Investigating the role of transient adenosine to modulate neurotransmitters through electrochemical and fluorescence imaging techniques

Adenosine is a ubiquitously present local neuromodulator in the brain, generated through the enzymatic hydrolysis of adenosine triphosphate (ATP), and it plays a dual role as a neuroprotector. It is crucial for maintaining neuronal homeostasis and rapidly responding to cellular stress. Adenosine’s regulatory actions are mediated via GPCR receptors, primarily A1 and A2A receptors, which exert opposing effects on neurotransmitter release.
Fast scan cyclic voltammetry (FSCV) allows for sub-second monitoring of adenosine and its neuromodulatory effects on neurotransmitter release. However, while FSCV offers exceptional temporal resolution, it is limited by its lack of spatial resolution, selectivity, and inability to detect non-electroactive neurotransmitters. This dissertation investigates the integration of FSCV with fluorescence imaging (iGluSnFR3) to enable real-time analysis of adenosine's modulation of neurotransmitters.
Chapter 1 introduces adenosine, fast scan cyclic voltammetry (FSCV), and various methods for measuring adenosine levels. It highlights techniques such as micro-dialysis, biosensors, and FSCV, focusing on their ability to capture adenosine dynamics on a rapid time scale. The strengths and limitations of these approaches are analyzed, particularly in the context of fast-acting adenosine. Studies involving electrically stimulated, spontaneously released, and mechanically stimulated adenosine are reviewed.
Chapter 2 focuses on transient adenosine neuromodulation of serotonergic neurons in the dorsal raphe nuclei (DRN), using FSCV. Exogenous adenosine was found to inhibit serotonin release by more than 50% within the first 20 seconds, with recovery occurring alongside adenosine clearance in wild-type (WT) slices. In contrast, this inhibitory effect was limited to the first 10 seconds in A1KO slices. Importantly, A1, A2A, and A3 receptors did not directly contribute to adenosine's inhibitory effect. Instead, adenosine inhibited serotonergic neurons via densely expressed 5HT1A autoreceptors.
Chapter 3 discusses the integration of multiplexing fast scan cyclic voltammetry (FSCV) and fluorescence imaging (iGluSnFR3) to enable simultaneous monitoring of multiple analytes and their interactions in real time. While FSCV is specifically designed to measure electroactive molecules, the addition of genetically encoded fluorescence sensors enhances the ability to monitor multiple electroactive and non-electroactive molecules simultaneously with improved specificity and spatial resolution. The study revealed an inverse correlation between electrically stimulated dopamine release and glutamate levels in the caudate putamen. Furthermore, adenosine was found to inhibit the release of both dopamine and glutamate, with this inhibition restricted to a 250 µm radius. Pharmacological studies using A1 receptor antagonists confirmed that this inhibitory effect of adenosine is mediated through A1 receptor activation.
Chapter 4 focuses on the application of multiplexing techniques to simultaneously study the effects of transient ischemia on neurotransmitters such as dopamine and glutamate. Deprivation of glucose and oxygen during oxygen-glucose deprivation (OGD) depletes cytosolic ATP, leading to a rapid rise in extracellular adenosine, which in turn modulates the release of other neurotransmitters. During OGD, both electrically stimulated dopamine and glutamate release were significantly reduced, and this suppression persisted for the first 30 minutes of reperfusion. While dopamine levels gradually recovered to baseline over the course of 120 minutes of reperfusion, glutamate levels remained at OGD-induced levels. Using transgenic mice (A2AKO) and pharmacological interventions (NMDA receptor antagonist and DPCPX), the study highlighted the critical neuroprotective roles of A1 and A2A receptors during OGD. Specifically, A1 receptor activation prevents excessive glutamate accumulation in extracellular spaces, thereby reducing excitotoxicity and minimizing neuronal damage.
Overall, this dissertation explored adenosine neuromodulation on other neurotransmitters by integrating fast scan cyclic voltammetry (FSCV) and fluorescence sensors. This integration allowed for simultaneous monitoring of both electroactive and non-electroactive molecules, along with their interactions, in real time with enhanced spatial resolution. Adenosine was shown to modulate dopamine and glutamate via A1 receptors in the striatum, with this effect being localized within a 250 µm range. Similarly, adenosine demonstrated neuromodulation of serotonergic neurons in the midbrain through densely expressed 5HT1A autoreceptors.
The combination of FSCV and fluorescence highlights the potential of multiplexing techniques to study complex neuromodulatory interactions, providing a powerful tool for understanding the dynamics of neurotransmitter systems in both normal and pathological conditions.