Application of substance P (SP) to intracardiac neurons of the guinea pig causes slow depolarization and increases neuronal excitability. The present study was done to determine the influence of SP on fast excitatory responses of intracardiac neurons to ACh. Intracellular recording methods were used to measure responses of intracardiac neurons in whole mount preparations of atrial ganglionated nerve plexus from guinea pig hearts. Local pressure ejection of 100 μM SP (1 s) from a glass micropipette caused slow depolarization of all neurons (n = 38) and triggered action potential generation in 47% of the cells tested. Bath application of SP (0.5-100 μM) caused a dose-dependent depolarization of intracardiac neurons but rarely evoked action potentials, even at the highest concentration. However, such treatment with SP enhanced nicotinic responses evoked by local pressure ejections of ACh (10 mM, 10- to 100-ms duration) in 77% of intracardiac neurons studied (n = 52). A significant increase in amplitude of ACh-evoked fast depolarization occurred during treatment with 0.5 μM SP (13.0 ± 1.8 mV for control vs. 17.7 ± 1.9 mV with SP present, n = 7, P = 0.019). At higher concentrations of SP, enhancement of the response to ACh resulted mainly in action potential generation. However, responses to ACh were attenuated by SP in 15% of the intracardiac neurons studied. This attenuation occurred primarily during exposure to 10 and 100 μM SP and was manifest as a reduction in amplitude of nicotinic fast depolarization or inhibition of ACh-evoked action potentials. These findings support the conclusion that SP could function as a neuromodulator and neurotransmitter in intracardiac ganglia of the guinea pig.

Opioid receptors have been a target of pharmacological manipulation in alcohol use disorder (AUD) recovery protocols for many years. Aerobic exercise, a common adjunct in AUD recovery, is known to modulate opioid receptors (ORs) both during both acute and long term exposure. The three subtypes of ORs: mu (MOR), delta (DOR), and kappa (KOR) are all expressed on neurons in the mesocorticolimbic circuitry. Kappa-opioid receptors are expressed directly on dopamine (DA) neuron terminals in the nucleus accumbens (NAc). Mu and Delta ORs are expressed on cholinergic interneurons (CINs) and GABA neurons in the NAc. In alcohol dependent rodents, KORs are hypersensitized. It is theorized that this hypersensitization contributes to EtOH seeking behavior. In contrast, aerobic exercise desensitizes the KORs. Given the high degree of pharmacological overlap between opioid receptors, it is also hypothesized that EtOH and aerobic exercise may have effects on MORs and DORs as well. Here, it is investigated whether a routine of voluntary aerobic exercise decreases EtOH induced changes to KOR modulation of dopamine (DA) release in the nucleus accumbens (NAc) along with possible mechanisms through which this might occur. The responsiveness of MORs and DORs in EtOH dependence, and how aerobic exercise modulates those effects is also investigated. Exercise attenuated EtOH induced hypersensitization of KORs in the NAc. Exercise decreases expression of KORs, which may account for the changes in KOR sensitization. The MOR agonist DAMGO decreased DA reuptake ex vivo, but not signal amplitude while DOR agonist DPDPE had no effect on either reuptake or signal amplitude. Overall, dependent animals that were allowed to exercise, consumed less EtOH in a drinking in the dark model. These data suggest that exercise is a useful adjunct to AUD recovery protocols, and that its effects are likely mediated by KORs. The findings related to MORs and DORs suggest that MORs, but not DORs, may act through acetyl choline receptors to modulate DA reuptake in the NAc, however much more work is needed to characterize this effect.

Animals respond to changes in the environment and internal states to modify their behavior. The basal ganglia, including the striatum contribute to action selection by integrating sensory, motor and reward information. Therefore, dysregulation of striatal function is common in many neuropsychiatric disorders, including Parkinson’s disease, Huntington disease, schizophrenia, and addiction. Here, using fiber photometry, pharmacology, and behavioral approaches in transgenic mice, I explored the cellular and circuit mechanisms underlying key striatal functions. In Chapter 1, I begin by presenting the existing literature on the anatomy and physiology of the striatum. Next, I review the important functions of the striatum. Within this general review, I highlight the specific roles that striatal (DA) and acetylcholine (ACh) play in striatal circuitry and function. In Chapter 2, I demonstrate the naturally evoked ACh dip has a DA component and a non-DA component. Specifically, I show that DA via cholinergic DA D2 receptors (D2Rs) modulate the length of the ACh dip and rebound ACh levels following the dip. In addition, I show that DA coordinates the activity between DA and ACh during behavior. Finally, I present data that supports a role for ACh in motivated behavior. In Chapter 3, I show that cholinergic D2Rs are not necessary for reward learning but do facilitate reversal learning in a probabilistic choice task. In addition, I show that changes in DA and ACh levels contribute to reversal learning in a probabilistic choice task. Finally, in Chapter 4, I discuss the general conclusions and study implications, as well as future directions.

Cognitive flexibility is an important mechanism enabling organisms to adapt to their changing environment. Different brain structures are involved in this complex process. It has been repeatedly shown that the striatum is one of the key structures controlling cognitive flexibility. Striatum receives rich input from different brain regions while its output is rather uniform. Striatal functions and signalling are greatly modulated by dopamine and acetylcholine. A number of studies have shown involvement of striatal acetylcholine and its receptors in the control of cognitive flexibility but very little is known about the role of M4 muscarinic acetylcholine receptors. These receptors are inhibitory, and they have been shown to induce long-term depression in striatal medium spiny neurons, therefore opposing the action of the dopamine D1 receptors. We hypothesize that the inhibitory effect of M4 muscarinic acetylcholine receptors may supress spiny projection neurons coding for outdated and no longer effective behavioural strategy and thus they may be necessary for the flexible change of behaviour. In the present thesis, I investigated the effects of pharmacological antagonism of M4 receptors on cognitive flexibility of mice tested in a simple reversal learning paradigm. Key words: striatum; cholinergic...

Purpose: Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. The purpose of this project is to quantify ACh release from isolated murine splenocytes when challenged with different mediators that stimulate T cells in this pathway. Methods:Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.01 - 5 micrograms/ml (0.068 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine (0.5 mM) to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours. Splenocytes were challenged with saline (control) or 1 mM (-) isoproterenol for 4 and 24 hours in the next set of experiments, and ACh in the medium was quantified. We also evaluated separate and combined effects of isoproterenol and activation of T cells with CD3 and CD28 antibodies on ACh release. Results:Correlation coefficients (R2) indicate linearity for ACh and Ch in culture media in the calibration range. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated in cell culture media for 4 and 24 hours, with and without splenocytes, showed a small but statistically significant loss of ACh after 24 h compared to 0 time media controls. However, the average loss of ACh was less than 10% and was not affected by the presence of splenocytes, suggesting that it was due to chemical hydrolysis. Incubation for 4 hr with and without splenocytes did not affect recovery of ACh. Treatment of splenocytes with isoproterenol for 4 hours did not cause significant release of ACh. However, significant release of ACh was detected after 24 hours exposure to isoproterenol or T cell activation. Media from untreated splenocytes had an ACh concentration of 0.14 +/- 0.07 mcg/mL. Isoproterenol treated had 0.28 +/- 0.14 mcg/mL, T-cell activated had 0.32 +/- 0.17 mg/mL, and isoproterenol + T-cell activation had 0.47 +/- 0.16 mcg/mL. Using a 1-way analysis of variance, statistically significant differences were detected between each of these groups. Conclusion: The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway in isolated splenocytes. Statistically significant differences in ACh release between control splenocytes and those treated with isoproterenol and T-cell activation can be detected. Quantitative investigation of this pathway helps provide an improved understanding of ACh dynamics as a mediator released from leukocytes. Further studies using this model and methodology will provide novel insights into cholinergic anti-inflammatory mechanisms and other immunomodulatory actions of non-neuronal ACh.

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