Combining Old and New Tricks: The Study of Genes, Neurons, and Behavior in Crayfish.

For over a century the nervous system of decapod crustaceans has been a workhorse for the neurobiology community. Many fundamental discoveries including the identification of electrical and inhibitory synapses, lateral and pre-synaptic inhibition, and the Na+/K+-pump were made using lobsters, crabs, or crayfish. Key among many advantages of crustaceans for neurobiological research is the unique access to large, accessible, and identifiable neurons, and the many distinct and complex behaviors that can be observed in lab settings. Despite these advantages, recent decades have seen work on crustaceans hindered by the lack of molecular and genetic tools required for unveiling the cellular processes contributing to neurophysiology and behavior. In this perspective paper, we argue that the recently sequenced marbled crayfish, Procambarus virginalis, is suited to become a genetic model system for crustacean neuroscience. P. virginalis are parthenogenetic and produce genetically identical offspring, suggesting that germline transformation creates transgenic animal strains that are easy to maintain across generations. Like other decapod crustaceans, marbled crayfish possess large neurons in well-studied circuits such as the giant tail flip neurons and central pattern generating neurons in the stomatogastric ganglion. We provide initial data demonstrating that marbled crayfish neurons are accessible through standard physiological and molecular techniques, including single-cell electrophysiology, gene expression measurements, and RNA-interference. We discuss progress in CRISPR-mediated manipulations of the germline to knock-out target genes using the 'Receptor-mediated ovary transduction of cargo' (ReMOT) method. Finally, we consider the impact these approaches will have for neurophysiology research in decapod crustaceans and more broadly across invertebrates.

The dynamic range of voltage-dependent gap junction signaling is maintained by Ih-induced membrane potential depolarization.

Like their chemical counterparts, electrical synapses show complex dynamics such as rectification and voltage dependence that interact with other electrical processes in neurons. The consequences arising from these interactions for the electrical behavior of the synapse, and the dynamics they create, remain largely unexplored. Using a voltage-dependent electrical synapse between a descending modulatory projection neuron (MCN1) and a motor neuron (LG) in the crustacean stomatogastric ganglion, we find that the influence of the hyperpolarization-activated inward current (Ih) is critical to the function of the electrical synapse. When we blocked Ih with CsCl, the apparent voltage dependence of the electrical synapse shifted by 18.7 mV to more hyperpolarized voltages, placing the dynamic range of the electrical synapse outside of the range of voltages used by the LG motor neuron (-60.2 mV to -44.9 mV). With dual electrode current- and voltage-clamp recordings, we demonstrate that this voltage shift is not due to a change in the properties of the gap junction itself, but is a result of a sustained effect of Ih on the presynaptic MCN1 axon terminal membrane potential. Ih-induced depolarization of the axon terminal membrane potential increased the electrical postsynaptic potentials and currents. With Ih present, the axon terminal resting membrane potential is depolarized, shifting the dynamic range of the electrical synapse toward the functional range of the motor neuron. We thus demonstrate that the function of an electrical synapse is critically influenced by a voltage-dependent ionic current (Ih).NEW & NOTEWORTHY Electrical synapses and voltage-gated ionic currents are often studied independently from one another, despite mounting evidence that their interactions can alter synaptic behavior. We show that the hyperpolarization-activated inward ionic current shifts the voltage dependence of electrical synaptic transmission through its depolarizing effect on the membrane potential, enabling it to lie within the functional membrane potential range of a motor neuron. Thus, the electrical synapse's function critically depends on the voltage-gated ionic current.

Neuropeptide Modulation Increases Dendritic Electrical Spread to Restore Neuronal Activity Disrupted by Temperature.

Peptide neuromodulation has been implicated to shield neuronal activity from acute temperature changes that can otherwise lead to loss of motor control or failure of vital behaviors. However, the cellular actions neuropeptides elicit to support temperature-robust activity remain unknown. Here, we find that peptide neuromodulation restores rhythmic bursting in temperature-compromised central pattern generator (CPG) neurons by counteracting membrane shunt and increasing dendritic electrical spread. We show that acutely rising temperatures reduced spike generation and interrupted ongoing rhythmic motor activity in the crustacean gastric mill CPG. Neuronal release and extrinsic application of Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), a substance-P-related peptide, restored rhythmic activity. Warming led to a significant decrease in membrane resistance and a shunting of the dendritic signals in the main gastric mill CPG neuron. Using a combination of fluorescent calcium imaging and electrophysiology, we observed that postsynaptic potentials and antidromic action potentials propagated less far within the dendritic neuropil as the system warmed. In the presence of CabTRP Ia, membrane shunt decreased and both postsynaptic potentials and antidromic action potentials propagated farther. At elevated temperatures, CabTRP Ia restored dendritic electrical spread or extended it beyond that at cold temperatures. Selective introduction of the CabTRP Ia conductance using a dynamic clamp demonstrated that the CabTRP Ia voltage-dependent conductance was sufficient to restore rhythmic bursting. Our findings demonstrate that a substance-P-related neuropeptide can boost dendritic electrical spread to maintain neuronal activity when perturbed and reveals key neurophysiological components of neuropeptide actions that support pattern generation in temperature-compromised conditions.SIGNIFICANCE STATEMENT Changes in body temperature can have detrimental consequences for the well-being of an organism. Temperature-dependent changes in neuronal activity can be especially dangerous if they affect vital behaviors. Understanding how temperature changes disrupt neuronal activity and identifying how to ameliorate such effects is critically important. Our study of a crustacean circuit shows that warming disrupts rhythmic neuronal activity by increasing membrane shunt and reducing dendritic electrical spread in a key circuit neuron. Through the ionic conductance activated by it, substance-P-related peptide modulation restored electrical spread and counteracted the detrimental temperature effects on rhythmic activity. Because neuropeptides are commonly implicated in sustaining neuronal activity during perturbation, our results provide a promising mechanism to support temperature-robust activity.

Temperature-robust activity patterns arise from coordinated axonal Sodium channel properties.

Action potentials are a key component of neuronal communication and their precise timing is critical for processes like learning, memory, and complex behaviors. Action potentials propagate through long axons to their postsynaptic partners, which requires axons not only to faithfully transfer action potentials to distant synaptic regions but also to maintain their timing. This is particularly challenging when axons differ in their morphological and physiological properties, as timing is predicted to diverge between these axons when extrinsic conditions change. It is unknown if and how diverse axons maintain timing during temperature changes that animals and humans encounter. We studied whether ambient temperature changes cause different timing in the periphery of neurons that centrally produce temperature-robust activity. In an approach combining modeling, imaging, and electrophysiology, we explored mechanisms that support timing by exposing the axons of three different neuron types from the same crustacean (Cancer borealis) motor circuit and involved in the same functional task to a range of physiological temperatures. We show that despite substantial differences between axons, the effects of temperature on action potential propagation were moderate and supported temperature-robust timing over long-distances. Our modeling demonstrates that to maintain timing, the underlying channel properties of these axons do not need to be temperature-insensitive or highly restricted, but coordinating the temperature sensitivities of the Sodium activation gate time constant and the maximum Sodium conductance is required. Thus, even highly temperature-sensitive ion channel properties can support temperature-robust timing between distinct neuronal types and across long distances.

Physiologists turned Geneticists: Identifying transcripts and genes for neuronal function in the Marbled Crayfish, Procambarus virginalis.

The number of undergraduate researchers interested in pursuing neurophysiological research exceeds the research laboratory positions and hands-on course experiences available because these types of experiments often require extensive experience or expensive equipment. In contrast, genetic and molecular tools can more easily incorporate undergraduates with less time or training. With the explosion of newly sequenced genomes and transcriptomes, there is a large pool of untapped molecular and genetic information which would greatly inform neurophysiological processes. Classically trained neurophysiologists often struggle to make use of newly available genetic information for themselves and their trainees, despite the clear advantage of combining genetic and physiological techniques. This is particularly prevalent among researchers working with organisms that historically had no or only few genetic tools available. Combining these two fields will expose undergraduates to a greater variety of research approaches, concepts, and hands-on experiences. The goal of this manuscript is to provide an easily understandable and reproducible workflow that can be applied in both lab and classroom settings to identify genes involved in neuronal function. We outline clear learning objectives that can be acquired by following our workflow and assessed by peer-evaluation. Using our workflow, we identify and validate the sequence of two new Gamma Aminobutyric Acid A (GABAA) receptor subunit homologs in the recently published genome and transcriptome of the marbled crayfish, Procambarus virginalis. Altogether, this allows undergraduate students to apply their knowledge of the processes of gene expression to functional neuronal outcomes. It also provides them with opportunities to contribute significantly to physiological research, thereby exposing them to interdisciplinary approaches.

State-Dependent Modification of Sensory Sensitivity via Modulation of Backpropagating Action Potentials.

Neuromodulators play a critical role in sensorimotor processing via various actions, including pre- and postsynaptic signal modulation and direct modulation of signal encoding in peripheral dendrites. Here, we present a new mechanism that allows state-dependent modulation of signal encoding in sensory dendrites by neuromodulatory projection neurons. We studied the impact of antidromic action potentials (APs) on stimulus encoding using the anterior gastric receptor (AGR) neuron in the heavily modulated crustacean stomatogastric ganglion (STG). We found that ectopic AP initiation in AGR's axon trunk is under direct neuromodulatory control by the inferior ventricular (IV) neurons, a pair of descending projection neurons. IV neuron activation elicited a long-lasting decrease in AGR ectopic activity. This modulation was specific to the site of AP initiation and could be mimicked by focal application of the IV neuron co-transmitter histamine. IV neuron actions were diminished after blocking H2 receptors in AGR's axon trunk, suggesting a direct axonal modulation. This local modulation did not affect the propagation dynamics of en passant APs. However, decreases in ectopic AP frequency prolonged sensory bursts elicited distantly near AGR's dendrites. This frequency-dependent effect was mediated via the reduction of antidromic APs, and the diminishment of backpropagation into the sensory dendrites. Computational models suggest that invading antidromic APs interact with local ionic conductances, the rate constants of which determine the sign and strength of the frequency-dependent change in sensory sensitivity. Antidromic APs therefore provide descending projection neurons with a means to influence sensory encoding without affecting AP propagation or stimulus transduction.

Changes in Dorsal Root Ganglion Gene Expression in Response to Spinal Cord Stimulation.

BACKGROUND AND OBJECTIVES: Spinal cord stimulation (SCS) has been shown to influence pain-related genes in the spinal cord directly under the stimulating electrodes. There is limited information regarding changes occurring at the dorsal root ganglion (DRG). This study evaluates gene expression in the DRG in response to SCS therapy. METHODS: Rats were randomized into experimental or control groups (n = 6 per group). Experimental animals underwent spared-nerve injury, implantation of lead, and continuous SCS (72 hours). Behavioral assessment for mechanical hyperalgesia was conducted to compare responses after injury and treatment. Ipsilateral DRG tissue was collected, and gene expression quantified for interleukin 1b (IL-1b), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), GABA B receptor 1 (GABAbr1), substance P (subP), Integrin alpha M (ITGAM), sodium/potassium ATP-ase (Na/K ATPase), fos proto-oncogene (cFOS), serotonin receptor 3A (5HT3r), galanin (Gal), vasoactive intestinal peptide (VIP), neuropeptide Y (NpY), glial fibrillary acidic protein (GFAP), and brain derived neurotropic factor (BDNF) via quantitative polymerase chain reaction. Statistical significance was established using analysis of variance (ANOVA), independent t tests, and Pearson correlation tests. RESULTS: Expression of IL-1b and IL-6 was reversed following SCS therapy relative to the increase caused by the injury model. Both GABAbr1 and Na/K ATPase were significantly up-regulated upon implantation of the lead, and SCS therapy reversed their expression to within control levels. Pearson correlation analyses reveal that GABAbr1 and Na/K ATPase expression was dependent on the stimulating current intensity. CONCLUSIONS: Spinal cord stimulation modulates expression of key pain-related genes in the DRG. Specifically, SCS led to reversal of IL-1b and IL-6 expression induced by injury. Interleukin 6 expression was still significantly larger than in sham animals, which may correlate to residual sensitivity following continuous SCS treatment. In addition, expression of GABAbr1 and Na/K ATPase was down-regulated to within control levels following SCS and correlates with applied current.

Extracellular Axon Stimulation.

This is a detailed protocol explaining how to perform extracellular axon stimulations as described in Städele and Stein, 2016. The ability to stimulate and record action potentials is essential to electrophysiological examinations of neuronal function. Extracellular stimulation of axons traveling in fiber bundles (nerves) is a classical technique in brain research and a fundamental tool in neurophysiology (Abbas and Miller, 2004; Barry, 2015; Basser and Roth, 2000; Cogan, 2008). It allows for activating action potentials in individual or multiple axons, controlling their firing frequency, provides information about the speed of neuronal communication, and neuron health and function.

Axonal Conduction Velocity Measurement.

Action potential conduction velocity is the speed at which an action potential (AP) propagates along an axon. Measuring AP conduction velocity is instrumental in determining neuron health, function, and computational capability, as well as in determining short-term dynamics of neuronal communication and AP initiation (Ballo and Bucher, 2009; Bullock, 1951; Meeks and Mennerick, 2007; Rosenthal and Bezanilla, 2000; Städele and Stein, 2016; Swadlow and Waxman, 1976). Conduction velocity can be measured using extracellular recordings along the nerve through which the axon projects. Depending on the number of axons in the nerve, AP velocities of individual or many axons can be detected. This protocol outlines how to measure AP conduction velocity of (A) stimulated APs and (B) spontaneously generated APs by using two spatially distant extracellular electrodes. Although an invertebrate nervous system is used here, the principles of this technique are universal and can be easily adjusted to other nervous system preparations (including vertebrates).

Genomics of the Effect of Spinal Cord Stimulation on an Animal Model of Neuropathic Pain.

BACKGROUND: Few studies have evaluated single-gene changes modulated by spinal cord stimulation (SCS), providing a narrow understanding of molecular changes. Genomics allows for a robust analysis of holistic gene changes in response to stimulation. METHODS: Rats were randomized into six groups to determine the effect of continuous SCS in uninjured and spared-nerve injury (SNI) animals. After behavioral assessment, tissues from the dorsal quadrant of the spinal cord (SC) and dorsal root ganglion (DRG) underwent full-genome microarray analyses. Weighted Gene Correlation Network Analysis (WGCNA), and Gene Ontology (GO) analysis identified similar expression patterns, molecular functions and biological processes for significant genes. RESULTS: Microarray analyses reported 20,985 gene probes in SC and 19,104 in DRG. WGCNA sorted 7449 SC and 4275 DRG gene probes into 29 and 9 modules, respectively. WGCNA provided significant modules from paired comparisons of experimental groups. GO analyses reported significant biological processes influenced by injury, as well as the presence of an electric field. The genes Tlr2, Cxcl16, and Cd68 were used to further validate the microarray based on significant response to SCS in SNI animals. They were up-regulated in the SC while both Tlr2 and Cd68 were up-regulated in the DRG. CONCLUSIONS: The process described provides highly significant interconnected genes and pathways responsive to injury and/or electric field in the SC and DRG. Genes in the SC respond significantly to the SCS in both injured and uninjured animals, while those in the DRG significantly responded to injury, and SCS in injured animals.