|Home | About | Journals | Submit | Contact Us | Français|
During development, spontaneous retinal activity is required for the refinement of connections throughout the developing visual system. However, the cellular mechanisms by which activity causes long-term changes in axonal morphology are not yet identified. One likely candidate is activation of second messenger pathways. Here we use live-cell indicators to observe evoked and spontaneous changes in cAMP levels and PKA activity in retinal neurons. We observed an immediate increase in cAMP levels and PKA activity to short applications of solutions containing forskolin or high potassium concentration, indicating this second messenger pathway can be activated quickly in response to neural activity. Expression of a PKA indicator in neonatal rat retinal explants revealed spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. These findings demonstrate for the first time that spontaneous activity in developing circuits induces oscillations in kinase activity.
Neuronal activity is linked to long-term changes in cells, such as activity-dependent expression of genes, by activation of second messenger pathways (West et al., 2002). Though several such pathways have been identified, there is a short list of second messengers, such as calcium, cAMP, cGMP, DAG, and IP3 to mediate these effects. Therefore the specificity of the activated pathways is not likely to be determined by concentrations of second messengers but rather by their spatial localization and dynamic properties. Oscillations have been detected in both calcium and cAMP concentrations, and there is growing evidence that these oscillations are “tuned” to drive particular cellular events (Zaccolo and Pozzan, 2003; Spitzer et al., 2004; Fields et al., 2005; Dyachok et al., 2006).
The developing visual system is a model for the study of how activity induces long lasting changes in neurons. Prior to the development of vision, spontaneous activity in the retina, called retinal waves, is critical for the normal refinement of retinal ganglion cell axonal projections to both the superior colliculus and the lateral geniculate nucleus of the thalamus (for reviews, see (Grubb and Thompson, 2004; Ruthazer and Cline, 2004; Torborg and Feller, 2005). In addition, knockout mice lacking adenylate cyclase (AC1) exhibit defects in retinotopic and eye-specific refinement (Ravary et al., 2003; Plas et al., 2004), indicating that activation of the cAMP/PKA pathway is critical for refinement of retinal projections. This hypothesis is supported by the observations that a knockdown of the transcription factor CREB, a known target of PKA, leads to a reduction in eye-specific segregation (Pham et al., 2001). Although spontaneous patterned activity and the cAMP/PKA pathway are important for refinement of retinal projections, how retinal waves affect the cAMP/PKA second messenger cascades either in RGCs or their targets remains unknown.
Here we used two genetically encoded fluorescence-based indicators to monitor the cAMP/PKA pathway in both dissociated retinal neurons and the intact retina. The first indicator is a PKA based cAMP sensor (CS) (Zaccolo et al., 2002). The second is an A-Kinase Activity Reporter, AKAR2.2 (Zhang et al., 2001; Zhang et al., 2005), which undergoes changes in its fluorescent properties in response to phosphorylation by PKA. To demonstrate the utility of CS and AKAR2.2, we compared their kinetics of response to direct activation of adenylate cyclase and solutions containing high concentrations of potassium. We also monitored spontaneous oscillations in PKA activity levels in the intact retina and determined their relationship to retinal waves.
Dissociated neurons and glia from postnatal day 1 (P1) rat retina were plated on coverslips, with a subset of cultures placed on superior colliculus cultures from litter mates plated the day before as described previously (Colicos et al., 2004, modified from Meyer-Franke et al., 1995; Goldberg et al., 2002a). During imaging experiments, cultures were perfused continuously with external media (5 mM KCl, 123 mM NaCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH was adjusted with NaOH to 7.3). Transfection of cultured neurons was performed by electroporation using an exponentially decaying pulse.
Acutely isolated whole retinas were mounted on filter paper precoated with 100 μg/μL ploy-D-lysine and 50 μg/μL laminin. They were incubated in dissection solution (described in Colicos et al., 2004) containing plasmid DNA (concentration ~ 0.2 μg/μL) for 10 min and then electroporated with two square wave pulses with an electric field strength of 6.75V/mm, then allowed to recover for 10 min in dissection solution. They were then cultured in the serum-free culture media (also described in Colicos et al.,2004), with Neurobasal media replaced by Neurobasal-A media (Invitrogen). Before imaging, electroporated retinas were transferred to media without forskolin. For recordings, explants are placed ganglion cell layer up in a temperature-controlled chamber (30°C, Warner Systems), and perfused continuously with artificial cerebrospinal fluid (ACSF: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl2, 1.0 mM K2HPO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, 11 mM D-glucose) bubbled with 95% O2/5% CO2. Explant health was assayed using whole cell recording to measure resting potentials, action potentials and spontaneous depolarizations.
Live imaging of FRET indicators was performed on an upright Zeiss Axioskop 2, using a 60x AchroPlan water immersion lens. Individual FRET channel detection was accomplished by using a Dual-View image splitter with appropriate yellow and cyan channel filters. Images were captured on a Photometrics CoolSnap HQ CCD camera and analyzed in MetaMorph v6.3. Background fluorescence was subtracted from both channels and CFP bleedthrough into the YFP channel was corrected (FYFP = FFRET 0.51* FCFP)(Gordon et al., 1998; van Rheenen et al., 2004).
High potassium solution was the same as external media with 105 mM KCl substituted for an equivalent amount of NaCl. Short applications of high K+ external solution were delivered through a glass electrode (approximately 4μm tip size, located less than 20 microns from transfected neuron) using a PV830 Pneumatic PicoPump at 2–10 psi, 500 msec pulse duration at 1 Hz to dissociated RGCs, and 50 msec pulse duration at 4 Hz to RGCs in retinal explants.
Whole-cell recordings from retinal explants were made using an Axopatch 200B amplifier and pClamp6 software (Axon Instruments). The intracellular solution consisted of 98.3 mM potassium-gluconate, 1.7 mM KCl, 0.6 mM EGTA, 5 mM MgCl2, 2 mM Na2-ATP, 0.3 mM GTP, 40 mM HEPES, pH 7.25 with KOH. The calculated ECl was −60 mV with the ACSF. All pharmacological agents were purchased from Sigma.
To monitor cAMP levels and PKA activity in living mammalian neurons, we utilized two different genetically encoded indicators dependent upon fluorescence energy resonance transfer (FRET). The first, which we will refer to as the cAMP sensor (CS), consists of CFP fused to a regulatory subunit and YFP fused to the catalytic subunit of protein kinase A (Zaccolo et al., 2002). An increase in cAMP concentration results the dissociation of the regulatory and catalytic subunits, resulting in a decrease in FRET efficiency. Therefore, for CS, increases in cAMP levels are reported as the changes in the ratio of the emission fluorescence of CFP to emission fluorescence of YFP (FCFP/FYFP). The second indicator, AKAR2.2 (Zhang et al., 2005) consists of a fusion of CFP, a phosphothreonine-binding domain (FHA1), a PKA consensus phosphorylation target, and the YFP variant citrine (Griesbeck et al., 2001). When PKA is activated, the catalytic subunit of PKA phosphorylates the target in AKAR2.2, causing a conformational change that increases FRET efficiency. Therefore, increases in PKA activity are reported as changes in the ratio FYFP/FCFP for this indicator.
To characterize the physiological responses of both CS and AKAR2.2, the indicators were expressed in dissociated retinal neurons. Retinas from P0–P1 rats were dissociated and electroporated with plasmids encoding either AKAR2.2 or CS. After 2–4 days in vitro, the indicators were strongly expressed throughout the cell and formed functional FRET pairs as assayed with a partial photobleach assay (See Supplemental Figure).
We next determined the response of the indicators to direct elevation of intracellular levels of cAMP. Neurons expressing either AKAR2.2 or CS exhibited immediate responses to bath application of the adenylate cyclase activator forskolin (10 μM) alone, or in combination with the phosphodiesterase inhibitor IBMX (100 μM) to strongly stimulate the cAMP/PKA pathway (Figures 1A and 1B). The response to forskolin alone was significantly smaller than the response to the combination of forskolin and IBMX for both AKAR2.2 (ΔRFSK=0.044±0.014, n=10 vs. ΔRFSK+IBMX=0.118±0.040, n=8, p<0.01) and the CS (ΔRFSK=0.012±0.005, n=10 vs. ΔRFSK+IBMX=0.099±0.048, n=8, p<0.01), demonstrating that indicator response was correlated with the strength of activation of the cAMP/PKA pathway (Figure 1D). We found no correlation between the starting ratio and the size of the response, indicating a lack of saturation of the indicator while in the basal state or with forskolin alone (data not shown).
One notable difference between the two indicators was that the response of CS was much slower than that of AKAR2.2. Possible explanations for the slower CS response include the following: 1) Activation of a small fraction of endogenous PKA may be sufficient to saturate AKAR, whereas saturation of the CS requires full dissociation of both endogenous and exogenous PKA; 2) Endogenous PKA may be anchored in close proximity to adenylate cyclase whereas the exogenous CS is either anchored some distance away or diffusible throughout the cell (Zhang et al., 2005); 3) cAMP may activate PKA kinase activity before completely dissociating the regulatory and catalytic subunits (Yang et al., 1995; Nikolaev et al., 2004). In summary, we conclude that in our system, AKAR2.2 functioned as a more sensitive reporter for monitoring brief activation of the cAMP/PKA pathway.
We next investigated whether the cAMP/PKA pathway is activated by depolarization in dissociated retinal neurons. Short applications of external solution containing high concentrations of KCl to neurons expressing AKAR2.2 led to immediate and reproducible changes in FRET efficiency in a subset of transfected cells (Figure 1C, ΔR=0.013±0.006, n=14/23 neurons). The amplitude of the FRET ratio changes measured in response to short applications of high-K+ solution were significantly smaller than those measured for bath application of forskolin + IBMX but comparable to bath application of forskolin alone (Figure 1E). These findings indicate that in some cells, the cAMP/PKA pathway is activated by depolarization, likely through a calcium-dependent adenylate cyclase (Ferguson and Storm, 2004).
We introduced the AKAR2.2 plasmid into neonatal rat retinal explants using electroporation. Whole cell current clamp recordings from individual RGCs exhibited periodic spontaneous depolarizations, consistent with the persistence of retinal waves after 48 hours in culture (interevent interval: 1.68 ± 0.89 min, n=29 RGCs, 26 retina explants). After 20 hours, AKAR2.2 was strongly expressed in RGCs, readily identified by the appearance of axons at the inner retinal surface (Figure 2A). In a subset of transfected RGCs, spontaneous decreases in FCFP simultaneous with increases in FYFP were detected, indicating spontaneous oscillations in PKA activity (ΔR=0.025 ± 0.015, n = 12/114 RGCs, Figure 2B). The magnitude of FRET ratio changes were similar in amplitude and kinetics (rise time measured trough to peak: τspontaneous=20.5 ± 4.6, n=12; τevoked=18.2±10.3, n=14, p>0.5) to those induced by acute application of potassium in dissociated retinal neurons (Figure 2E), suggesting that retinal waves may lead to cAMP transients via depolarization.
Several observations indicate that spontaneous oscillations in PKA activity were correlated with retinal waves. First, the spontaneous oscillations in PKA occurred with a periodicity similar to that of spontaneous depolarizations associated with retinal waves (2.05 ± 0.94 min, n=12, peak-to-peak interval, Figure 2C, p=0.173). Second, spontaneous oscillations in PKA activity were blocked by bath application of the nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine (10–20μM, DHβE), which reliably blocks retinal waves (n = 5, Figure 2D). Third, in one instance where we observed two transfected RGCs in the same field of view, we observed correlated spontaneous oscillations in PKA activity.
To determine whether the spontaneous PKA transients exhibit a strong temporal relationship with retinal waves, we conducted simultaneous AKAR imaging and whole cell voltage clamp recordings from a nearby cell to detect the compound synaptic currents associated with retinal waves (n=38 waves from 4 cells, Figure 3A). We computed the “wave-triggered” AKAR response by averaging the segment of the FRET ratio for AKAR2.2 from 5 seconds preceding to 40 seconds following the compound PSC. We found that, on average, the FRET ratio of AKAR2.2 increased within 20 seconds following a barrage of synaptic current (Figure 3B). For comparison, we computed the wave-triggered fura-2 response using simultaneous whole cell recording and calcium imaging. In contrast to the AKAR2.2 response, the calcium response in nearby cells occurred within a few seconds following a compound synaptic event (Figures 4C and D, n= 21 waves from 5 cells). This is consistent with previous measurements that indicate increases in intracellular calcium associated with retinal waves are driven by influx through voltage–gated channels during spontaneous depolarizations (Penn et al., 1998). Combined with the observation that blockade of retinal waves also blocks PKA activity, the increase in PKA activity following a retinal wave is consistent with the hypothesis that retinal waves drive PKA activity transients in a time-locked manner.
It is important to note that in PKA also plays a role in the generation of retinal waves. Retinal waves are generated by a network of starburst amacrine cells (Zheng et al., 2004; Zheng et al., 2006). Bath application of the PKA inhibitor Rp-cAMP blocks retinal waves (Stellwagen et al., 1999) and cAMP levels in starburst amacrine cells influences the frequency of retinal waves (Zheng et al., 2006). However, the data does not have implications for wave generation because we recorded PKA activity exclusively in RGCs, which are not thought to participate in the generation of retinal waves (T. Mon, L. Gabn, and Z. J. Zhou, Society for Neuroscience, 2005). Whether cAMP levels or PKA activity spontaneously oscillate in developing starburst amacrine cells is yet to be determined.
Why do we detect depolarization-induced PKA transients in only a subset of RGCs? In transfected RGCs that did not exhibit spontaneous oscillations in the FRET ratio, we measured substantial ratio changes in response to bath application of forskolin (10μM, ΔR = 0.091 ± 0.058, N = 5/5 neurons), demonstrating that the absence of oscillations is not due to a lack of functional AKAR2.2. In addition, we also found that 52% of non-oscillating cells had detectable FRET ratio changes in response to strong depolarization via short applications of KCl (105mM, ΔR = 0.011 ± .007, N = 12/23 neurons). These evoked FRET ratio changes and risetimes are similar to those due to spontaneous PKA oscillations. In separate experiments, similar KCl applications induced calcium transients larger in amplitude than the spontaneous calcium transients induced by retinal waves (ΔF/FKCl: ΔF/Fspontaneous = 2.22 ± 0.90, N = 9, data not shown). Based on these observations, we conclude that under our experimental conditions, AKAR detected the RGCs that exhibited the strongest depolarizations during waves.
We demonstrate that live indicators for cAMP levels and PKA activity can reliably measure activation of this second messenger pathway in neurons in the intact circuitry of the developing retina. In addition, our findings correlate spontaneous neural activity in developing networks with temporal oscillations in kinase activity. These findings have implications for identifying the plasticity mechanisms that convert spontaneous firing patterns into long-term changes in cellular morphology in the visual system.
Temporal coding of second messenger pathway activation may be critical for the onset and timing of downstream development processes that are driven by spontaneous neural activity (Berridge et al., 2000; Spitzer et al., 2004). For example, spontaneous activity in the developing spinal cord is frequency-tuned to modulate the expression of proteins critical for pathfinding of motor neurons (Hanson and Landmesser, 2004).
Whether periodic activation of retinal ganglion cells is required for normal development of the visual system is not yet determined. Though it is well established that spontaneous retinal activity patterns are critical for the refinement of retinal projections to central targets, the precise role of correlated activity patterns is the subject of debate. In a knockout mouse that has uncorrelated retinal ganglion cell firing instead of retinal waves (Bansal et al., 2000; McLaughlin et al., 2003), it has recently been shown that several features of both retinocollicular projections (McLaughlin et al., 2003; Mrsic-Flogel et al., 2005) and retinogeniculate projections (Rossi et al., 2001; Muir-Robinson et al., 2002; Grubb et al., 2003) were altered. In contrast, both pharmacological manipulations of activity patterns in ferret and a different knockout mouse with altered retinal patterns develop normal refinement of retinal projections (Stellwagen and Shatz, 2002; Huberman et al., 2003; Torborg et al., 2005). By comparing the retinal firing patterns that drive or permit segregation to those that do not, we identified several candidate activity features that may be essential for this refinement process (Torborg et al., 2005). The primary hypothesis that arose from these studies is that RGCs must fire high frequency bursts with a slow periodicity for normal eye-specific refinement. Such an activity pattern would strongly activate both calcium-dependent and cAMP-dependent second messenger cascades.
There is growing evidence that the cAMP/PKA pathway may be a primary component of refinement of retinal projections. AC1−/− mice lack eye-specific layers and have poor retinotopic refinement (Ravary et al., 2003; Plas et al., 2004). Recent evidence indicates that adenylate cyclase is acting presynaptically, perhaps by regulating the response of RGC growth cones to guidance molecules (Nicol et al., 2006) or presynaptic function (Lu et al., 2006). Indeed, periodic activation of isolated RGCs and the spontaneous activity in retinal explants profoundly increases the rate of axonal outgrowth in response to BDNF (Goldberg et al., 2002b). These findings indicate that the endogenous pattern of retinal activity regulated axon outgrowth via this second messenger cascade, possibly by increasing the surface expression of neurotrophin receptors (Meyer-Franke et al., 1998; Nagappan and Lu, 2005).
In conclusion, we have observed that retinal waves drive oscillations in PKA activity of RGCs. Visualization of second messenger cascade kinetics is an important step in elucidating the link between activity and the mechanisms by which it influences development of neural circuits.
A/B. Fluorescence image of cultured retinal neuron expressing AKAR2.2 (A) and the cAMP sensor (B). Scale bar is 20μm.
C. Summary of partial acceptor photobleaching experiments. Five images before and after photobleach are averaged to measure the change in intensity.