Functional Recovery of a Locomotor Network after Injury: Plasticity beyond the Central Nervous System.

Many animals depend on descending information from the brain for the initiation and proper execution of locomotion. Interestingly, after injury and the loss of such inputs, locomotor function can sometimes be regained without the regrowth of central connections. In the medicinal leech, Hirudo verbana, we have shown that crawling reemerges after removal of descending inputs. Here, we studied the mechanisms underlying this return of locomotion by asking if central pattern generators (CPGs) in crawl-recovered leeches are sufficient to produce crawl-specific intersegmental coordination. From recovered animals, we treated isolated chains of ganglia with dopamine to activate the crawl CPGs (one crawl CPG per ganglion) and observed fictive crawl-like bursting in the dorsal-longitudinal-excitor motoneuron (DE-3), an established crawl-monitor neuron. However, these preparations did not exhibit crawl-specific coordination across the CPGs. Although the crawl CPGs always generated bidirectional activation of adjacent CPGs, we never observed crawl-appropriate intersegmental phase delays. Because central circuits alone were unable to organize crawl-specific coordination, we tested the coordinating role of the peripheral nervous system. In transected leeches normally destined for recovery, we removed afferent information to the anterior-most (lead) ganglion located below the nerve-cord transection site. In these dually treated animals, overt crawling was greatly delayed or prevented. After filling the peripheral nerves with Neurobiotin tracer distal to the nerve-root lesion, we found a perfect correlation between regrowth of peripheral neuronal fibers and crawl recovery. Our study establishes that during recovery after injury, crawl-specific intersegmental coordination switches to a new dependence on afferent information.

An annotated CNS transcriptome of the medicinal leech, Hirudo verbana: De novo sequencing to characterize genes associated with nervous system activity.

The medicinal leech is one of the most venerated model systems for the study of fundamental nervous system principles, ranging from single-cell excitability to complex sensorimotor integration. Yet, molecular analyses have yet to be extensively applied to complement the rich history of electrophysiological study that this animal has received. Here, we generated the first de novo transcriptome assembly from the entire central nervous system of Hirudo verbana, with the goal of providing a molecular resource, as well as to lay the foundation for a comprehensive discovery of genes fundamentally important for neural function. Our assembly generated 107,704 contigs from over 900 million raw reads. Of these 107,704 contigs, 39,047 (36%) were annotated using NCBI's validated RefSeq sequence database. From this annotated central nervous system transcriptome, we began the process of curating genes related to nervous system function by identifying and characterizing 126 unique ion channel, receptor, transporter, and enzyme contigs. Additionally, we generated sequence counts to estimate the relative abundance of each identified ion channel and receptor contig in the transcriptome through Kallisto mapping. This transcriptome will serve as a valuable community resource for studies investigating the molecular underpinnings of neural function in leech and provide a reference for comparative analyses.

The stomatogastric nervous system of the medicinal leech: its anatomy, physiology and associated aminergic neurons.

Blood feeding is an essential and signature activity of the medicinal leech species Hirudo verbana. Despite keen interest in understanding the neuronal substrates of this behavior, a major component of the nervous system associated with feeding has remained overlooked. In this study, for the first time, we report on the presence and characteristics of five stomatogastric ganglia (STGs) comprising the visceral stomatogastric nervous system (STN) of the leech. Although a brief report was published by Ruth Hanke in 1948 indicating that a ring of three ganglia (not five) was associated with the cephalic ganglia, this information was never integrated into subsequent neurobiological studies of feeding. Here, the anatomical features of the STGs are described, as are the morphological and electrophysiological characteristics of neurons originating in them. We also determined that two of the five STGs (STG-1 and STG-3) each contained two relatively large (ca. 40 µm diameter) serotonergic neurons. The STN was also enriched with dopaminergic and serotonergic arborizations; however, no intrinsic dopaminergic somata were observed. The trajectory of the serotonergic large lateral (LL) neuron, a command-like cell for feeding, was documented to project directly to the STN and not to the jaw and pharyngeal musculature as previously reported, thus reopening the important question of how the LL cell activates and coordinates biting activity with pharyngeal swallowing. Additional studies revealed that the LL cell is excited by blood serum applied to the lip and is strongly inhibited by dopamine. These findings provide a new foundation for understanding the regulation and modulation of neural networks involved in feeding.

Motoneurons regulate the central pattern generator during drug-induced locomotor-like activity in the neonatal mouse.

Motoneurons are traditionally viewed as the output of the spinal cord that do not influence locomotor rhythmogenesis. We assessed the role of motoneuron firing during ongoing locomotor-like activity in neonatal mice expressing archaerhopsin-3 (Arch), halorhodopsin (eNpHR), or channelrhodopsin-2 (ChR2) in Choline acetyltransferase neurons (ChAT+) or Arch in LIM-homeodomain transcription factor Isl1+ neurons. Illumination of the lumbar cord in mice expressing eNpHR or Arch in ChAT+ or Isl1+ neurons, depressed motoneuron discharge, transiently decreased the frequency, and perturbed the phasing of the locomotor-like rhythm. When the light was turned off motoneuron firing and locomotor frequency both transiently increased. These effects were not due to cholinergic neurotransmission, persisted during partial blockade of gap junctions and were mediated, in part, by AMPAergic transmission. In spinal cords expressing ChR2, illumination increased motoneuron discharge and transiently accelerated the rhythm. We conclude that motoneurons provide feedback to the central pattern generator (CPG) during drug-induced locomotor-like activity.

Compensatory plasticity restores locomotion after chronic removal of descending projections.

Homeostatic plasticity is an important attribute of neurons and their networks, enabling functional recovery after perturbation. Furthermore, the directed nature of this plasticity may hold a key to the restoration of locomotion after spinal cord injury. Here we studied the recovery of crawling in the leech Hirudo verbana after descending cephalic fibers were surgically separated from crawl central pattern generators shown previously to be regulated by dopamine. We observed that immediately after nerve cord transection leeches were unable to crawl, but remarkably, after a day to weeks, animals began to show elements of crawling and intersegmental coordination. Over a similar time course, excessive swimming due to the loss of descending inhibition returned to control levels. Additionally, removal of the brain did not prevent crawl recovery, indicating that connectivity of severed descending neurons was not essential. After crawl recovery, a subset of animals received a second transection immediately below the anterior-most ganglion remaining. Similar to their initial transection, a loss of crawling with subsequent recovery was observed. These data, in recovered individuals, support the idea that compensatory plasticity directly below the site of injury is essential for the initiation and coordination of crawling. We maintain that the leech provides a valuable model to understand the neural mechanisms underlying locomotor recovery after injury because of its experimental accessibility, segmental organization, and dependence on higher-order control involved in the initiation, modulation, and coordination of locomotor behavior.

Necessary, sufficient and permissive: a single locomotor command neuron important for intersegmental coordination.

In this report we posed the overarching question: What multiple contributions can a single neuron have on controlling the behavior of an animal, especially within a given context? To address this timely question, we studied the neuron R3b-1 in the medicinal leech. This bilaterally paired neuron descends from the cephalic ganglion and projects uninterrupted through the segmental ganglia comprising the nerve cord; its terminal arbors invade each hemi-ganglion. We discovered that a single R3b-1 neuron functions as a command neuron in the strictest sense, as it was both necessary and sufficient for fictive crawling behavior. Aside from these command-related properties, we determined that R3b-1 modulates the cycle period of crawl motor activity. R3b-1 has previously been shown to activate swimming behavior, but when the CNS was exposed to dopamine (DA), crawling became the exclusive locomotor pattern produced by R3b-1. DA exposure also led to bursting in R3b-1 that matched periods observed during fictive crawling, even when potential ascending inputs from crawl oscillators were removed. Although the above attributes render R3b-1 an intriguing cell, it is its ability to permit the coordination of the segmentally distributed crawl oscillators that makes this multifunctional neuron so notable. To our knowledge, this cell provides the first biological example of a single command neuron that is also vital for the intersegmental coordination of a locomotor behavior. Furthermore, our study highlights the importance of DA as an internal contextual cue that can integrate functional layers of the nervous system for adaptive behavior.

Nanowires precisely grown on the ends of microwire electrodes permit the recording of intracellular action potentials within deeper neural structures.

AIMS: Nanoelectrodes are an emerging biomedical technology that can be used to record intracellular membrane potentials from neurons while causing minimal damage during membrane penetration. Current nanoelectrode designs, however, have low aspect ratios or large substrates and thus are not suitable for recording from neurons deep within complex natural structures, such as brain slices. MATERIALS & METHODS: We describe a novel nanoelectrode design that uses nanowires grown on the ends of microwire recording electrodes similar to those frequently used in vivo. RESULTS & DISCUSSION: We demonstrate that these nanowires can record intracellular action potentials in a rat brain slice preparation and in isolated leech ganglia. CONCLUSION: Nanoelectrodes have the potential to revolutionize intracellular recording methods in complex neural tissues, to enable new multielectrode array technologies and, ultimately, to be used to record intracellular signals in vivo.

Keeping it together: mechanisms of intersegmental coordination for a flexible locomotor behavior.

The coordination of multiple neural oscillators is key for the generation of productive locomotor movements. In the medicinal leech, we determined that activation and coordination of the segmental crawl oscillators, or unit burst generators, are dependent on signals descending from the cephalic ganglion. In nearly intact animals, removing descending input (reversibly with a sucrose block) prevented overt crawling, but not swimming. Cephalic depolarization was sufficient for coordination. To determine whether descending signals were necessary for the generation and maintenance of posterior-directed intersegmental phase delays, we induced fictive crawling in isolated whole nerve cords using dopamine (DA) and blocked descending inputs. After blockade, we observed a significant loss of intersegmental coordination. Appropriate phase delays were also absent in DA-treated chains of ganglia. In chains, when one ganglion was removed from its neighbors, crawling in that ganglion emerged robust and stable, underscoring that these oscillators operate best with either all or none of their intersegmental inputs. To study local oscillator coupling, we induced fictive crawling (with DA) in a single oscillator within a chain. Although appropriate intersegmental phase delays were always absent, when one ganglion was treated with DA, neighboring ganglia began to show crawl-like bursting, with motoneuron spikes/burst greatest in untreated posterior ganglia. We further determined that this local excitatory drive excluded the swim-gating cell, 204. In conclusion, both long-distance descending and local interoscillator coupling contribute to crawling. This dual contribution helps to explain the inherent flexibility of crawling, and provides a foundation for understanding other dynamic locomotor behaviors across animal groups.

Dopamine activates the motor pattern for crawling in the medicinal leech.

Locomotion in segmented animals is thought to be based on the coupling of "unit burst generators," but the biological nature of the unit burst generator has been revealed in only a few animal systems. We determined that dopamine (DA), a universal modulator of motor activity, is sufficient to activate fictive crawling in the medicinal leech, and can exert its actions within the smallest division of the animal's CNS, the segmental ganglion. In the entire isolated nerve cord or in the single ganglion, DA induced slow antiphasic bursting (approximately 15 s period) of motoneurons known to participate in the two-step elongation-contraction cycle underlying crawling behavior. During each cycle, the dorsal (DE-3) and ventral (VE-4) longitudinal excitor motoneurons fired approximately 180 degrees out of phase from the ventrolateral circular excitor motoneuron (CV), which marks the elongation phase. In many isolated whole nerve cords, DE-3 bursting progressed in an anterior to posterior direction with intersegmental phase delays appropriate for crawling. In the single ganglion, the dorsal (DI-1) and ventral (VI-2) inhibitory longitudinal motoneurons fired out of phase with each DE-3 burst, further confirming that the crawl unit burst generator exists in the single ganglion. All isolated ganglia of the CNS were competent to produce DA-induced robust fictive crawling, which typically lasted uninterrupted for 5-15 min. A quantitative analysis indicated that DA-induced crawling was not significantly different from electrically evoked or spontaneous crawling. We conclude that DA is sufficient to activate the full crawl motor program and that the kernel for crawling resides within each segmental ganglion.