Relationships among membrane currents allow central pattern generator (CPG) neurons to reliably drive motor programs. We hypothesize that continually active CPG neurons utilize activity-dependent feedback to correlate expression of ion channel genes to balance essential membrane currents. However, episodically activated neurons experience absences of activity-dependent feedback and, thus, presumably employ other strategies to coregulate the balance of ionic currents necessary to generate appropriate output after periods of quiescence. To investigate this, we compared continually active pyloric dilator (PD) neurons with episodically active lateral gastric (LG) CPG neurons of the stomatogastric ganglion (STG) in male Cancer borealis crabs. After experimentally activating LG for 8 h, we measured three potassium currents and abundances of their corresponding channel mRNAs. We found that ionic current relationships were correlated in LG's silent state, but ion channel mRNA relationships were correlated in the active state. In continuously active PD neurons, ion channel mRNAs and ionic currents are simultaneously correlated. Therefore, two distinct relationships exist between channel mRNA abundance and the ionic current encoded in these cells: in PD, a direct correlation exists between Shal channel mRNA levels and the A-type potassium current it carries. Conversely, such channel mRNA-current relationships are not detected and appear to be temporally uncoupled in LG neurons. Our results suggest that ongoing feedback maintains membrane current and channel mRNA relationships in continually active PD neurons, while in LG neurons, episodic activity serves to establish channel mRNA relationships necessary to produce the ionic current profile necessary for the next bout of activity.

Postganglionic neurons of the autonomic nervous system lie outside of the central nervous system and innervate specific target effectors such as organs or glands. The major pelvic ganglion (MPG) is one such ganglion that plays a significant role in controlling bladder function in rodents. However, because of technical and physical constraints in recording electrophysiological signals from these neurons in vivo, the functional neural activity in MPG is mostly unknown. Transgenic animal models expressing genetically encoded calcium indicators now provide opportunities to monitor the activity of populations of neurons in vivo to overcome these challenges related to traditional electrophysiological methods. However, like many peripheral neurons, the MPG is not conducive to conventional fluorescent microscopy techniques, as it is located in the pelvic cavity, thus limiting robust optical access by benchtop microscopes. Here, we present an endoscopic approach based on a custom miniscope system (UCLA V3) that allows for effective in vivo monitoring of neural activity in the MPG for the first time. We show that our imaging approach can monitor activity of hundreds of MPG neurons simultaneously during the filling and emptying of the bladder in a urethaneanesthetized transgenic mouse line expressing GCaMP6s in cholinergic MPG neurons. By using custom analysis scripts, we isolated the activity of hundreds of individual neurons and show that populations of neurons have distinct phasic activation patterns during sequential bladder filling and voiding events. Our imaging approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models. NEW & NOTEWORTHY The functional activity and information processing within autonomic ganglia is mostly unknown because of technical and physical constraints in recording electrophysiological signals from these neurons in vivo. Here, we use a microendoscopic approach to measure in vivo functional activity patterns from a population of autonomic neurons controlling bladder function for the first time. This approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.

Recently, activity has been proposed as a primary feedback mechanism used by continuously bursting neurons to coordinate ion channel mRNA relationships that underlie stable output. However, some neuron types only have intermittent periods of activity and so may require alternative mechanisms that induce and constrain the appropriate ion channel profile in different states of activity. To address this, we used the pyloric dilator (PD; constitutively active) and the lateral gastric (LG; periodically 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 LG neurons. The same stimulus does not significantly alter PD activity. Activation of LG upregulated ion channel mRNAs and lead to a greater number of positively correlated pairwise channel mRNA relationships. 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. In addition, we found that ion channel mRNA correlations induced by the active state of LG are influenced by a combination of activity- and neuromodulator-dependent feedback mechanisms. Interestingly, some of these same correlations are maintained by distinct mechanisms in PD, suggesting that these motor networks use distinct feedback mechanisms to coordinate the same mRNA relationships across neuron types.NEW & NOTEWORTHY Neurons use various feedback mechanisms to adjust and maintain their output. Here, we demonstrate that different neurons within the same network can use distinct signaling mechanisms to regulate the same ion channel mRNA relationships.

The central nervous systems of decapod crustaceans contains numerous amines, amino acids, and peptides that modulate the central pattern generating (CPG) circuits found within its stomatogastric nervous system (STNS) and cardiac ganglion (CG); over 30 modulators have been identified in one species of crab (C. borealis). CPGs in the STNS are responsible for internally chewing (Gastric Mill CPG) and filtering food (Pyloric CPG) into the midgut, while the CG drives the heartbeat. Neuromodulation allows these CPGs to flexibly produce a variety of output patterns; however, the variety of modulators exceeds the variety of induced output, leading us to ask why so many modulators are present. Although modulatory capacity, a measure of the extent to which a neuronal circuit can be modulated, may be solely an inherited trait, we hypothesize a functional correlation: decapods that need more behavioral flexibility utilize a broader range of modulators than those with more stereotyped behaviors. We thus predicted that decapods with broad diets would have a larger modulatory capacity in the STNS, but not the CG, than those with limited diets. To test this, we compared responses of the STNS and CG CPGs in three species of majoid crabs (Pugettia producta, Libinia emarginata, and Chionoecetes opilio) to the same neuromodulators. L. emarginata and C. opilio both have broad diets, while P. producta primarily eats kelp. With one exception, each species' CG exhibited similar responses to all modulators. The STNS of crabs with broader diets responded similarly to all modulators, whereas the P. producta STNS showed limited responses to the same set of neuromodulators. CG responses did not follow this trend, as all crabs showed varied CG response to modulation. Modulators: Cancer borealisTachykinin Related Peptide (CabTRP), Crustacean Cardioactive Peptide (CCAP), Red Pigment Concentrating Hormone (RPCH), Myosuppressin (Myo), Oxotremorine (Oxo), Dopamine (DA), Proctolin (Proc).

Spinal cord injury (SCI) has substantial impacts on autonomic function. In part, SCI results in loss of normal autonomic activity that contributes to injury-associated pathology such as neurogenic bladder, bowel, and sexual dysfunction. Yet little is known of the impacts of SCI on peripheral autonomic neurons that directly innervate these target organs. In this study, we measured changes in synaptic properties of neurons of the mouse major pelvic ganglion (MPG) associated with acute and chronic SCI. Our data show that functional and physiological properties of synapses onto MPG neurons are altered after SCI and differ between acute and chronic injury. After acute injury excitatory postsynaptic potentials (EPSPs) show increased rise and decay time constants leading to overall broader and longer EPSPs, whereas in chronic-injured animals EPSPs are reduced in amplitude and show faster rise and decay leading to shorter EPSPs. Synaptic depression and low-pass filtering are also altered in injured animals. Finally, cholinergic currents are smaller in acute-injured animals but larger in chronic-injured animals relative to control animals. These changes in synaptic properties are associated with differences in nicotinic receptor subunit expression as well. MPG CHRNA3 mRNA levels decreased after injury, whereas CHRNA4 mRNAs increased. Furthermore, changes in the correlations of α- and β-subunit mRNAs suggest that nicotinic receptor subtype composition is altered after injury. Taken together, our data demonstrate that peripheral autonomic neurons are fundamentally altered after SCI, suggesting that longer-term therapeutic approaches could target these neurons directly to potentially help ameliorate neurogenic target organ dysfunction.NEW & NOTEWORTHY Spinal cord injury (SCI) has substantial impacts on autonomic function, yet little is known of the impacts of SCI on autonomic neurons that directly innervate effectors impacted by injury. Here we investigated changes at the cellular level associated with SCI in neurons that are "downstream" of the central injury. An understanding of these off-target impacts of SCI ultimately will be critical in the context of effective restoration of function through neuromodulation of pharmacological therapeutic approaches.

Central pattern generators produce many rhythms necessary for survival (e.g., chewing, breathing, locomotion), and doing so often requires coordination of neurons through electrical synapses. Because even neurons of the same type within a network are often differentially tuned, uniformly applied neuromodulators or toxins can result in uncoordinated activity. In the crab (Cancer borealis) cardiac ganglion, potassium channel blockers and serotonin cause increased depolarization of the five electrically coupled motor neurons as well as loss of the normally completely synchronous activity. Given time, compensation occurs that restores excitability and synchrony. One of the underlying mechanisms of this compensation is an increase in coupling among neurons. However, the salient physiological signal that initiates increased coupling has not been determined. Using male C. borealis, we show that it is the loss of synchronous voltage signals between coupled neurons that is at least partly responsible for plasticity in coupling. Shorter offsets in naturalistic activity across a gap junction enhance coupling, whereas longer delays depress coupling. We also provide evidence on why a desynchronization-specific potentiation or depression of the synapse could ultimately be adaptive through using a hybrid network created by artificially coupling two cardiac ganglia. Specifically, a stray neuron may be brought back in line by increasing coupling if its activity is closer to the remainder of the network. However, if a the activity of a neuron is far outside network parameters, it is detrimental to increase coupling, and therefore depression of the synapse removes a potentially harmful influence on the network. SIGNIFICANCE STATEMENT Understanding how neural networks maintain output over years despite environmental and physiological challenges requires understanding the regulatory principles of these networks. Here, we study how cells that are synchronously active at baseline respond to becoming desynchronized. In this system, a loss of synchrony causes different parts of the heart to receive uncoordinated stimulation. We find a calcium-dependent control mechanism that alters the strength of electrical connections between motor neurons. Although others have described similar control mechanisms, here, we demonstrate that voltage changes are sufficient to elicit regulation. Furthermore, we demonstrate that strong connections in a sufficiently perturbed network can prevent any neuron from producing its target activity, thus suggesting why the connections are not constitutively as strong as possible.

Neuroscience is a highly interdisciplinary field, but more collaboration among STEM disciplines is needed to advance undergraduate neuroscience education. This paper reports the development of code-based virtual laboratories to cross-foster ideas from engineering and biology in the neuroscience classroom. The simulations use a combination of NEURON and Python code to model neurophysiological processes. We report that the use of computational tools in the classroom increases student self-reported comfort in participating in a computational research project. The tools we developed have potential to increase persistence and retention of undergraduate students by encouraging interdisciplinary thinking and reducing barriers to entry.

ABSTRACT The crustacean cardiac ganglion (CG) network coordinates the rhythmic contractions of the heart muscle to control the circulation of blood. The network consists of 9 cells, 5 large motor cells (LC1-5) and 4 small endogenous pacemaker cells (SCs). We report a new three-compartmental biophysical model of an LC that is morphologically realistic and includes provision for inputs from the SCs via a gap-junction coupled spike-initiation-zone (SIZ) compartments. To determine physiologically viable LC models in this realistic configuration, maximal conductances in three compartments of an LC are determined by random sampling from a biologically-characterized 9D-parameter space, followed by a three-stage rejection protocol that checks for conformity with electrophysiological features from single cell traces. LC models that pass the single cell rejection protocol are then incorporated into a network model which is then used in a final rejection protocol stage. Using disparate experimental data, the study provides hitherto unknown structure-function insights related to the crustacean cardiac ganglion large cell, including predictions about morphology including the role of its SIZ, and the differential roles of active conductances in the three compartments. Further, we extend analyses of emergent conductance relationships and correlations in model neurons relative to their biological counterparts, allowing us to make inferences both with respect to the biological system as well as the implications of the ability to detect such relationships in populations of model neurons going forward.

This special issue is dedicated to ‘Future Perspectives of Neurogenetics’ in honor of Troy D. Zars (1967–2018). It is intended to offer insight and inspiration for our understanding of how adaptive...

I knew Troy for nearly 15 years, and in that time I don't recall hearing any childhood stories like those in seemingly every personal statement I've read from aspiring scientists or medical students. No stories about hours spent gazing at an anthill. I don't recall hearing about shelves crowded with insects collected on Styrofoam, or animal skulls kept in a shoebox under his bed. If these collected crania existed, it was more likely because Troy was a crack shot with a pellet gun than a need to know adaptations in the dentition of local squirrel populations. I don't recall hearing about science projects taken to the Iowa State Capitol to share with politely interested legislators. But I do recall hearing about spending the entirety of the daylight hours in the summer, with his brother Doug, finding where the crappie were biting. About crystal clear water on a lake in Minnesota that you didn't quite need to know the exact location of, just in case you were thinking of going and plundering the walleye within. I definitely heard about triumphs as a starting lineman not only for his high school football team, but the mighty Norse of Luther College. I heard about summer warehouse jobs in sweltering Iowa Julys. And I saw, firsthand, love and commitment and family. Troy's story demonstrates that the finest scientists are not just cultivated in narrow STEM curricula that begin at age 5. They are just as likely to be football-playing fishermen, fathers, husbands, and friends who can navigate an operant conditioning paradigm during the week, and dance a polka and produce a magnificent smoked pork shoulder on Saturday. Nature and an independent spirit and a little bit of mischief is a different kind of Magnet school. And it gave us truly one of the best.

Altered microglia function contributes to loss of CNS homeostasis during aging in the brain. Few studies have evaluated age-related alterations in spinal cord microglia. We previously demonstrated that lumbar spinal cord microglial expression of inducible nitric oxide synthase (iNOS) was equivalent between aging, neurologically normal dogs and dogs with canine degenerative myelopathy (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157). This unexpected finding suggested that microglia in aging spinal cord have a pro-inflammatory polarization. In this study, we reexamined our microglial results (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157) within the context of aging rather than disease by comparing microglia in aging versus young adult dogs. For both aging and young adult dogs, the density of microglia was significantly higher closest to the motor neuron cell body. However, there was no difference in densities between aging versus young adult dogs at all distances except for the furthest distance analyzed. The number of motor neurons with polarized microglia was higher in aging dogs; yet, the density per motor neuron of arginase-1-expressing microglia was reduced in aging dogs compared with young adult dogs. Finally, aging dogs had increased steady-state mRNA levels for genes consistent with activated microglia compared with young adult dogs. However, altered mRNA levels were limited to the lumbar spinal cord. These data suggested that aging dog spinal cord microglia exhibit regional immunophenotypic differences, which may render lumbar motor neurons more susceptible to age-related pathological insults.

Central pattern generators (CPGs) are neuronal circuits that can autonomously generate rhythmic behaviors such as breathing, locomotion and vocalization (Marder and Bucher 2001). The study of these circuits is important because they are essential for survival. However, their investigation has also been prioritized for pragmatic reasons: because they can produce naturalistic rhythmic patterns without sensory feedback, they often continue to function in a quasi-normal way in ex vivo preparations (Rhodes et al., 2007. Many CPGs, such as the stomatogastric ganglion (STG) circuit that controls crustacean stomach movements, represent some of the best understood neuronal circuits in all of neuroscience, and have provided fundamental insights into nervous system mechanisms such as neuromodulation, synaptic plasticity, and activity-dependent homeostasis (Daur, Nadim, and Bucher 2016).

1. Overview The lower urinary tract (LUT) in mammals consists of the urinary bladder, external urethral sphincter (EUS) and the urethra. Control of the LUT is achieved via a neural circuit which integrates two distinct components. One component of the neural circuit is ‘reflexive’ in that it relies solely on input from sensory neurons in the bladder and urethra that is fed back via spinal neurons to the LUT. The second neural component is termed ‘top-down’ and is a conditioned input that comes from structures such as Pontine Micturition center (PMC), and Pontine storage center (PSC), that also receive the afferent sensory input from the LUT relayed through periaqueductal gray (PAG). The reader is referred to excellent references such as [1-3] from de Groat’s group for the top down control, which is not discussed here. This chapter focuses primarily on outlining the challenges in the development of computational model of the neural circuit that controls the LUT. Section 2 describes the anatomy of the LUT with primary focus on the neural anatomy. We describe the overall efferent and afferent neural pathways of LUT and briefly discuss the neurotransmitters involved and known details related to species/sex differences and developmental changes. Section 3 provides a brief summary of efforts to model the various neural components of the LUT for the purpose of understanding how they might participate in control. We list challenges in modeling the control of LUT at cellular and network levels and finally provide a brief description of an on-going effort to develop a biophysical model of the system.

This special issue is dedicated to ‘Future Perspectives of Neurogenetics’ in honor of Troy D. Zars (1967–2018). It is intended to offer insight and inspiration for our understanding of how adaptive...

Dopamine provides crucial neuromodulatory functions in several insect and rodent learning and memory paradigms. However, an early study suggested that dopamine may be dispensable for aversive place memory in Drosophila. Here we tested the involvement of particular dopaminergic neurons in place learning and memory. We used the thermogenetic tool Gr28bD to activate protocerebral anterior medial (PAM) cluster and non-PAM dopaminergic neurons in an operant way in heat-box place learning. We show that activation of PAM neurons influences performance during place learning, but not during memory testing. These findings provide a gateway to explore how dopamine influences place learning.

Many neurons receive synchronous input from heterogeneous presynaptic neurons with distinct properties. An instructive example is the crustacean stomatogastric pyloric circuit pacemaker group, consisting of the anterior burster (AB) and pyloric dilator (PD) neurons, which are active synchronously and exert a combined synaptic action on most pyloric follower neurons. Although the stomatogastric system of the crab Cancer borealis has become a preferred model system for exploration of cellular and synaptic basis of circuit dynamics, in this species, the identity of the PD neuron neurotransmitter and its contribution to the total pacemaker group synaptic output remain unexplored. We examined the synaptic properties of the crab PD neuron using a combination of single cell mRNA analysis, electrophysiology and pharmacology. The crab PD neuron expresses high levels of choline acetyltransferase and the vesicular acetylcholine transporter mRNAs, hallmarks of cholinergic neurons. Conversely, the AB neuron does not express either of these cholinergic markers, and expresses high levels of vesicular glutamate transporter mRNA, consistent with a glutamatergic phenotype. Notably, in the combined synapses to the LP and PY neurons, the major contribution is from the glutamatergic AB neuron and only between 25-30% of the synaptic strength is due to the PD neuron. However, there was no difference between the short-term synaptic plasticity in the total pacemaker synapse compared to that of the PD neuron alone. These findings provide a guide for similar explorations of heterogeneous synaptic connections in other systems and a baseline in this system for the exploration of the differential influence of neuromodulators.

The many roles of innexins, the molecules that form gap junctions in invertebrates, have been explored in numerous species. Here, we present a summary of innexin expression and function in two small, central pattern generating circuits found in crustaceans: the stomatogastric ganglion and the cardiac ganglion. The two ganglia express multiple innexin genes, exhibit varying combinations of symmetrical and rectifying gap junctions, as well as gap junctions within and across different cell types. Past studies have revealed correlations in ion channel and innexin expression in coupled neurons, as well as intriguing functional relationships between ion channel conductances and electrical coupling. Together, these studies suggest a putative role for innexins in correlating activity between coupled neurons at the levels of gene expression and physiological activity during development and in the adult animal.

Central pattern generator (CPG) networks rely on a balance of intrinsic and network properties to produce reliable, repeatable activity patterns. This balance is maintained by homeostatic plasticity where alterations in neuronal properties dynamically maintain appropriate neural output in the face of changing environmental conditions and perturbations. However, it remains unclear just how these neurons and networks can both monitor their ongoing activity and use this information to elicit homeostatic physiological responses to ensure robustness of output over time. Evidence exists that CPG networks use a mixed strategy of activity-dependent, activity-independent, modulator-dependent, and synaptically regulated homeostatic plasticity to achieve this critical stability. In this review, we focus on some of the current understanding of the molecular pathways and mechanisms responsible for this homeostatic plasticity in the context of central pattern generator function, with a special emphasis on some of the smaller invertebrate networks that have allowed for extensive cellular-level analyses that have brought recent insights to these questions.

A new genetically encoded system manipulates the pH inside cells to detect whether they are coupled to each other.