithms in an attempt to decipher the molecular underpinnings of motor neuron degeneration caused by mutant astrocytes. To allow such analyses, first, mutant SOD1-induced toxicity on purified embryonic stem cell-derived motor neurons was validated and characterized. This was followed by the validation of signaling pathways identified by bioinformatics in purified embryonic stem cell-derived motor neurons, using both pharmacological and genetic techniques, leading to the discovery that nuclear factor kappa B (NF-κB) is instrumental in the demise of motor neurons exposed to mutant astrocytes in vitro. These findings demonstrate the usefulness of this novel genomic approach to study neurodegeneration and to point to NF-κB as a potential valuable therapeutic target for ALS.

Variation in adult rat maternal behavior is predicted by the experience of maternal licking and grooming (LG) in infancy, such that females that experience high levels of LG (High LG) during postnatal development typically themselves become High LG dams, and females that experience low levels of LG become Low LG dams. Experience of high maternal LG also predicts elevated estrogen receptor-alpha (ERalpha) and oxytocin receptor levels in brain regions critical for maternal behavior such as the medial preoptic area of the hypothalamus. The first series of experiments within this thesis demonstrates that these neuroendocrine differences in ERalpha-immunoreactivity and mRNA emerge in offspring during the postnatal period (postnatal days 0-21). These studies also show postnatal emergence of epigenetic alterations of the ERalpha gene (Esr1), including DNA methylation and chromatin remodeling, in response to maternal care. Furthermore, this research reveals sensitive periods during postnatal development for maternal LG to affect gene expression and onset of maternal behavior in juvenile offspring. The mesolimbic dopamine system is also critically implicated in adult maternal behaviors, and was hypothesized to be responsive to variation in maternal LG. A second series of studies demonstrate that low or high levels of maternal LG predict levels of dopamine neurons in the ventral tegmental area, an effect that emerges during the postnatal period and lasts through adulthood. These neurobiological changes within the ventral tegmental area may be shaped by maternal LG-associated effects on postnatal levels of transcription factors that contribute to development of the mesolimbic dopamine system. Reward-directed behaviors known to be dependent upon mesolimbic dopamine function were also found to be different among offspring of High or Low LG dams. Finally, a third series of experiments reveal that over-expressing ERalpha in the medial preoptic area beginning early in postnatal development is sufficient to enhance maternal behaviors, and increase the level of dopaminergic cells in the ventral tegmental area in offspring reared by Low LG dams to the level of those reared by High LG dams. This finding suggests that ERalpha is a mediating factor for the effect of maternal LG on offspring maternal behavior. Together, these studies show that the quality of the maternal environment early in life programs long-lasting alterations in two brain systems critical for complex behaviors such as maternal and reward-directed behaviors.

Normal motor signaling in the basal ganglia requires regulating which movements to suppress and which to enact. In Parkinson's disease, loss of dopamine levels due to loss of dopaminergic neurons results in unbalanced basal ganglia output and loss of motor control. Motor sequences are thought to be triggered by cortical inputs as these glutamatergic inputs provide the main excitatory drive to the striatal output neurons. Dopamine is a crucial modulator of corticostriatal activity and loss of its normal function plays an important role in the pathophysiology of Parkinson's disease. We hypothesize that the functional reorganization of the cortical inputs to the striatum following long-term dopamine depletion as well as the response to dopamine replacement therapies has important functional implications in the pathogenesis and treatment of Parkinson's disease motor symptoms. To address this hypothesis, we adapted an optical technique using lipophilic dye, FM 1-43, to characterize the activities of the two major classes of corticostriatal projection neurons - the ipsilateral and contralateral cortical projections - and compared the influence of dopamine D2 receptors on these inputs. We found that both cortical projections shared similar patterns of terminal release and were both inhibited by D2 receptor activation. A D2 receptor-mediated inhibition specifically targeted the least active (slow-releasing) corticostriatal inputs with low probability of release. This "filtering" effect by D2 receptors confirmed a role for dopamine in modulating excitatory cortical inputs that could be crucial to selection of proper motor functions. To study the loss of motor control during conditions of chronic dopamine depletion, we employed a classic Parkinson's disease rodent model in which dopamine is depleted from one hemisphere using the neurotoxin, 6-hydroxydopamine. Behavior tests confirmed lateralized motor response due to loss of function in the forelimb contralateral to the side of lesion. The effect of chronic dopamine depletion on corticostriatal synaptic activity was assessed by comparing the activity between the dopamine-intact and dopamine-lesioned hemispheres. We proposed that in the dopamine-intact hemisphere, D2 receptor activation exerted selective inhibitory influence or "filtering" on corticostriatal signaling through two mechanisms: presynaptic D2 receptors directly inhibiting glutamate release and postsynaptic D2 receptor-mediated retrograde endocannabinoid inhibition activating presynaptic CB1 receptors. However, in the dopamine-lesioned hemisphere, there was a supersensitive inhibition by D2 receptor activation and the "filtering" effect was lost: the "filtering" was partially restored by concurrently activating D2 receptors and inhibiting CB1 receptors. We then tested whether this endocannabinoid-mediated restoration of D2 receptor "filtering" in corticostriatal inputs had an effect on motor function in vivo. We examined changes in motor function and corticostriatal activity in 6-OHDA lesioned mice following DA replacement therapy with L-DOPA in combination with modulators of endocannabinoid transmission. We found that treatment with L-DOPA alone or with L-DOPA + URB597 (an inhibitor of endocannabinoid breakdown) reduced contralateral akinesia and in fact led to a contralateral limb use preference. Following L-DOPA treatment, corticostriatal presynaptic activity was depressed in the lesioned striata and D2 receptor-mediated inhibition was occluded. Treatment of L-DOPA with the CB1 receptor antagonist, AM251, completely normalized motor function. This treatment regime also completely normalized basal corticostriatal activity on the lesioned hemisphere, and the D2 receptor "filtering" effect was restored. Our findings confirm that dopamine modulates excitatory corticostriatal activity presynaptically via D2 receptor activation, a portion of which is due to cannabinoid effects. Furthermore, a correlation between dopamine-induced loss of motor function and loss of corticostriatal "filtering" by D2 receptors was demonstrated by the fact that dopamine replacement treatment that restores behavior also preserves this "filtering" mechanism in corticostriatal inputs. These findings suggest that dopaminergic "filtering" of particular corticostriatal synaptic activity contributes to motor commands.

The development of the mammalian neocortex requires the careful balancing of proliferation, migration, and differentiation. The cellular machinery coordinating these events includes molecular motor proteins such as dynein. Regulation of dynein activity is particularly important, since it is the major microtubule minus-end directed motor in cells. Dynein is a large, complex structure comprising several subunits and binding partners. Its function is critical for multiple stages of brain development. The dynein regulatory proteins NudE and NudEL have been implicated in several aspects of dynein function, including brain development. Originally identified as nuclear distribution (nud) factors in the dynein pathway, NudE and NudEL are now known to have diverse roles in mitosis, cell migration, and intracellular trafficking. Mice null for Nde1, the gene encoding NudE, have microcephaly, whereas mice null for Ndel1, which encodes NudEL, are embryonic lethal. Additionally, Nde1 mutations have recently been shown to result in microcephaly and lissencephaly in human patients. NudE and NudEL are functionally related paralogs that are more than 70% similar. Both bind to dynein and LIS1, another dynein regulatory protein involved in brain development. In addition to serving as recruitment factors, NudE and NudEL impact dynein force production and allow dynein to serve as a persistent motor under high load. This would be particularly important during the proliferation of neural progenitors, which undergo cell cycle-linked nuclear oscillations. These oscillations, termed interkinetic nuclear migration (INM), require forces acting upon the nucleus to drive upward (basal) and downward (apical) movement in the proliferative ventricular zone (VZ) of the brain. Research from our lab has identified dynein, along with LIS1, as being responsible for apical movement, and the unconventional kinesin Kif1a as the driving force behind basal movement. The aim of this thesis has been to understand the mechanisms by which NudE and NudEL regulate dynein function in brain development. We identify a role for NudE, but not NudEL, in INM and radial progenitor mitosis. Additionally, we find that both NudE and NudEL are involved in the multipolar-to-bipolar transition of neurons, and that NudEL has a role in bipolar neuronal migration. Our results provide an additional molecular explanation for microcephaly resulting from Nde1 mutations, implicating a block in INM as a cause for reduced proliferation, since cells are unable to reach the ventricular surface where they normally undergo mitosis. NudEL has previously been implicated in having a role in neurite extension and axon elongation. We found that NudE/EL localized to a single neurite of a Stage 2 hippocampal neuron as well as the axon tip of a Stage 3 neuron. In addition, the Stage 2 localization was coincident with the appearance of established early markers of neuronal polarity. We studied the role of NudE/EL in establishing neuronal polarity and found that Nde1 and Ndel1 RNAi inhibited axon formation. Overexpression of NudEL did not result in noticeable changes in axon formation. We conclude that in addition to the role of NudEL in axon extension and outgrowth, NudE/EL serve as early markers of neuronal polarity and are required, though not necessarily sufficient, for axon specification.

Genetic variation in the dystrobrevin binding protein 1 (DTNBP1) gene has been linked to cognition and neurological diseases such as schizophrenia and bipolar disorder. Unfortunately, the neuronal function of the encoded product, dysbindin, is poorly understood. Many reports have claimed that dysbindin deficiency leads to defects in synaptic transmission. However, the specific impairments reported have been variable and inconsistent even at the same synapse. I conducted an independent functional characterization of hippocampal synapses in sandy mice, which contain a spontaneous deletion in the DTNBP 1 gene resulting in the loss of expression of dysbindin, the protein that it encodes. I had observed enhanced excitatory basal synaptic transmission at the CA3-CA1 connection of juvenile mice, which has not been reported in the literature. To understand this novel phenomenon in better detail, a series of experiments was performed in hippocampal slices and cultures to functionally dissect the specific molecular constituent underlying this synaptic impairment. Several experiments for pre-synaptic function conducted in slices and cultures measuring paired-pulse responses in slices, and the rate of synaptic vesicle exocytosis and miniature frequency in cultures revealed the absence of alterations in the release of neurotransmitter. However, experimentation of post-synaptic function revealed an enhancement in excitatory current from spontaneous miniature events in cultures and an enhancement in evoked AMPA receptor transmission from CA1 cells in slices. Thus, the enhanced CA3-CA1 basal synaptic transmission was due to a post-synaptic defect originating from an enhancement in CA1 AMPA receptor current. An increase in the ratio of surface/intracellular expression for the GluR2 and GluR3 subunits in hippocampal slices suggested that the enhancement of CA1 AMPA receptor transmission was due to an increase in the number of surface AMPA receptors. Furthermore, the increase in CA1 AMPA receptor transmission was likely to be due to a defect in the re-distribution of AMPA receptors between the surface membrane and intracellular compartments because no changes were observed in the total expression of the GluR2 and GluR3 subunits in sandy mice. The composition of the increased number of surface AMPA receptors appeared subunit specific because neither changes in the ratio of surface/intracellular expression for the GluR1 and GluR4 subunits, nor changes in GluR1S845, which is associated with GluR1 surface expression, were observed in hippocampal preparations. Enhanced single channel conductance in the AMPA receptor did not appear to contribute to the increase of evoked CA1 AMPA receptor transmission. Indeed neither changes in the proportion of AMPA receptors lacking GluR2 subunit, which have a high single channel conductance, nor in the phosphorylation of GluR1S831, which is associated with high single channel conductance, were found. These data favor a model whereby loss of dysbindin likely leads to enhanced recycling of AMPA receptors to the membrane surface from an intracellular compartment. Consistent with this model, CA3-CA1 LTP, a type of synaptic plasticity that is thought to underlie learning and memory and is known to involve AMPA receptor trafficking to the synapse, was enhanced in sandy juvenile mice. Taken together, these results demonstrate that dysbindin expression plays a key role in modulating the sub-cellular distribution of AMPA receptors at the CA3-CA1 synapse which likely influences both basal synaptic transmission and plasticity.