Imaging second messenger dynamics in developing neural circuits

Abstract

A characteristic feature of developing neural circuits is that they are spontaneously active. There are several examples, including the retina, spinal cord, and hippocampus, where spontaneous activity is highly correlated among neighboring cells, with large depolarizing events occurring with a periodicity on the order of minutes. One likely mechanism by which neurons can "decode" these slow oscillations is through activation of second messenger cascades that either influence transcriptional activity or drive posttranslational modifications. Here, we describe recent experiments where imaging has been used to characterize slow oscillations in the cAMP/PKA second messenger cascade in retinal neurons. We review the latest techniques in imaging this specific second messenger cascade, its intimate relationship with changes in intracellular calcium concentration, and several hypotheses regarding its role in neurodevelopment.

Figure 1. Schematics of genetically encoded, FRET based cAMP level and PKA activity sensors

A. LEFT: Schematic of the PKA-based cAMP sensors. CFP and YFP are C-terminal fusions to the regulatory and catalytic subunits of PKA (Lissandron, 2005). MIDDLE: Fluorescence image of a dissociated retinal neuron expressing PKA based cAMP sensor. RIGHT: Time course of F_CFP (blue), F_YFP (yellow), and the FRET ratio (red) of F_YFP/F_CFP for the PKA-based cAMP sensor during application of both the adenylyl cyclase activator forskolin (10µM) and phosphodiesterase inhibitor IBMX (100µM). Bar represents time of drug applications. All ratios are corrected for CFP bleedthrough into YFP channel and differential bleaching of the two fluorophores. Elevation of cAMP leads to decreases in FRET ratio. Ratio trace is inverted to show increases in cAMP as upward deflections. B. LEFT: Schematics of EPAC-based sensors: Different truncations of the exchange protein activated by CAMP (Epac) are sandwiched between a CFP/YFP FRET pair. The different variations have different binding affinities for cAMP (Ponsioen, 2004). MIDDLE: Fluorescence image of a dissociated retinal neuron expressing ICUE2. RIGHT: Time course of F_CFP (blue), F_YFP (yellow), and the FRET ratio (red) of F_YFP/F_CFP for ICUE2 where elevation of cAMP leads to decreases in FRET ratio. Ratio trace is inverted to show increases in cAMP as upward deflections. C. LEFT: Schematic of the PKA activity reporter, AKAR3. Modified from Zhang, 2001. A-kinase-activity reporter (AKAR) is a fusion of CFP and YFP to either a FHA1 or 14-3-3 phosphobinding region and PKA target substrate. Different variations have varying dynamic range and dephosphorylation rates. MIDDLE: Fluorescence image of a dissociated retinal neuron expressing AKAR2.2. RIGHT: Time course of F_CFP (blue), F_YFP (yellow), and the FRET ratio (red) of F_YFP/F_CFP for the AKAR2.2 where elevation of PKA activity leads to increases in FRET ratio. MIDDLE and RIGHT, Modified from Dunn, Wang et al. 2006

Fig. 2. Spontaneous changes in cAMP levels and PKA activity in retinal ganglion cells during retinal waves

A. Time course of F_CFP (blue), F_YFP (yellow) recorded simultaneously and averaged over the cell body of a RGC expressing ICUE2. The ICUE2 FRET ratio is computed as F_YFP/F_CFP, inverted to show cAMP increases as upward deflections, corrected for CFP bleedthrough into YFP channel, and corrected for differential bleaching of CFP and YFP. B. Time course of F_CFP (blue), F_YFP (yellow) recorded simultaneously and averaged over the cell body of a RGC expressing AKAR2.2. The ratio is computed as F_YFP/F_CFP, corrected for CFP bleedthrough into YFP channel and corrected for differential bleaching. From Dunn, Wang et al., 2006.

Figure 3. Spontaneous oscillations in cAMP are driven by certain patterns of calcium spikes in spinal neurons

A. Surface plot images show a spontaneous, transient increase in cAMP concentration in an immature spinal neuron loaded with the ratiometric dye, FlCRhR. Color scale represents the 520 nm/580 nm fluorescence ratio. (s) soma, (a) axon, (gc) growth cone. Scale bar, 10 µm. B-D. Fluorescence ratio traces of F₅₂₀/F₅₈₀ in a spinal neuron loaded with FlCRhR show cAMP response to induced calcium influx. Arrows indicate stimulated Ca²⁺ spikes. (A) single spikes, (B) three spikes, (C) five spikes. From Gorbunova and Spitzer, 2002.