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 activity-dependent 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, PKA-dependent 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.

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 (G max) of the LP transient potassium current (I A) 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 (I h) 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 (I A) and activity-dependent (I h) 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).

Activation of high and low affinity dopamine receptors generates a closed loop that maintains a conductance ratio and its activity correlate.

Neuromodulators alter network output and have the potential to destabilize a circuit. The mechanisms maintaining stability in the face of neuromodulation are not well described. Using the pyloric network in the crustacean stomatogastric nervous system, we show that dopamine (DA) does not simply alter circuit output, but activates a closed loop in which DA-induced alterations in circuit output consequently drive a change in an ionic conductance to preserve a conductance ratio and its activity correlate. DA acted at low affinity type 1 receptors (D1Rs) to induce an immediate modulatory decrease in the transient potassium current (IA) of a pyloric neuron. This, in turn, advanced the activity phase of that component neuron, which disrupted its network function and thereby destabilized the circuit. DA simultaneously acted at high affinity D1Rs on the same neuron to confer activity-dependence upon the hyperpolarization activated current (Ih) such that the DA-induced changes in activity subsequently reduced Ih. This DA-enabled, activity-dependent, intrinsic plasticity exactly compensated for the modulatory decrease in IA to restore the IA:Ih ratio and neuronal activity phase, thereby closing an open loop created by the modulator. Activation of closed loops to preserve conductance ratios may represent a fundamental operating principle neuromodulatory systems use to ensure stability in their target networks.

Tonic nanomolar dopamine enables an activity-dependent phase recovery mechanism that persistently alters the maximal conductance of the hyperpolarization-activated current in a rhythmically active neuron.

The phases at which network neurons fire in rhythmic motor outputs are critically important for the proper generation of motor behaviors. The pyloric network in the crustacean stomatogastric ganglion generates a rhythmic motor output wherein neuronal phase relationships are remarkably invariant across individuals and throughout lifetimes. The mechanisms for maintaining these robust phase relationships over the long-term are not well described. Here we show that tonic nanomolar dopamine (DA) acts at type 1 DA receptors (D1Rs) to enable an activity-dependent mechanism that can contribute to phase maintenance in the lateral pyloric (LP) neuron. The LP displays continuous rhythmic bursting. The activity-dependent mechanism was triggered by a prolonged decrease in LP burst duration, and it generated a persistent increase in the maximal conductance (G(max)) of the LP hyperpolarization-activated current (I(h)), but only in the presence of steady-state DA. Interestingly, micromolar DA produces an LP phase advance accompanied by a decrease in LP burst duration that abolishes normal LP network function. During a 1 h application of micromolar DA, LP phase recovered over tens of minutes because, the activity-dependent mechanism enabled by steady-state DA was triggered by the micromolar DA-induced decrease in LP burst duration. Presumably, this mechanism restored normal LP network function. These data suggest steady-state DA may enable homeostatic mechanisms that maintain motor network output during protracted neuromodulation. This DA-enabled, activity-dependent mechanism to preserve phase may be broadly relevant, as diminished dopaminergic tone has recently been shown to reduce I(h) in rhythmically active neurons in the mammalian brain.

Tonic dopamine induces persistent changes in the transient potassium current through translational regulation.

Neuromodulatory effects can vary with their mode of transmission. Phasic release produces local and transient increases in dopamine (DA) up to micromolar concentrations. Additionally, since DA is released from open synapses and reuptake mechanisms are not nearby, tonic nanomolar DA exists in the extracellular space. Do phasic and tonic transmissions similarly regulate voltage-dependent ionic conductances in a given neuron? It was previously shown that DA could immediately alter the transient potassium current (I(A)) of identified neurons in the stomatogastric ganglion of the spiny lobster Panulirus interruptus. Here we show that DA can also persistently alter I(A), and that the immediate and persistent effects of DA oppose one another. The lateral pyloric (LP) neuron exclusively expresses type 1 DA receptors (D1Rs). Micromolar DA produces immediate depolarizing shifts in the voltage dependence of LP I(A), whereas tonic nanomolar DA produces a persistent increase in LP I(A) maximal conductance (G(max)) through a translation-dependent mechanism involving target of rapamycin (TOR). The pyloric dilator (PD) neuron exclusively expresses D2Rs. Micromolar DA produces an immediate hyperpolarizing shift in PD I(A) voltage dependence of activation, whereas tonic DA persistently decreases PD I(A) G(max) through a translation-dependent mechanism not involving TOR. The persistent effects on I(A) G(max) do not depend on LP or PD activity. These data suggest a role for tonic modulators in the regulation of voltage-gated ion channel number; and furthermore, that dopaminergic systems may be organized to limit the amount of change they can impose on a circuit.

Cell specific dopamine modulation of the transient potassium current in the pyloric network by the canonical D1 receptor signal transduction cascade.

Dopamine (DA) modifies the motor pattern generated by the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, by directly acting on each of the circuit neurons. The 14 pyloric neurons fall into six cell types, and DA actions are cell type specific. The transient potassium current mediated by shal channels (I(A)) is a common target of DA modulation in most cell types. DA shifts the voltage dependence of I(A) in opposing directions in pyloric dilator (PD) versus lateral pyloric (LP) neurons. The mechanism(s) underpinning cell-type specific DA modulation of I(A) is unknown. DA receptors (DARs) can be classified as type 1 (D1R) or type 2 (D2R). D1Rs and D2Rs are known to increase and decrease intracellular cAMP concentrations, respectively. We hypothesized that the opposing DA effects on PD and LP I(A) were due to differences in DAR expression patterns. In the present study, we found that LP expressed somatodendritic D1Rs that were concentrated near synapses but did not express D2Rs. Consistently, DA modulation of LP I(A) was mediated by a Gs-adenylyl cyclase-cAMP-protein kinase A pathway. Additionally, we defined antagonists for lobster D1Rs (flupenthixol) and D2Rs (metoclopramide) in a heterologous expression system and showed that DA modulation of LP I(A) was blocked by flupenthixol but not by metoclopramide. We previously showed that PD neurons express D2Rs, but not D1Rs, thus supporting the idea that cell specific effects of DA on I(A) are due to differences in receptor expression.

D(2) receptors receive paracrine neurotransmission and are consistently targeted to a subset of synaptic structures in an identified neuron of the crustacean stomatogastric nervous system.

Dopamine (DA) modulates motor systems in phyla as diverse as nematodes and arthropods up through chordates. A comparison of dopaminergic systems across a broad phylogenetic range should reveal shared organizing principles. The pyloric network, located in the stomatogastric ganglion (STG), is an important model for neuromodulation of motor networks. The effects of DA on this network have been well characterized at the circuit and cellular levels in the spiny lobster, Panulirus interruptus. Here we provide the first data about the physical organization of the DA signaling system in the STG and the function of D(2) receptors in pyloric neurons. Previous studies showed that DA altered intrinsic firing properties and synaptic output in the pyloric dilator (PD) neuron, in part by reducing calcium currents and increasing outward potassium currents. We performed single cell reverse transcriptase-polymerase chain reaction (RT-PCR) experiments to show that PD neurons exclusively expressed a type 2 (D(2alphaPan)) DA receptor. This was confirmed by using confocal microscopy in conjunction with immunohistochemistry (IHC) on STG whole-mount preparations containing dye-filled PD neurons. Immunogold electron microscopy showed that surface receptors were concentrated in fine neurites/terminal swellings and vesicle-laden varicosities in the synaptic neuropil. Double-label IHC experiments with tyrosine hydroxylase antiserum suggested that the D(2alphaPan) receptors received volume neurotransmissions. Receptors were further mapped onto three-dimensional models of PD neurons built from Neurolucida tracings of confocal stacks from the IHC experiments. The data showed that D(2alphaPan) receptors were selectively targeted to approximately 40% of synaptic structures in any given PD neuron, and were nonuniformly distributed among neurites.