Neurotransmitter identity and electrophysiological phenotype are genetically coupled in midbrain dopaminergic neurons.

Most neuronal types have a well-identified electrical phenotype. It is now admitted that a same phenotype can be produced using multiple biophysical solutions defined by ion channel expression levels. This argues that systems-level approaches are necessary to understand electrical phenotype genesis and stability. Midbrain dopaminergic (DA) neurons, although quite heterogeneous, exhibit a characteristic electrical phenotype. However, the quantitative genetic principles underlying this conserved phenotype remain unknown. Here we investigated the quantitative relationships between ion channels' gene expression levels in midbrain DA neurons using single-cell microfluidic qPCR. Using multivariate mutual information analysis to decipher high-dimensional statistical dependences, we unravel co-varying gene modules that link neurotransmitter identity and electrical phenotype. We also identify new segregating gene modules underlying the diversity of this neuronal population. We propose that the newly identified genetic coupling between neurotransmitter identity and ion channels may play a homeostatic role in maintaining the electrophysiological phenotype of midbrain DA neurons.

Removal of endogenous neuromodulators in a small motor network enhances responsiveness to neuromodulation.

We studied the changes in sensitivity to a peptide modulator, crustacean cardioactive peptide (CCAP), as a response to loss of endogenous modulation in the stomatogastric ganglion (STG) of the crab Cancer borealis Our data demonstrate that removal of endogenous modulation for 24 h increases the response of the lateral pyloric (LP) neuron of the STG to exogenously applied CCAP. Increased responsiveness is accompanied by increases in CCAP receptor (CCAPr) mRNA levels in LP neurons, requires de novo protein synthesis, and can be prevented by coincubation for the 24-h period with exogenous CCAP. These results suggest that there is a direct feedback from loss of CCAP signaling to the production of CCAPr that increases subsequent response to the ligand. However, we also demonstrate that the modulator-evoked membrane current (IMI) activated by CCAP is greater in magnitude after combined loss of endogenous modulation and activity compared with removal of just hormonal modulation. These results suggest that both receptor expression and an increase in the target conductance of the CCAP G protein-coupled receptor are involved in the increased response to exogenous hormone exposure following experimental loss of modulation in the STG.NEW & NOTEWORTHY The nervous system shows a tremendous amount of plasticity. More recently there has been an appreciation for compensatory actions that stabilize output in the face of perturbations to normal activity. In this study we demonstrate that neurons of the crustacean stomatogastric ganglion generate apparent compensatory responses to loss of peptide neuromodulation, adding to the repertoire of mechanisms by which the stomatogastric nervous system can regulate and stabilize its own output.

Neuropeptide receptor transcript expression levels and magnitude of ionic current responses show cell type-specific differences in a small motor circuit.

We studied the relationship between neuropeptide receptor transcript expression and current responses in the stomatogastric ganglion (STG) of the crab, Cancer borealis. We identified a transcript with high sequence similarity to crustacean cardioactive peptide (CCAP) receptors in insects and mammalian neuropeptide S receptors. This transcript was expressed throughout the nervous system, consistent with the role of CCAP in a range of different behaviors. In the STG, single-cell qPCR showed expression in only a subset of neurons. This subset had previously been shown to respond to CCAP with the activation of a modulator-activated inward current (IMI), with one exception. In the one cell type that showed expression but no IMI responses, we found CCAP modulation of synaptic currents. Expression levels within STG neuron types were fairly variable, but significantly different between some neuron types. We tested the magnitude and concentration dependence of IMI responses to CCAP application in two identified neurons, the lateral pyloric (LP) and the inferior cardiac (IC) neurons. LP had several-fold higher expression and showed larger current responses. It also was more sensitive to low CCAP concentrations and showed saturation at lower concentrations, as sigmoid fits showed smaller EC50 values and steeper slopes. In addition, occlusion experiments with proctolin, a different neuropeptide converging onto IMI, showed that saturating concentrations of CCAP activated all available IMI in LP, but only approximately two-thirds in IC, the neuron with lower receptor transcript expression. The implications of these findings for comodulation are discussed.

Activity-dependent feedback regulates correlated ion channel mRNA levels in single identified motor neurons.

Neurons generate cell-specific outputs via interactions of conductances carried by ion channel proteins that are homeostatically regulated to maintain key quantitative relationships among subsets of conductances. Given the challenges of both normal channel protein turnover and short-term plasticity, how is the balance of membrane conductances maintained over long-term timescales to ensure stable electrophysiological phenotype? One possible mechanism is to dynamically regulate production of channel protein via feedback that constrains relationships at the channel mRNA level. Recent modeling work has postulated that such mRNA relationships could emerge as a result of activity-dependent homeostatic tuning rules that ensure an appropriate ratio of mRNA for key ion channels is maintained to preserve robust cellular output. Yet, this has never been demonstrated in biological neurons. In this study, we quantified multiple ion channel mRNAs from single identified motor neurons of the stomatogastric ganglion to determine whether correlations among channel mRNAs are actively maintained, and, if so, by what form of feedback. In these neurons, we identified correlations among mRNAs for voltage-gated calcium and potassium channels. By performing experiments that decoupled activity, synaptic connectivity, and neuromodulatory state, we determined that correlated channel mRNAs are maintained by an activity-dependent process. This is the first study to demonstrate that distinct relationships across channel mRNAs are dynamically maintained in an activity-dependent manner. This feedback from cellular activity to coordinated transcriptome-level interactions represents a novel aspect of regulation of neuronal output with implications for long-term stability of neuron function.

Characterization of inward currents and channels underlying burst activity in motoneurons of crab cardiac ganglion.

Large cell motoneurons in the Cancer borealis cardiac ganglion generate rhythmic bursts of action potentials responsible for cardiac contractions. While it is well known that these burst potentials are dependent on coordinated interactions among depolarizing and hyperpolarizing conductances, the depolarizing currents present in these cells, and their biophysical characteristics, have not been thoroughly described. In this study we used a combined molecular biology and electrophysiology approach to look at channel identity, expression, localization, and biophysical properties for two distinct high-voltage-activated calcium currents present in these cells: a slow calcium current (ICaS) and a transient calcium current (ICaT). Our data indicate that CbCaV1 is a putative voltage-gated calcium channel subunit in part responsible for an L-type current, while CbCaV2 (formerly cacophony) is a subunit in part responsible for a P/Q-type current. These channels appear to be localized primarily to the somata of the motoneurons. A third calcium channel gene (CbCaV3) was identified that encodes a putative T-type calcium channel subunit and is expressed in these cells, but electrophysiological studies failed to detect this current in motoneuron somata. In addition, we identify and characterize for the first time in these cells a calcium-activated nonselective cationic current (ICAN), as well as a largely noninactivating TTX-sensitive current reminiscent of a persistent sodium current. The identification and further characterization of these currents allow both biological and modeling studies to move forward with more attention to the complexity of interactions among these distinct components underlying generation of bursting output in motoneurons.

Neuromodulation independently determines correlated channel expression and conductance levels in motor neurons of the stomatogastric ganglion.

Neuronal identity depends on the regulated expression of numerous molecular components, especially ionic channels, which determine the electrical signature of a neuron. Such regulation depends on at least two key factors, activity itself and neuromodulatory input. Neuronal electrical activity can modify the expression of ionic currents in homeostatic or nonhomeostatic fashion. Neuromodulators typically modify activity by regulating the properties or expression levels of subsets of ionic channels. In the stomatogastric system of crustaceans, both types of regulation have been demonstrated. Furthermore, the regulation of the coordinated expression of ionic currents and the channels that carry these currents has been recently reported in diverse neuronal systems, with neuromodulators not only controlling the absolute levels of ionic current expression but also, over long periods of time, appearing to modify their correlated expression. We hypothesize that neuromodulators may regulate the correlated expression of ion channels at multiple levels and in a cell-type-dependent fashion. We report that in two identified neuronal types, three ionic currents are linearly correlated in a pairwise manner, suggesting their coexpression or direct interactions, under normal neuromodulatory conditions. In each cell, some currents remain correlated after neuromodulatory input is removed, whereas the correlations between the other pairs are either lost or altered. Interestingly, in each cell, a different suite of currents change their correlation. At the transcript level we observe distinct alterations in correlations between channel mRNA amounts, including one of the cell types lacking a correlation under normal neuromodulatory conditions and then gaining the correlation when neuromodulators are removed. Synaptic activity does not appear to contribute, with one possible exception, to the correlated expression of either ionic currents or of the transcripts that code for the respective channels. We conclude that neuromodulators regulate the correlated expression of ion channels at both the transcript and the protein levels.