Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist

Abstract

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Feedback Control of Neuronal Excitability and Epileptiform Bursting using a Photocaged Adenosine A1Agonist

“…The observed third order binding properties (Hill coefficient 3) imply that there is only a narrow therapeutic window for A1R medication: as an example, the difference between 20% and 80% effect is from 28 to 70 nM, while for a first order kinetics that range would be 10 to 176 nM. However, the fast activation (below the time resolution of our measurements, so at least in the range of seconds) and the slow recovery (time constant around 22 mins, determined in Craey et al [14]), make the A1Rs ideally suited for pulsed application and modulation of the CPA concentration.…”

“…DPCPX and DMSO were purchased from Sigma-Aldrich (Missouri, USA). cCPA was synthesized and provided by the Laboratory of Medicinal Chemistry (Ghent University, Belgium) [14]. Stock solutions of CPA (1, 30, 40, 50, 100 and 1000 µM) and of cCPA (3 mM) were made in DMSO and stored at -20 • C. The final perfusion solution always contained 0.1% DMSO.…”

“…In a previous study the recovery time constant (1/Poff) was determined to be 22±1 mins [14] The CPA-concentration dependent EPSP suppression was determined by perfusing the slice with increasing CPA concentrations (1, 30, 50 and 1000nM) in a cumulative sequence starting from baseline. At least 10 minutes after switching the perfusion solution to a higher CPA concentration, three SRcurves were recorded that related EPSP amplitudes to thirteen stimulus intensities (500 to 3500 mV in steps of 250 mV) between threshold and saturation (Figure 2A).…”

“…Recently a photocaged derivative of the A1R agonist N 6 -cyclopentyladenosine (CPA) was synthetized by conjugating its 6-amino group to 7-(diethylamino)-4-(hydroxymethyl)coumarin (DEACM) [14]. The resulting caged CPA (cCPA) had a more than 1000-fold decreased affinity for the A1R and was irreversibly released by 405 nm light.…”

“…Micromolar concentrations of cCPA did not affect the FPs while flashes of 405 nm light resulted in fast decrease and slow recovery of FP amplitude and thus induced a transient reduction of neurotransmission. Based on these observations an on-off closed-loop control paradigm was implemented, where repeatedly evoked FPs were monitored and a hardware system delivered light flashes that released CPA on pre-set level crossing [14]. The system was able to stabilize the FP at an amplitude that was 50% of its baseline value.…”