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Methylphenidate (MPH) is the most commonly prescribed drug to treat Attention Deficit Hyperactivity Disorder (ADHD). ADHD is now considered a life-long disorder; therefore, patients take MPH from adolescence into adulthood, highlighting the need for research studying chronic MPH use. MPH increases dopamine and norepinephrine within the synaptic cleft; therefore, chronic use of MPH may lead to changes within important dopaminergic pathways. One pathway, the mesolimbic pathway, includes the hippocampus, an area where adult neurogenesis occurs. We investigated the effects of chronic low and high doses of MPH on neurogenesis and examined levels of a few key proteins linked to cell proliferation in the hippocampus. Low dose MPH appears to increase cell proliferation and cell survival in the hippocampus, and these effects are accompanied by increases in vascular endothelial growth factor (VEGF), the receptor for brain-derived neurotrophic factor (TrkB), and beta-catenin. While high dose MPH may initially increase neuronal proliferation, newly-generated neurons are unable to survive long-term, and decreases in VEGF, TrkB, and beta-catenin are observed with chronic high dose MPH. Another major dopaminergic pathway is the nigrostriatal pathway, which is involved in motor control and degenerates with Parkinson’s disease. Chronic use of MPH appears to sensitize dopaminergic neurons within this pathway to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but the cause of this sensitization is unknown. The autooxidation of excess dopamine forms dopamine-quinones that lead to free radical production, but the antioxidant, glutathione, can protect neurons. However, we showed that chronic MPH increases dopamine-quinone formation and causes a subsequent glutathione depletion within the striatum. Therefore, oxidative stress may sensitize dopamine neurons to MPTP. We also assessed the vulnerability of dopaminergic neurons in the nigrostriatal pathway to MPTP after chronic MPH in females. Interestingly, proestrus (high estrogen) females were more sensitive to MPTP than anestrus (low estrogen) females. Similar to males, chronic MPH caused a depletion in glutathione that was further decreased following MPTP exposure. However, chronic MPH did not significantly alter dopaminergic neuronal numbers or quinone formation in females. These studies highlight some of the potential effects of chronic MPH use.

The mammalian hippocampus is responsible for many crucial brain functions such as learning, memory, and neurogenesis in adults. Its degeneration is a pathology associated with the early stages of Alzheimer’s disease. A variety of genes have been associated with both neuroprotection and neurogenesis in the brain, some of which include progesterone membrane component 1 (Pgrmc1) and kit ligand (KitL). Pgrmc1 is recognized for mediating hormonal functions in both the ovary and neuroendocrine regions such as the anteroventral periventricular nucleus (AVPV), but its functions in the hippocampus are not well known. Both Pgrmc1 and KitL share downstream targets, the most strongly supported being genes in the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway. I hypothesized that Pgrmc1 regulates neural targets through KitL/c-Kit signaling. To investigate this hypothesis I used a variety of in vivo and in vitro techniques. These techniques included mapping both KitL and receptor c-Kit in the adult female rat brain using in situ hybridization. I used Pgrmc1 silencing with siRNA in hippocampal-derived mHe-18 cells and Pgrmc1/2 double conditional knock out mouse brains to study Pgrmc1 regulation of KitL synthesis. To determine common downstream targets of KitL and Pgrmc1 I then treated mHe-18 cells with soluble KitL protein. Finally, to determine whether c-Kit mediated effects of Pgrmc1, I treated cells with both Pgrmc1 siRNA and AG-1296, a c-Kit inhibitor. The results show that Pgrmc1 regulates KitL expression, as well as downstream targets Pias1, 2, 3, and 4. However, AG-1296 did not abrogate Pgrmc1 regulation of the downstream targets, demonstrating regulation independent of KitL signaling. Taken together, these results suggest that while Pgrmc1 alters KitL expression and regulates the same genes as KitL/c-Kit, the mechanism of action likely differs. Considering that these two genes are involved in neurogenesis and neuroprotection, as well as memory and learning, a better understanding of the pathways may help lead the way in treating neurodegenerative diseases in the future.

Chlorpyrifos is a widely used organophosphate insecticide, functioning through the inhibition of acetylcholinesterase. Recent studies report negative long-lasting biochemical and behavioral effects at levels without acetylcholinesterase inhibition. Our lab studies have identified the endocannabinoid system as a target for OP low-dose neurotoxicity. This thesis identifies the proteins and their associated neurotransmitter systems in the hippocampus that have been affected by low dose developmental exposure to the OP insecticide CPF. Male rat pups were treated from postnatal day 10 (PND) - PND16 with either corn oil (vehicle), 0.75 mg/kg of CPF, or 0.02 mg/kg of PF-04457845, a specific fatty acid amide hydrolase (FAAH) inhibitor. On PND38, rats were sacrificed for hippocampal extraction, and shotgun proteomics was used for protein expression. DAVID and Ingenuity Pathway Analysis software detected differentially expressed proteins such as Neuroligin-2 and Synaptotagmin 2, and disrupted signaling pathways such as ephrin B signaling, synaptogenesis signaling, and glutamate receptor signaling. Taken together, our data suggests that CPF reduces glutaminergic signaling pathways, greatly reducing long-term potentiation, prohibiting proper synapse formation, and therefore disrupting the proper functioning of the hippocampus.

Patients with schizophrenia show a number of cognitive deficits that may be related to abnormal hippocampal physiology and function. One such cognitive deficit is in transitive inference. Simply stated, transitive inference is the ability to infer A > C after directly learning A > B and B > C. The hippocampus has been implicated in transitive inference as lesions of the hippocampus in C57BL/6 mice after initial training and testing impairs transitive inference. Likewise, lesions of the hippocampus in rats prior to training also impair transitive inference. However, lesions of the whole hippocampus are not able to specifically examine the role of the dorsal versus ventral hippocampus in this task. This is important because studies suggest that the dorsal and ventral poles of the hippocampus may be functionally different. The present experiment used reversible inactivation of the dorsal and ventral hippocampus to examine the role of these structures in transitive inference. Mice were trained to learn that A>B, B>C, C>D, and D>E during training phases and then were tested to show if they learned that A>E (the novel control pairing) and that B>D (the novel pairing which requires transitive inference) during test sessions. Following these test sessions, cannulae were inserted into the hippocampus and the mice were allowed 5 days to recover. After the recovery period, mice underwent 4 more test sessions. The GABAA agonist muscimol or saline was infused into the dorsal or ventral hippocampus thirty minutes before each test session. The mice which received muscimol infusion into the dorsal hippocampus performed similarly to controls on the novel control pairing (A>E) but were significantly impaired on the novel pairing (B>D) which required transitive inference. The DBA/2 strain of mice have altered hippocampal function and has been used to model schizophrenia. The study also compared performance of DBA/2J and C57BL/6J inbred mice in TI, and foreground and background fear conditioning, which both involve the hippocampus. Separate mice were then trained with two different fear conditioning paradigms. For background fear conditioning, mice are trained with two paired presentations of a conditioned stimulus (CS, 30 second, 85 dB white noise) and an unconditioned stimulus (US, 2 second, 0.57 mA foot shock). Mice are then tested the next day for both freezing to the training context. Foreground fear conditioning differed in that the mice were presented with only the shocks during training. DBA/2J mice performed significantly worse than the C57BL/6J in both foreground and background fear conditioning and transitive inference. These results provide further support for the role of the dorsal hippocampus in transitive inference. Moreover, these results may help provide a better understanding of the cognitive deficits associated with schizophrenia. / Psychology

Approximately 1 in 5 Americans smoke despite the widely known negative health consequences of the habit. One factor that contributes to the high rates of nicotine addiction and its continued use is the ability of the drug to alter long-term memory. Learning in the presence of nicotine results in changes to the cellular and molecular processes that support the formation and storage of long-term memories. The consolidation of long-term memory requires a number of mechanisms, such as gene transcription. Previous work has found that learning a contextual fear conditioning task in the presence of nicotine results in the upregulation of the c-jun N-terminal kinase (JNK1) gene in the hippocampus and that JNK protein activation is necessary for the nicotine induced enhancement of contextual conditioning. The present study examines the transcription factors involved in the transcriptional regulation of jnk1 in the hippocampi of mice following learning in the presence of nicotine. The hypothesis that cAMP response element binding protein (CREB) regulates jnk1 transcription was examined. Further, a protein/DNA transcription factor array was used as an unbiased examination of changes in transcription factor activity following learning in the presence of nicotine. Using chromatin immunoprecipitation (ChIP), transcription factors identified from the array and CREB were examined for changes in their binding to the jnk1 promoter following fear conditioning in the presence of nicotine. An increase in the binding of phosphorylated CREB was found in the jnk1 promoter of mice trained in the presence of nicotine. This implicates CREB activation in the increase of jnk1 transcription following learning in the presence of nicotine. Additionally, data from the transcription factor array suggest other factors such as PARP, TR, USF-1 and E2F-1 as potentially playing a role in the cognitive effects of nicotine. These findings are discussed with respect to how they inform our understanding of the signaling cascades and genetics involved in the memory enhancing effects of this addictive drug. / Psychology

In order to continue the decline of smoking prevalence, it is imperative to understand the factors that contribute to the development of nicotine and tobacco addiction, such as adolescent initiation of nicotine use, stress, and the interaction of adolescent stress and adolescent nicotine. Both clinical and preclinical literature indicates that adolescent, but not adult, nicotine administration leads to long-lasting impairments in learning and memory and affect. Specifically, chronic nicotine treatment beginning in early adolescence or late adolescence resulted in adult deficits in contextual fear learning. However, the current study demonstrated that these adult learning deficits did not occur solely from nicotine administration. Rather, an interaction between adolescent nicotine and adolescent stress resulted in adult learning deficits in contextual fear. Additionally, it was found that dietary choline supplementation that began immediately following cessation of adolescent nicotine treatment and continued through a protracted abstinence period lasting thirty days reversed adult deficits in contextual fear. Finally, the current study found that adolescent nicotine exposure beginning at either early adolescence or late adolescence increased depressive-like behaviors, but not anxiety-like behaviors, following a protracted abstinence period. In contrast, chronic nicotine treatment in adult increased anxiety-like behaviors measured by the elevated plus-maze following a protracted abstinence period. The work encompassed in this dissertation suggests that the interactions between adolescent stress and adolescent nicotine increases the risk for developing cognitive and affective impairments, which may promote continued use of nicotine in adulthood. / Psychology

Caffeine is the most widely used psychoactive substance in the world. While findings suggest that chronic caffeine exerts negligible effects on cognition in adults, the effects of chronic caffeine on cognition in children and adolescents are not well understood. The hippocampus, a brain region important for learning and memory, undergoes extensive structural and functional modifications during pre-adolescence and adolescence. As a result, chronic caffeine may have differential effects on hippocampus-dependent learning and memory in pre-adolescents and adolescents compared to adults. The present study characterized the effects of chronic caffeine and withdrawal from chronic caffeine on hippocampus-dependent (contextual) and hippocampus-independent (cued) fear conditioning in pre-adolescent, adolescent, and adult mice. In addition, we investigated whether exposure to chronic caffeine during pre-adolescence, adolescence, or adulthood had long-lasting effects on conditioning in adulthood. Results indicate that exposure to chronic caffeine during pre-adolescence and adolescence either enhances or impairs contextual conditioning in a concentration-dependent manner. However, withdrawal from chronic caffeine impairs contextual conditioning in pre-adolescent mice only. In addition, exposure to chronic caffeine during pre-adolescence either enhances or impairs retention of contextual memories in adulthood in a concentration-dependent manner. In contrast, exposure to chronic caffeine during adolescence impairs cued conditioning in adulthood. These findings support the hypothesis that exposure to chronic caffeine during pre-adolescence and adolescence compromises hippocampus-dependent learning and memory. Furthermore, exposure to chronic caffeine during adolescence may produce long-lasting deficits in learning and memory in adulthood. / Psychology

Adult hippocampal neurogenesis, or the process of creating new neurons in the dentate gyrus (DG) of the hippocampus, underlies learning and memory capacity. This cognitive ability is essential for humans to operate in their everyday lives, but cognitive disruption can occur in response to traumatic insult such as brain injury. Previous findings in rodent models have characterized the effect of moderate traumatic brain injury (TBI) on neurogenesis and found learning and memory shortfalls correlated with limited neurogenic capacity. While there are no substantial changes after one mild TBI, research has yet to determine if neurogenesis contributes to the worsened cognitive outcomes of repetitive mild TBI. Here, we examined the effect of neurogenesis on cognitive decline following repetitive mild TBI by utilizing AraC to limit the neurogenic capacity of the DG. Utilizing a BrdU fate-labeling strategy, we found a significant increase in the number of immature neurons that correlate learning and memory impairment. These changes were attenuated in AraC-treated animals. We further identified endothelial cell (EC)-specific EphA4 receptor as a key mediator of aberrant neurogenesis. Taken together, we conclude that increased aberrant neurogenesis contributes to learning and memory deficits after repetitive mild TBI. / Doctor of Philosophy / In the United States, millions of people experience mild traumatic brain injuries, or concussions, every year. Patients often have a lower ability to learn and recall new information, and those who go on to receive more concussions are at an increased risk of developing long-term memory-associated disorders such as dementia and chronic traumatic encephalopathy. Despite the high number of athletes and military personnel at risk for these disorders, the underlying cause of long-term learning and memory shortfalls associated with multiple concussions remains ill defined. In the brain, the hippocampus play an important role in learning and memory and is one of only two regions in the brain where new neurons are created from neural stem cells through the process of neurogenesis. Our study seeks to address the role of neurogenesis in learning and memory deficits in mice. These findings provide the foundation for future, long-term mechanistic experiments that uncover the aberrant or uncontrolled processes that derail neurogenesis after multiple concussions. In short, we found an increase in the number of newborn immature neurons that we classify as aberrant neurogenesis. Suppressing this process rescued the learning and memory problems in a rodent model of repeated concussion. These findings improve our understanding of the processes that contribute to the pathophysiology of TBI.