The pathophysiology of Parkinson's disease (PD) has long been attributed to dopamine (DA) loss in the striatum. However, it remains unclear whether simple underactivation of striatal DA receptors is sufficient to induce parkinsonian signs. To test this hypothesis, we performed unilateral infusions of cis-flupenthixol (cis-flu; D1/D2 antagonist) into the macaque putamen, while the macaque performed a reaching task. Twenty-six cis-flu and three saline infusions were performed across three hemispheres in two macaques. Neuronal and local field potential activity was recorded simultaneously from cortex, globus pallidus externa (GPe), and globus pallidus interna (GPi) during most infusions. The reaching task required each macaque to make visually-cued reaching movements to a target for a reward. The macaque was then required to return its hand to a home position without external cues. Injection-related slowing of movement initiation or execution was thought to reflect akinetic- or bradykinetic-like effects, respectively. Following 8/26 cis-flu infusions, macaques exhibited a marked slowing in the initiation of self-generated return movements (95% increase). This was the most severe behavioral effect of cis-flu infusions. The initiation and execution of externally-cued movements were also prolonged following 9/26 and 6/26 injections, but only by 20% and 15% respectively. In general, akinetic-like effects occurred twice as often as bradykinetic-like effects (p<0.05, 2= 4.1). Interestingly, akinetic and bradykinetic effects could be elicited independently. In addition to affecting behavior, intrastriatal DA receptor blockade also reduced resting and peri-movement activity in the cortex and suppressed resting GPe activity. Burstiness, synchrony, and oscillatory activity in cortex were increased following intrastriatal DA receptor blockade as well. Oscillatory activity was also increased in the GPe and GPi. In conclusion, suppression of striatal DA activity was sufficient to induce akinetic-like signs, most severely affecting movement initiation during self-generated movements. Furthermore, distinct parkinsonian-like signs could be elicited independently, suggesting that separate signs may have unique pathophysiologic substrates. Intrastriatal DA receptor blockade also induced changes in cortical and BG activity that were consistent with findings in the parkinsonian state. Interestingly, many of these neuronal activity changes were specific to cortex, implicating an important role for cortical activity in the development of akinetic parkinsonian signs.

Human brain aging is associated with robust normal functional, structural, and molecular changes that underlie changes in cognition, memory, mood and motor function, amongst other processes. Normal aging is also a requirement for onset of many neurological diseases, ranging from later onset neurodegenerative diseases such as Alzheimers(AD) and Parkinsons diseases(PD), to earlier onset psychiatric disorders such as bipolar disorder(BPD) and schizophrenia(SCHZ). Understanding the molecular mechanisms and genetic underpinnings of normal age-related brain changes would have profound consequences for prevention and treatment of age-related impairments and disease. Here I introduce current knowledge of these functional changes, their structural and molecular underpinnings, their genetic modulators, and the contribution of normal aging to age-related neurological disease. I then present my contribution to this field in the form of three papers on genetic modulation of mammalian brain molecular aging. These studies demonstrate and investigate mechanisms underlying the causal modulation of molecular brain aging rates by Brain Derived Neurotrophic Factor (BDNF) and Serotonin (5-HT) in knock-out (KO) mice, and associative modulation by the putative longevity gene, Sirtuin 5, in humans (novel low-expressing promoter polymorphism (Sirt5prom2)). In humans we additionally investigate the potential mechanism(s) underlying neurological disease gating by normal aging, providing supporting evidence for molecular aging being a genetically controlled transcriptional program that progressively promotes age-regulated neurological diseases. In the discussion, I place these studies in a broader context within the field, detailing their implications and future directions.

Traumatic brain injury (TBI) represents a significant cause of death and disability in industrialized countries. Of particular importance to patients is the chronic effect that TBI has on cognitive function. Therapeutic strategies have been difficult to evaluate because of the complexity of injuries and variety of patient presentations within a TBI population. Experimental therapies based upon cortical and hippocampal neuroprotection have not translated clinically. However, pharmacotherapies targeting dopamine (DA) have consistently shown benefits in attention, behavioral outcome, executive function, and memory. Striatal damage causes deficits in executive function, learning, and memory. Dopamine and cAMP regulated phosphoprotein 32 (DARPP-32), expressed within striatal medium spiny neurons, is known to regulate several substrates of cognition. We found that controlled cortical impact injury in rats produces a chronic decrease in DARPP-32 threonine-34 phosphorylation and increase in protein phosphatase-1 activity. There is no effect of injury on threonine-75 phosphorylation or DARPP-32 protein. Amantadine has known benefits on post-TBI cognitive deficits and when given daily for two weeks reversed the DARPP-32 and protein phosphatase-1 changes. Amantadine also decreased the phosphorylation of threonine-75 consistent with activity as a partial N-methyl-D-aspartic acid receptor antagonist and partial dopamine agonist. FK-506, also known as tacrolimus, is a calcineurin inhibitor that has been shown to decrease cell death in the hippocampus following a fluid percussion experimental TBI. Calcineurin is also an important regulator of DARPP-32 phosphorylation in the striatum. We evaluated the effect of FK-506 on the hippocampus and DARPP-32 in the striatum to better detail its effects after a TBI. An acute administration of FK-506 following controlled cortical impact reversed the effects of TBI on DARPP-32 phosphorylation seen chronically. We then evaluated the effect of a combined drug therapy on cognitive deficits post TBI. An acute treatment with FK-506 post TBI followed by chronic Amantadine therapy demonstrated an improvement in both motor behavior and Morris water maze deficits seen following TBI. Neither drug produced benefit when given alone. These data demonstrate that DARPP-32 represents a promising new therapeutic target for TBI induced cognitive deficits.

Immediate early genes (IEGs) typically are the first genetic responders to a variety of cellular activations. The IEG that encodes activity-regulated cytoskeleton-associated protein (arc/arg3.1) has attracted much interest because its mRNA is transported to and translated near activated synapses. Moreover, arc has been implicated in both long-term potentiation (LTP) and long-term depression (LTD). However, little is known about the time course of altered arc expression during LTP and LTD. Here we characterized arc mRNA levels in area CA1 of the adult rat hippocampus in vivo after LTP- and LTD-inducing stimulations that were identical except for the temporal patterning of the stimulation pulses. We observed a persistent increase in arc mRNA level during LTP. In contrast, during LTD, arc mRNA level first was decreased and then transiently increased relative to control level. These findings demonstrate that arc mRNA is regulated differently during LTP and LTD, and they provide evidence for stimulation-induced down-regulation of mRNA availability during LTD. Findings of abbreviated LTD when transcription was inhibited indicate that the prolonged maintenance of the NMDA receptor-dependent LTD studied here requires de novo transcription. Furthermore, findings of no LTD-associated change in the mRNA level of the IEG zif268 show that the decrease in arc mRNA during LTD is not a general genetic response. Thus, the regulation of arc expression not only differs between LTP and LTD but also diverges from that of other IEGs implicated in activity-dependent synaptic plasticity.

A corticolimbic circuit including the amygdala and medial prefrontal cortex (mPFC) affects sensitivity to threat, related aspects of personality and risk for psychopathology. Serotonin (5‐HT) is a potent neuromodulator of this circuit, however, 5‐HT receptors mediating these effects and genetic sources of variability in 5‐HT receptor availability are not understood. We determined the association between 5‐HT1A and 5‐HT2A binding and the response to threat within this corticolimbic circuit using a multimodal neuroimaging strategy in humans in vivo. Corticolimbic circuit function was assessed with a threat‐related faces matching paradigm using functional magnetic resonance imaging (fMRI). Regional 5‐HT1A and 5‐HT2A binding was assessed with [11C]WAY100635 and [18F]altanserin PET, respectively. We evaluated the association between receptor binding and common polymorphisms (rs6295, rs6311 and 5‐HTTLPR) in 5‐HT related genes. In Study 1 we found that 5‐HT1A binding within the dorsal raphe nucleus was inversely associated with threat‐related amygdala reactivity. This is consistent with 5‐HT1A autoreceptors negatively regulating 5‐HT release, which within the amygdala potentiates its response to threat. In Study 2 we found that mPFC 5‐HT2A binding was inversely associated with threat‐related amygdala reactivity and positively associated with amygdala habituation and amygdala‐mPFC functional connectivity. In Study 3 we found that mPFC 5‐HT1A binding significantly moderated the inverse association between mPFC 5‐HT2A binding and amygdala reactivity. These findings are consistent with the co‐localization of 5‐HT1A and 5‐HT2A on glutamatergic neurons within mPFC indicating the 5‐HT2A receptor is localized to facilitate regulation of the amygdala and the 5‐HT1A receptor is localized to moderate its effects within mPFC. In Study 4 we found that 5‐HTTLPR genotype predicted 5‐HT1A and 5‐HT2A binding in brain regions within this circuit such that the S and LG alleles were associated with reduced 5‐HT1A and 5‐HT2A binding. These findings provide novel insight into mechanisms that mediate the effects of 5‐HT signaling on the response to threat of a key corticolimbic circuit in humans. Our findings indicate that 5‐HT1A and 5‐HT2A receptors contribute significantly to threat‐related corticolimbic circuit function in humans. Furthermore, the 5‐HTTLPR may contribute to individual variability in neural and behavioral sensitivity to threat by biasing 5‐HT1A and 5‐HT2A availability.

The disease signature of major depressive disorder is distributed across multiple physical scales and investigative specialties, including genes, cells and brain regions. No single mechanism or pathway currently implicated in depression can reproduce its diverse clinical presentation, which compounds the difficulty in finding consistently disrupted molecular functions. We confront these key roadblocks to depression research - multi-scale and multi-factor pathology - by conducting parallel investigations at the levels of genes, neurons and brain regions, using transcriptome networks to identify collective patterns of dysfunction. Our findings highlight how the collusion of multi-system deficits can form a broad-based, yet variable pathology behind the depressed phenotype. For instance, in a variant of the classic lethality-centrality relationship, we show that in neuropsychiatric disorders including major depression, differentially expressed genes are pushed out to the periphery of gene networks. At the level of cellular function, we develop a molecular signature of depression based on cross-species analysis of human and mouse microarrays from depression-affected areas, and show that these genes form a tight module related to oligodendrocyte function and neuronal growth/structure. At the level of brain-region communication, we find a set of genes and hormones associated with the loss of feedback between the amygdala and anterior cingulate cortex, based on a novel assay of interregional expression synchronization termed gene coordination. These results indicate that in the absence of a single pathology, depression may be created by dysynergistic effects among genes, cell-types and brain regions, in what we term the floodgate model of depression. Beyond our specific biological findings, these studies indicate that gene interaction networks are a coherent framework in which to understand the faint expression changes found in depression and complex neuropsychiatric disorders.

Traumatic brain injury (TBI) is a widespread problem in the United States affecting thousands of individuals annually. Due to our lack of understanding of the mechanisms of TBI, medical management of the functional deficits in these patients is difficult. In this study, injury induced deficits in striatal dopamine neurotransmission was studied using rats injured by controlled cortical impact. We identified specific decrease in the levels of phosphorylated tyrosine hydroxylase (TH) measured by Western blots in the striatum and substantia nigra at 1 week following TBI, suggesting a decrease in TH activity. A direct measurement of TH activity by an in-vivo TH activity assay showed a correlating deficit in the injured animals. Striatal dopamine release evoked by potassium stimulus using microdialysis probes was decreased in injured animals compared to shams at 1 week. These results suggest deficits in presynaptic dopamine synthesis and release. To reverse these deficits, nicotine which was previously demonstrated to enhance striatal dopamine signaling, was administered for 1 week following injury. Rats that were treated with nicotine showed recovery of dopamine release and TH activity deficits. We have previously identified that TBI induces deficits in phosphorylation of striatal posynaptic protein: dopamine and cAMP regulated phosphoprotein 32 (DARPP-32), an important regulator of striatal dopamine signaling. To assess if nicotine treatment can also reverse this deficit in DARPP-32 phosphorylation at threonine 34 (pDARPP-32-T34), Western blot was used. There was no enhancement of pDARPP-32-T34 levels by nicotine treatment compared to saline controls. Also, phosphorylation levels of molecules downstream of pDARPP-32-T34: extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) were not affected by nicotine treatment. Behavioral experiments testing motor function by Beam Balance Test and Beam Walking Test and cognitive function by Morris Water Maze test showed no benefits of nicotine treatment. The molecular results in this study suggest that nicotine may lead to activativation of multiple receptor signaling pathways that have opposite modulation of pDARPP-32-T34. This study gives us a better understanding of the complex signaling pathways of striatal dopamine neurotransmission in the setting of TBI.

Precise neuronal connections are crucial for normal brain function. Often this is accomplished during development, as initially imprecise connections are refined in a manner that depends on neural activity, both spontaneous and sensory-evoked. In the auditory system, many connections are topographically organized according to frequency, or tonotopically, an organizational scheme important for processing information about sound. In this thesis, I investigated the development of precise tonotopy in the inhibitory connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), a pathway in the auditory brainstem involved in sound localization. Although MNTB-LSO connections exhibit tonotopy from the outset, tonotopic precision increases during development through a process of silencing imprecise inputs and strengthening maintained connections before hearing onset, followed by anatomical pruning after hearing. I teased apart the relationship between functional and anatomical refinement, as well as the degree to which spontaneous and sound-evoked activity play a role in each. Finally, I attempted to link the tonotopic specificity of this circuit to a specific aspect of auditory perception, frequency discrimination. In Chapter 2, I mapped the tonotopic precision of individual MNTB axons in the LSO over the first three weeks of postnatal development and showed that pruning does not take place before hearing onset, indicating that functional and anatomical refinement take place during distinct developmental periods. In Chapter 3, I showed that anatomical refinement after hearing onset depends on efferent cholinergic transmission in the cochlea, most likely due to its role in patterning pre-hearing spontaneous activity and the functional refinement of connections. In Chapter 4, I showed that eliminating the normal spectrotemporal structure of sound-evoked activity by rearing animals in pulsed white noise does not disrupt pruning. Finally, in Chapter 5, I showed that the loss of tonotopic precision that results from the elimination of cochlear cholinergic transmission is also accompanied by impaired frequency discrimination, providing a link between tonotopic refinement, the efferent system, and auditory perception. I discuss the results in the context of a model of tonotopic refinement and a new role of the efferent system during development.

The advent of reliable simultaneous recording of the activity of many neurons has enabled the study of interactions between neurons at a large scale: the number of observed pairwise interactions is proportional to the square of the number of recorded neurons. The dominant phenomenon in these pairwise interactions is synchronization, reflecting a system where many observed variables have in common a smaller set of latent variables. This permits the possibility that the complex signals observed in the brain might be reducible to a simpler system. We used this insight to design a better signal processing scheme for neuroprosthetics; to identify the same neurons in many recording sessions from their pairwise interactions; to show that the tuning functions of neurons in motor and premotor cortex do not reflect simple coordinate frame models; and to identify error as a dominant signal during continuous movements.

Amyotrophic Lateral Sclerosis (ALS) is a rapidly fatal neurological disease characterized by the degeneration of motor neurons involved in voluntary muscle control. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective long-term treatments. The identification and characterization of novel protein biomarkers could improve the speed and accuracy of disease diagnosis, and assist in predicting and tracking disease progression. Additionally, the utilization of such biomarkers could also expedite the development of effective treatments by both providing insight into disease pathogenesis, and improving the efficacy of clinical trials. In this work, I examined two cerebrospinal fluid (CSF) proteins, cystatin C and free hemoglobin, for biomarker utility and functional relationships with disease pathogenesis. Cystatin C is a constitutively expressed cysteine protease inhibitor that appears to be reduced in the CSF of ALS patients. I evaluated cystatin C concentration by ELISA in CSF and plasma samples from ALS patients, normal controls, and neurological disease controls. These data were used to evaluate cystatin C as a diagnostic, surrogate, and prognostic biomarker in ALS. Plasma cystatin C was equally elevated in ALS patients and disease controls, demonstrating no biomarker utility. However, CSF levels were confirmed to be lower in ALS patients than in healthy controls, and may possess diagnostic utility when used in conjunction with other biomarkers. CSF cystatin C also exhibited potential for one surrogate biomarker application, and for prognostic biomarker utility. The trends in CSF cystatin C abundance suggest a neuroprotective role for cystatin C in ALS. Accordingly, reductions in CSF cystatin C may contribute to disease development through the loss of a protective function mediated by cysteine protease inhibition. CSF free hemoglobin levels were also measured by ELISA, and were evaluated for utility as a biomarker of blood-CNS barrier damage in ALS. The proportion of ALS patients exhibiting elevated CSF free hemoglobin was higher than in either control group, suggesting that blood-CNS barrier damage may occur in this disease. Overall, the results of this work identify and clarify potential biomarker applications of two CSF proteins, and also provide new insight into potential pathogenic mechanisms of ALS.