Dopaminergic Tone Persistently Regulates Voltage-gated Ion Current Densities Through the D1R-PKA Axis, RNA Polymerase II Transcription, RNAi, mTORC1, and Translation

Long-term intrinsic and synaptic plasticity must be coordinated to ensure stability and flexibility in neuronal circuits. Coordination might be achieved through shared transduction components. Dopamine (DA) is a well-established participant in many forms of long-term synaptic plasticity. Recent work indicates that DA is also involved in both activity-dependent and -independent forms of long-term intrinsic plasticity. We previously examined DA-enabled long-term intrinsic plasticity in a single identified neuron. The lateral pyloric (LP) neuron is a component of the pyloric network in the crustacean stomatogastric nervous system (STNS). LP expresses type 1 DA receptors (D1Rs). A 1 h bath application of 5 nM DA followed by washout produced a significant increase in the maximal conductance (Gmax ) of the LP transient potassium current (IA ) that peaked ∼4 h after the start of DA application; furthermore, if a change in neuronal activity accompanied the DA application, then a persistent increase in the LP hyperpolarization activated current (Ih ) was also observed. Here, we repeated these experiments with pharmacological and peptide inhibitors to determine the cellular processes and signaling proteins involved. We discovered that the persistent, DA-induced activity-independent (IA ) and activity-dependent (Ih ) changes in ionic conductances depended upon many of the same elements that enable long-term synaptic plasticity, including: the D1R-protein kinase A (PKA) axis, RNA polymerase II transcription, RNA interference (RNAi), and mechanistic target of rapamycin (mTOR)-dependent translation. We interpret the data to mean that increasing the tonic DA concentration enhances expression of a microRNA(s) (miRs), resulting in increased cap-dependent translation of an unidentified protein(s)

Tonic 5nM DA Stabilizes Neuronal Output by Enabling Bidirectional Activity-Dependent Regulation of the Hyperpolarization Activated Current via PKA and Calcineurin

Volume transmission results in phasic and tonic modulatory signals. The actions of tonic dopamine (DA) at type 1 DA receptors (D1Rs) are largely undefined. Here we show that tonic 5nM DA acts at D1Rs to stabilize neuronal output over minutes by enabling activitydependent regulation of the hyperpolarization activated current (I h). In the presence but not absence of 5nM DA, I h maximal conductance (G max) was adjusted according to changes in slow wave activity in order to maintain spike timing. Our study on the lateral pyloric neuron (LP), which undergoes rhythmic oscillations in membrane potential with depolarized plateaus, demonstrated that incremental, bi-directional changes in plateau duration produced corresponding alterations in LP I hG max when preparations were superfused with saline containing 5nM DA. However, when preparations were superfused with saline alone there was no linear correlation between LP I hGmax and duty cycle. Thus, tonic nM DA modulated the capacity for activity to modulate LP I h G max; this exemplifies metamodulation (modulation of modulation). Pretreatment with the Ca2+-chelator, BAPTA, or the specific PKA inhibitor, PKI, prevented all changes in LP I h in 5nM DA. Calcineurin inhibitors blocked activity-dependent changes enabled by DA and revealed a PKA-mediated, activity-independent enhancement of LP I hG max. These data suggested that tonic 5nM DA produced two simultaneous, PKAdependent effects: a direct increase in LP I h G max and a priming event that permitted calcineurin regulation of LP I h. The latter produced graded reductions in LP I hG max with increasing duty cycles. We also demonstrated that this metamodulation preserved the timing of LP’s first spike when network output was perturbed with bath-applied 4AP. In sum, 5nM DA permits slow wave activity to provide feedback that maintains spike timing, suggesting that one function of low-level, tonic modulation is to stabilize specific features of a dynamic output

Monoaminergic Tone Supports Conductance Correlations and Stabilizes Activity Features in Pattern Generating Neurons of the Lobster, Panulirus interruptus

Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (IA ) and the hyperpolarization activated current (Ih ) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between IA and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC- dependent positive correlation between IA and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between IA and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term

Dopaminergic Tone Regulates Transient Potassium Current Maximal Conductance Through a Translational Mechanism Requiring D1Rs, cAMP/PKA, Erk and mTOR

Background: Dopamine (DA) can produce divergent effects at different time scales. DA has opposing immediate and long-term effects on the transient potassium current (IA) within neurons of the pyloric network, in the Panulirus interruptus stomatogastric ganglion. The lateral pyloric neuron (LP) expresses type 1 DA receptors (D1Rs). A 10 min application of 5-100 μM DA decreases LP IA by producing a decrease in IA maximal conductance (Gmax) and a depolarizing shift in IA voltage dependence through a cAMP-Protein kinase A (PKA) dependent mechanism. Alternatively, a 1 hr application of DA (≥5 nM) generates a persistent (measured 4 hr after DA washout) increase in IA Gmax in the same neuron, through a mechanistic target of rapamycin (mTOR) dependent translational mechanism. We examined the dose, time and protein dependencies of the persistent DA effect. Results: We found that disrupting normal modulatory tone decreased LP IA. Addition of 500 pM-5 nM DA to the saline for 1 hr prevented this decrease, and in the case of a 5 nM DA application, the effect was sustained for \u3e4 hrs after DA removal. To determine if increased cAMP mediated the persistent effect of 5nM DA, we applied the cAMP analog, 8-bromo-cAMP alone or with rapamycin for 1 hr, followed by wash and TEVC. 8-bromo-cAMP induced an increase in IA Gmax, which was blocked by rapamycin. Next we tested the roles of PKA and guanine exchange factor protein activated by cAMP (ePACs) in the DA-induced persistent change in IA using the PKA specific antagonist RpcAMP and the ePAC specific agonist 8-pCPT-2′-O-Me-cAMP. The PKA antagonist blocked the DA induced increases in LP IA Gmax, whereas the ePAC agonist did not induce an increase in LP IA Gmax. Finally we tested whether extracellular signal regulated kinase (Erk) activity was necessary for the persistent effect by co-application of Erk antagonists PD98059 or U0126 with DA. Erk antagonism blocked the DA induced persistent increase in LP IA. Conclusions: These data suggest that dopaminergic tone regulates ion channel density in a concentration and time dependent manner. The D1R- PK

Tonic 5nM DA endows LP I_h with activity dependence.

<p><b>(A) Experimental protocol</b>. Activity was recorded with extracellular electrodes throughout the experiment. Measures of activity were obtained at t = -10min. The diagram shows how these measures were used in conjunction with TEVC to create a recurring voltage step that mimicked slow oscillatory activity at t = -10min. A change in LP burst duration was created by varying the length of the depolarizing step, as shown. Note that cycle period was maintained regardless of the change in burst duration; i.e., a change in burst duration was accompanied by a corresponding change in interburst duration. Duty cycle is defined as burst duration/cycle period; thus, our manipulations altered burst duration and duty cycle to the same extent. At t = -5min TTX was added to the superfusate. At t = 0, LP I_h was measured with TEVC (black asterisk). Afterward, from t = 0 to 10min, 5nM DA was <b>(B)</b> or was not <b>(C)</b> added to the superfusate and TEVC was used to either hold the cell at its initial membrane potential in TTX (-100), or implement a recurrent voltage step (-75 to +50). At t = 10min, LP I_h was again measured with TEVC (black asterisk). The experiment terminated at this point and a single preparation was not used further for additional experiments. <b>(B-C) LP I</b>_<b>h</b><b>activity dependence curves</b>. For every experiment in 5nM DA <b>(B)</b> or saline <b>(C)</b>, the fold-change in LP I_h G_max at t = 10min (i.e. G_max at t = 10÷G_max at t = 0) was plotted against the % change in LP duty cycle and a linear regression was used to fit the data. Each data point on the plots represents one experiment and 51 animals were used to obtain the data shown in the plots; -100 on the <i>x</i>-axis represents experiments in TTX without a recurring step, i.e., complete activity blockade. Each line represents a linear regression analysis and the resulting R² and p values are shown on the graph. Red asterisk indicates that LP I_h G_max was significantly different at-100 and +50 in the presence but not absence of 5nM DA as determined using one-way ANOVAs with Tukey’s multiple comparisons post hoc tests to analyze the-100, 0, and +50 groups (<b>5nM DA</b>: F(2,17) = 8.464; p = 0.0035; <b>saline</b>: F(2,22) = 2.326; p = 0.1236).</p

LP I_h metamodulation is rapid and reversible.

<p><b>(A) The time course for metamodulation in 5nM DA</b>. Experiments measured the fold-change in LP I_h G_max at 1, 2, 5 and 10min, and every 10min thereafter, up to 60min after addition of 5nM DA. The protocol is as follows: At t = -5min TTX was added to the superfusate bathing the preparation diagramed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g001" target="_blank">Fig. 1A</a>. LP I_h was measured at t = 0. The TTX containing superfusate was then supplemented with 5nM DA, and LP I_h was re-measured at the indicated time point. One time point was obtained per experiment for 1,2, and 5min. Data for10–60 min represent repeated measures from the same experiment (i.e., in one experiment I_h was measured every 10min beginning at t = 10min and ending at t = 60min). Data were plotted as the mean±SEM, n≥4 per time point. The data were best fitted with a double exponential equation yielding time constants of 24sec and 9.9min. <b>(B) Removing DA rapidly abolished LP I</b>_<b>h</b><b>metamodulation</b>. The experiment is diagrammed in the inset; d&i, dissection and cell identification; asterisks indicate measures of LP I_h with TEVC. The percent change in LP I_h G_max relative to t = 0 is plotted (mean±SEM, increases are positive, decreases are negative). Asterisks indicate significantly different from t = 30min as determined using a repeated measures ANOVA with Dunnett’s post-hoc tests that compared all time points to t = 30min, F(5,5) = 2.677, p = 0.0453.</p

The slope of the LP I_h activity-dependence curve in 5nM DA reflects changes in Ca²⁺.

<p>Experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g003" target="_blank">Fig. 3B</a> were repeated for-100 and +50, except that an additional drug(s) to disrupt Ca²⁺ dynamics was also continuously superfused beginning at t = -20min (BAPTA or xestospongin C + ryanodine) or t = -5min (CdCl₂). The fold-changes in LP I_h G_max (mean+SEM) were plotted for each of the three treatment groups. Linear regression analyses and paired t-tests for each treatment group showed that in every case, the slope of the line was not significantly different from zero and the fold changes at-100 and +50 were not statistically significant. The original experiment from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g003" target="_blank">Fig. 3B</a> (dashed line) is shown for comparison.</p

The simplest working model for how tonic nM DA acts through PKA and calcineurin to enable bi-directional, activity-dependent regulation of LP I_h G_max.

<p>The graph represents the LP Ih Gmax activity dependence curve in 5nM DA (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g003" target="_blank">Fig. 3B</a>). The line indicates the idealized fold change in LP Ih Gmax observed in response to a 10min alteration in LP duty cycle. The boxed inset shows the putative molecular mechanism that is activated by tonic 5nM DA. First, DA acts through LP D1Rs to activate PKA. In turn, PKA phosphorylates an unknown protein (blue cylinder) to increase LP Ih Gmax. PKA also phosphorylates calcineurin, a phosphatase that is regulated by the Ca2+-calmodulin complex. PKA phosphorylation permits calcineurin regulation of LP Ih Gmax. When calcineurin is activated by both PKA and Ca2+-calmodulin, it dephosphorylates the unknown protein to reduce LP Ih Gmax. In the absence of PKA, calcineurin cannot influence LP Ih Gmax. The phosphorylation state of the unknown protein is superimposed upon the LP Ih Gmax activity-dependence curve in 5nM DA. Ca2+ is lowest at-100 and highest at +50, and the gradient below the graph indicates how calcineurin activity changes with LP duty cycle. PKA activity is constant because 5nM DA is tonically present (red bar). The unknown protein is fully phosphorylated at-100 because PKA is active but calcineurin is not. At +50 the unknown protein is completely dephosphorylated because calcineurin activity is maximal. In the idealized state, the unknown protein is partially phosphorylated at baseline (0). This phosphorylation is not DA-dependent. Instead, DA acts to increase baseline phosphorylation of the unknown protein through PKA and to enable activity-dependent regulation of the unknown protein’s phosphorylation state by calcineurin. In the absence of DA, the unknown protein maintains its baseline phosphorylation regardless of LP activity.</p

The experimental model.

<p>(<b>A) <i>In situ</i> preparation</b>. The STNS is dissected & pinned in a dish. The commissural ganglia (CoGs) contain DA neurons that project to the STG (black) and L-cells, which are the source of neurohormonal DA (gold). The well surrounding the STG (blue rectangle) is continuously superfused with saline (in/out arrows). There are ~30 neurons in the STG; 2 are drawn: pyloric dilator (PD), lateral pyloric (LP). Network neurons interact locally within the STG neuropil and can project axons to striated muscles surrounding the foregut. The diagram shows that PD & LP neurons project their axons through identified nerves to innervate muscles (rectangles). <b>(B) Spontaneous pyloric network output</b>. The top 2 traces are intra-cellular recordings from the <i>in situ</i> preparation diagrammed in A. The bottom 2 traces represent extra-cellular recordings from identified motor nerves containing pyloric neuron axons. The spikes from three pyloric neurons are indicated on <i>lvn</i>. These simultaneous recordings demonstrate the spontaneous, recurrent, rhythmic motor pattern produced by the circuit; scale bars, 10mV & 500ms. <b>(C) The pyloric circuit</b>. The pyloric network comprises 14 neurons. The diagram represents pyloric neuron interactions within the STG. Open circles represent the 6 cell types, numbers indicate more than 1 cell within a cell type: anterior burster (AB), inferior cardiac (IC), ventricular dilator (VD); pyloric constrictor (PY); filled circles, inhibitory chemical synapses; resistors & diodes, electrical coupling; red, pacemaker kernel and its output connections. <b>(D) Two electrode voltage clamp experiment</b>. Top: Typical LP I_h recording; Bottom: voltage protocol; scale bars, 500ms and 5nA.</p

Calcineurin acts as a Ca²⁺ sensor for metamodulation of LP I_h in 5nM DA.

<p><b>(A) The effect of blocking calcineurin with bath applied FK506</b>. Experiments were performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g005" target="_blank">Fig. 5</a> using the calcineurin inhibitor, FK506. The percent change in LP I_h G_max at t = 10min relative to t = 0 (mean+SEM) was plotted. Paired t-tests compared t = 10min vs. t = 0 within each treatment group; green asterisk, p<0.05. Black asterisks indicate that a one-way ANOVA on the four DA-treatment groups with Tukey’s post hoc tests that made all pair wise comparisons showed that the decrease observed for the TTX + DA + (+50 OSC) treatment group was significantly different from the other three DA treatment groups, and that those three DA treatment groups [(TTX+DA, TTX+DA+FK506, TTX+DA+FK506+(+50 OSC)] were not significantly different from one another: (F (3,26) = 11.08; p = 0.0001). There were no significant differences between the 4 treatment groups that did not receive DA, One way ANOVA, F(3,24) = 0.7714, p = 0.5229. <b>(B) The effect of calcineurin autoinhibitory peptide (CiP) injections</b>. Experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117965#pone.0117965.g005" target="_blank">Fig. 5</a> were repeated except PKI was omitted and CiP was injected into LP at-20min. Paired t-tests compared t = 10min vs. t = 0 within each treatment group; green asterisk, p<0.05. Black asterisks indicate that a one-way ANOVA on the four DA-treatment groups with Tukey’s post hoc tests that made all pair wise comparisons showed that the decrease observed for the TTX + DA + (+50 OSC) treatment group was significantly different from the other three DA treatment groups, and that those three DA treatment groups [(TTX+DA, TTX+DA+CiP, TTX+DA+CiP+(+50 OSC)] were not significantly different from one another: (F (3,18) = 8.71; p = 0.0014). There were no significant differences between the 4 treatment groups that did not receive DA, One way ANOVA, F(3,24) = 1.024, p = 0.4019.</p