Disentangling the feedback signals coordinating the ion channel mRNA profile of motor neurons of the crustacean stomatogastric ganglion
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How do neural networks reliably produce motor behaviors over the course of an organism's life? Remarkably, neural networks are able to stabilize their outputs in spite of physiological perturbations such as changes in development (Marder E and Goaillard JM 2006), ion channel turnover (Conrad R et al. 2018), or pH (Haddad SA and Marder E 2018). To better understand the mechanisms that favor network output stability, we have utilized the stomatogastric ganglion (STG) of the crustacean crab Cancer borealis. This small motor network has allowed us to shed light on the feedback mechanisms by which single motor neurons can coordinate an ion channel mRNA profile conducive to stable electrical outputs. The STG is a central pattern generating network made up of 26 individually identifiable motor neurons, each of which innervates and drives the activity of striated muscles of the crab stomach (Marder E and Bucher D 2007). Each motor neuron belongs to either the pyloric or gastric network of neurons, that drive filtering and grinding of food respectively. The pyloric rhythm is continuously active for the duration of a crabs lifetime and is in part driven by PD neurons (Marder E and Bucher D 2007) which use activity dependent feedback to maintain correlated ion channel mRNAs (Santin JM and Schulz DJ 2019). The gastric rhythm is transiently active and is induced in vivo whenever food mechanically deforms receptors located in the stomach (Beenhakker MP and Nusbaum MP 2004); this in turn leads to the neuromodulatory excitation of the silent lateral gastric (LG) neuron and initiation of gastric activity (DeLong ND et al. 2009). Therefore, the gastric network offers us the ability to initiate the active state of neurons like LG in vitro (Beeenhakker MP, Blitz DM, and Nusbaum MP 2004). The firing properties of a motor neuron are in part determined by the precise coordination of ionic conductances (Marder E and Goaillard JM 2006). Interestingly, the ion channel mRNAs that encode these conductances have also been found to be correlated in expression in a cell-type specific manner in STG neurons (Schulz DJ, Goaillard JM, and Marder E 2007). The active co-regulation of these transcripts suggests they may be important to promote output stability (Temporal S et al. 2012; Santin JM and Schulz DJ 2019). Previous work in the STG has proposed activity (in the form of membrane voltage) (Santin JM and Schulz DJ 2019; Temporal S, Lett KM, and Schulz DJ 2014; O'Leary T et al. 2013) and neuromodulation (Temporal S et al. 2012) as two major feedback signals by which motor neurons can coordinate their ion channel mRNA profiles. More recently, it has been shown that pacemaking motor neurons of the STG coordinate 64 percent of their ion channel mRNAs via membrane voltage feedback loops (Santin JM and Schulz DJ 2019). However, in the absences any obvious activity-dependent feedback, neurons with transient states may necessitate other feedback solutions to coordinate the ion channel mRNA profile necessary to resume and recapitulate reliable outputs. To address this, we used the pyloric dilator (PD; constitutively active) and the lateral gastric (LG; transiently active) neurons of the stomatogastric ganglion (STG) of the crustacean Cancer borealis. We experimentally stimulated descending inputs to the STG to cause release of neuromodulators known to elicit the active state of LG neurons and quantified the mRNA abundances and pairwise relationships of 11 voltage gated ion channels in active and silent LGs. The same stimulus does not significantly alter PD activity. Activation of LG up-regulated ion channel mRNAs and lead to a greater number of positively correlated pairwise channel mRNA relationships in LG. Conversely, this stimulus did not induce major changes in ion channel mRNA abundances and relationships of PD cells, suggesting their ongoing activity is sufficient to maintain channel mRNA relationships even under changing modulatory conditions. Additionally, we found that ion channel mRNA correlations induced by the active state of LG are influenced by a combination of activity dependent and neuromodulatory dependent feedback mechanisms. Interestingly, some of these same correlations are maintained by distinct mechanisms in PD, suggesting that motor networks utilize multiple feedback solutions to coordinate the same mRNA relationships of across neuron types. While our previous results indicated that LG's active state induced changes to its ion channel mRNA profile, this did not address an important question: if LG is able to recapitulate its activity after periods of silence, then the ion channel profile necessary to do so should presumably already be present in its membrane. How is that ion channel profile getting there in the first place? Previous work has shown that continuously active STG neurons possess correlated ion channel mRNAs that are mirrored in time by their corresponding correlated ionic currents (Temporal S et al. 2012; Schulz DJ, Goaillard JM, and Marder E 2006; Ransdell JL, Nair SS, and Schulz DJ 2012), which suggests that actively co-regulating mRNA transcripts via activity dependent feedback (Santin JM and Schulz DJ 2019) may serve to also maintain the co-regulation of the corresponding currents. Thus, other regulatory strategies must exist that allow transiently active neurons to establish the appropriate ion channel profile conducive to stable outputs. We asked whether the purpose of inducing new ion channel mRNA correlations during LG's active state would serve to establish the corresponding ionic current profile. To investigate this, we measured 3 potassium currents and their corresponding mRNA transcripts in silent and active LG neurons. We found that ion channel mRNAs and currents change but do not track each other across time: mRNAs became coregulated only during the active state of LG, but the corresponding currents became coregulated only in its silent state. We then performed a meta-analysis where we compared these results to those published in constitutively active pyloric dilator (PD) neurons by Temporal et al, 2012. We found that ion channel mRNAs and ionic current are both coregulated in time and track each other only in the active state of PD, while silencing PD's activity eliminated tracking. These comparative results suggest that each neuron type uses a different strategy to regulate the interaction between their ion channel mRNAs and ionic currents that is cell-type dependent. Thus, we hypothesize that the active state of LG neurons may serve to build a coregulated pool of channel mRNAs that is translated into functional protein after LG activity ceases, in order to establish the ionic current profile needed by silent LG neurons to resume their outputs. Finally, some of the new ion channel mRNA correlations we observed in LG's active state, were induced and coordinated by neuromodulatory feedback. However, we know that the STG is modulated by over 200 neuropeptides, transmitters, and hormones from descending modulatory centers (Goaillard JM and Marder E 2021; Marder E and Bucher D 2007; Marder E 2012; Bargmann C 2012). Thus, we wanted to narrow down some of the specific neuropeptides that were inducing some of LG's neuromodulator dependent ion channel mRNA correlations. Additionally, we also wanted to know whether STG neuropeptides not only modulated the electrophysiological outputs of LG and other STG neurons (Swensen AM and Marder E 2001, 2000), but if they could also regulate their ion channel mRNAs. To investigate this, we first silenced the STG network activity and blocked neurotransmission from higher modulatory centers, after which we incubated the LG, PD, lateral pyloric (LP), and lateral posterior gastric (LPG) neurons in one of two endogenous Cancer borealis peptides for 8 hours: proctolin and the crustacean cardioactive peptide (CCAP. Each neuron type has known physiological responses to both, one, or none of the neuropeptides. We then dissected each neuron type, and we quantified the abundances and relationships of 11 voltage gated ion channel mRNAs We found that single neuropeptides are able to rescue and induce the coregulation of ion channel mRNAs in each of our four cell types. Surprisingly, each peptide was able to act on the ion channel mRNA expression and coregulation of some neuron types independent of whether that neuron was known to be electrophysiologically irresponsive to that peptide. These results suggest that while neuropeptides can modulate the electrical output of neurons in a target-specific way, their ability to regulate ion channel mRNAs may be more network wide. Additionally, some ion channel mRNA relationships can be coregulated by two different peptides, which suggests that some mRNA relationships may be critical to motor neurons and may necessitate redundant regulation. Finally, our results also show that peptides can differentially regulate the same transcript across distinct neuron types, which suggests that peptide-induced mRNA expression is cell-type dependent.
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Ph. D.