Associations Between Striatal Acetylcholine and In Vivo Pharmacology and Behavior Acetylcholine (ACh) has neuromodulatory effects on several different neurons and synapses in the striatum. While the roles of cholinergic neurons and receptors have been examined, we know less about how changes in extracellular ACh levels relate to in vivo effects of pharmacological agents and behavior. To address this issue we have expressed the genetically encoded intensity-based acetylcholine sensing fluorescent reporter (iAChSnFR, developed by the laboratory of Loren Looger at the Howard Hughes Medical Institute) in dorsomedial striatum (DMS) and used in vivo fiber photometry in awake, freely-moving mice. In mice moving about a small box we observe fluctuations in fluorescence intensity that consist of rapid changes (seconds in duration) and slower changes (>10 sec in duration). These fluctuations are not observed in photometric recordings from DMS in mice expressing a mutated (iAChSnFR-Null) version of the sensor that has >1000x lower ACh affinity. Anesthetizing mice with inhaled 5% isoflurane produced 50% decrease in overall fluorescence and largely eliminated the fluctuations in fluorescence. Smaller decreases were observed when mice were given an intraperitoneal injection of ethanol at a dose of 2 g/kg that reduced their mobility. During recovery from either isoflurane or ethanol exposure fluorescence increases lasting 10s of seconds were observed more frequently than in the pre-drug period. In contrast, intraperitoneal injection of 15 mg/kg cocaine produced a sustained increase in fluorescence (>60 min) with subtle effects on fast and slow fluctuations. These findings indicate that sedative drugs decrease intra-striatal ACh levels, while the stimulant cocaine has the opposite effect. We also examined changes in ACh in DMS during performance of different behaviors. When mice were trained on an accelerating rotarod motor skill task, several changes in fluorescence were observed that correlated with behavioral performance. There was a general increase in fluorescence that persisted for most or all of the time that mice were running on the rotarod, especially on early trials when mice were still improving performance on the task. A prominent peak of increased fluorescence that persisted for 10s of seconds was also observed just prior to mice falling from the rod, and this response was also more prominent early in training. There were also changes in fast fluorescence fluctuations that can be examined by frequency domain analysis. No such fluctuations were observed in mice expressing the iAChSnFR-Null construct despite the presence of readily detectable baseline fluorescence in the DMS of these mice. Thus, the behavior-associated fluorescent fluctuations are likely due to changes in extracellular ACh. We continue to analyze the fluorescent signals to determine the behavioral correlates of the different components of the ACh signal. We are also examining changes in ACh levels during performance of other learned behaviors thought to involve dorsal striatum. Ketone-Rich Diet Prevents Dopaminergic and Behavioral Impairments in a Mouse Model of Parkinson's Disease Parkinson's Disease (PD) is a devastating neurological disorder characterized by severe hypokinesia, tremors and other movement impairments that is eventually fatal. Gradual degeneration of dopaminergic neurons in the substantia nigra pars compacta is the main cause of PD, and replenishing dopamine alleviates symptoms but does not prevent disease progression. Many studies indicate that mitochondrial dysfunction in the dopaminergic neurons plays a role in their degeneration, and that correcting metabolic effects that impair mitochondria may slow neuron loss and disease progression. Changing diet to a low-carbohydrate ketogenic diet, or supplementation with ketones is one such metabolic treatment that has been suggested for PD and other neurological disorders. To determine if a ketone-based diet could alter progression of dopamine loss and motor function we used a mitochondria-based mouse PD model. For this purpose we chose the MitoPark mouse in which the mitochondrial protein TFAM is genetically removed from dopaminergic neurons using a Cre recombinase-based breeding strategy (i.e. breeding mice carrying a lox-P flanked TFAM allele with DAT-Cre mice). Indeed, both male and female mice homozygous for this neuron-specific knockout (MitoPark mice) showed a profound loss of afferent stimulation-induced striatal dopamine release at 20 weeks of age, as assessed by fast-scan cyclic voltammetry in dorsal striatal brain slices, when compared to littermates carrying the TFAM floxed allele but DAT-Cre negative (controls). Smaller but significant decreases were observed at earlier ages and male mice showed earlier onset of the deficit than females. The MitoPark mice also showed progressive impairment in performance on the accerelating rotarod motor skill test beginning at 12 weeks of age and peaking at 18-20 weeks. Control mice and mice heterozygous for this neuron-specific TFAM knockout did not show motor skill impairment. To determine if feeding mice a ketone-rich diet would alter the dopamine release or motor skill phenotypes, we fed both control and MitoPark mice ad libitum a diet rich in beta-hydroxybutyrate for 16 weeks beginning on postnatal week 4. The diet enhanced blood ketone levels in both male and female mice. The decrease in dopamine release at week 20 was reduced in the ketone diet-fed MitoPark relative to control diet-fed MitoPark animals, and increased in the ketone-fed control mice relative the control diet-fed mice. The ketone rich diet also prevented the impairment of rotarod performance in the MitoPark mice. These findings indicate that a ketone-rich diet can ameliorate both a mitochondrial-based neurobiological insult and associated motor skill deficit in this mouse model of dopaminergic cell loss. Experiments assessing other behaviors and the extent of anatomical changes in these mice are ongoing.
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