Regional Variations of Spontaneous, Transient Adenosine Release in Brain Slices

Abstract

Transient adenosine signaling has been recently discovered in vivo, where the concentration is on average 180 nM and the duration only 3-4 s. In order to rapidly screen different brain regions and mechanisms of formation and regulation, here we develop a rat brain slice model to study adenosine transients. The frequency, concentration, and duration of transient adenosine events were compared in the prefrontal cortex (PFC), hippocampus (CA1), and thalamus. Adenosine transients in the PFC were similar to those in vivo, with a concentration of 160 ± 10 nM, and occurred frequently, averaging one every 50 ± 5 s. In the thalamus, transients were infrequent, occurring every 280 ± 40 s, and lower concentration (110 ± 10 nM), but lasted twice as long as in the PFC. In the hippocampus, adenosine transients were less frequent than those in the PFC, occurring every 79 ± 7 s, but the average concentration (240 ± 20 nM) was significantly higher. Adenosine transients are largely maintained after applying 200 nM tetrodotoxin, implying they are not activity dependent. The response to adenosine A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) differed by region; DPCPX had no significant effects in the PFC, but increased the average transient concentration in the thalamus and both the transient frequency and concentration in the hippocampus. Thus, the amount of adenosine available to activate receptors, and the ability to upregulate adenosine signaling with DPCPX, varies by brain region. This is an important consideration for designing treatments that modulate adenosine in order to cause neuroprotective effects.

Keywords: A1 receptor; Adenosine; brain slices; electrochemistry; fast-scan cyclic voltammetry.

Figure 1

Adenosine transient in the hippocampus. The color plot shows all data, with scanned voltage on the y-axis, time on the x-axis, and current depicted in color. A horizontal slice in the 3D color plot results in the i vs t plot (above) and a vertical slice gives the cyclic voltammogram at a given time (inset).

Figure 2

Concentration traces (top) and 3D color plots (bottom) for the (A) PFC, (B) thalamus, and (C) CA1. Adenosine transients are marked with stars in the concentration traces, which are all scaled the same to highlight the variety of concentrations in each region.

Figure 3

Differences in (A) interevent time (K–W test, p < 0.0001), (B) concentration (K–W test, p < 0.0001), and (C) duration (ANOVA, p < 0.0001) between the prefrontal cortex, thalamus, and CA1 region of the hippocampus. All are n = 8 slices.

Figure 4

Effect of TTX. (A) 200 μM TTX eliminated stimulated dopamine release in caudate of a sagittal slice. (B) In the same slices, but in the hippocampus, there was no effect of TTX on the mean interevent time or concentration, but there was a significant effect of TTX on the interevent time distribution. K–S test, **** p <0.0001. (C) There was no significant effect of TTX on the concentration of adenosine transients in the CA1 (K–S test).

Figure 5

Concentration changes (top) and 3D color plots (bottom) for the (A) PFC, (B) thalamus, and (C) CA1 when treated with 100 nM DPCPX. Verified adenosine transients are marked with a star in the concentration traces.

Figure 6

Cumulative distributions of the interevent times (top) and concentrations (bottom) of adenosine transients in the PFC (left), thalamus (middle), and CA1 (right). Control is black, and treated with 100 nM DPCPX is red. A K–S test was performed for all graphs, and significant differences are marked by asterisks, **p < 0.01, ****p < 0.0001.